InteractiveFly: GeneBrief

Dicer-1 : Biological Overview | Regulation & Characterization of RNAi Process | Developmental Biology | Effects of RNAi Depletion | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

Because of its ability to digest double-stranded RNA (dsRNA) into uniformly sized, small RNAs, this enzyme has been named Dicer. Dicer contains a region of homology to the RDE1/QDE2/ARGONAUTE family that has been genetically linked to RNAi (RNA interference). RNAi is a mechanism through which double-stranded RNAs silence cognate genes. In plants and animals, this can occur at both the transcriptional and the post-transcriptional levels. In both plants and animals, RNAi is characterized by the presence of RNAs of about 22 nucleotides in length that are homologous to the gene that is being suppressed. These 22-nucleotide sequences serve as guide sequences that instruct a multicomponent nuclease, RISC (RNA induced silencing complex), to destroy specific messenger RNAs. Dicer, an enzyme that can produce putative guide RNAs, is a member of the RNase III family of nucleases that specifically cleave double-stranded RNAs, and is evolutionarily conserved in worms, flies, plants, fungi and mammals. The enzyme has a distinctive structure, which includes a helicase domain (involved in unwinding RNA) and dual RNase III motifs (Bernstein, 2001).

Post-transcriptional gene silencing (PTGS) can be distinguished from RNAi by the fact that the target mRNA is not degraded but is translationally silenced. Biochemical studies have suggested that PTGS is accomplished, in part, by a mechanism similar to RNAi in that involves a nuclease that targets RNAs for degradation. Subsequently, an enzyme complex similar to RISC, targets mRNA for translational silencing. The specificity of this complex may derive from the incorporation of a small guide sequence that is homologous to the mRNA substrate. These ~22-nucleotide RNAs, originally identified in plants that were actively silencing transgenes, have been produced during RNAi in vitro using an extract prepared from Drosophila embryos. Putative guide RNAs can also be produced in extracts from Drosophila S2 cells. To investigate the mechanism of RNAi and PTGS, both biochemical fractionation and candidate gene approaches have been performed to identify the enzymes that execute each step of the related processes (Bernstein, 2001).

The enzyme complex RISC, an effector nuclease for RNAi [Hammond, 2000: see Characterization of RISC (RNA-induced silencing complex) in Drosophila] was isolated from Drosophila S2 cells in which RNAi had been initiated in vivo by transfection with double-stranded RNA (dsRNA). First, an investigation was carried out to discover whether the RISC enzyme, and the enzyme that initiates RNAi through processing of dsRNA into 22-nucleotide sequences, are distinct activities. RISC activity can be largely cleared from extracts by high-speed centrifugation (100,000g for 60 min), whereas the activity that produces 22-nucleotide sequences remains in the supernatant. This simple fractionation indicates that RISC and the 22-nucleotide sequence-generating activity may be separable. However, it seems probable that these enzymes interact at some point during the silencing process, and it remains possible that initiator and effector enzymes share common subunits (Bernstein, 2001).

RNase III family members are among the few nucleases that show specificity for dsRNA. Analysis of the Drosophila and Caenorhabditis elegans genomes revealed several types of RNase III enzymes: (1) the canonical RNase III, which contains a single RNase III signature motif and a dsRNA-binding domain (dsRBD); (2) a class represented by Drosha, a Drosophila enzyme that contains two RNase III motifs and a dsRBD (CeDrosha in C. elegans); (3) a class that contains two RNase III signatures and an amino-terminal helicase domain (for example, Drosophila CG4792 (Dicer 1) and CG6493 (Dicer 2) ; C. elegans K12H4.8), which have been proposed as potential RNAi nucleases. Representatives of all three classes were tested for the ability to produce discrete RNAs of ~22 nucleotides from dsRNA substrates (Bernstein, 2001).

To test the dual RNase III enzymes, variants of Drosha and CG4792 (subsequently called Dicer-1) tagged with the T7 epitope were prepared. These were expressed in transfected S2 cells and isolated by immunoprecipitation using antibody-agarose conjugates. Treatment of the dsRNA with the CG4792 immunoprecipitate yielded fragments of about 22 nucleotides, similar to those produced in either the S2 or embryo extracts. Neither the activity in extract nor that in immunoprecipitates depended on the sequence of the RNA substrate, since dsRNAs derived from several genes were processed equivalently. Negative results were obtained with Drosha and with immunoprecipitates of a DExH box helicase (Homeless). Western blotting confirmed that each of the tagged proteins was expressed and immunoprecipitated similarly. Thus, it is concluded that CG4792 may carry out the initiation step of RNAi by producing guide sequences of about 22 nucleotides from dsRNAs (Bernstein, 2001).

An antiserum directed against the carboxy terminus of the Dicer protein immunoprecipitates a nuclease activity (from either the Drosophila embryo extracts or from S2 cell lysates) that produces RNAs of about 22 nucleotides from dsRNA substrates. The putative guide RNAs that are produced by the Dicer enzyme precisely co-migrate with 22-nucleotide sequences that are produced in extract, and with 22-nucleotide sequences that are associated with the RISC enzyme. The enzyme that produces guide RNAs in Drosophila embryo extracts is ATP dependent. Depletion of ATP results in a roughly sixfold reduction of dsRNA cleavage rate and in the production of RNAs with a slightly lower mobility. Of note, both Dicer immunoprecipitates and extracts from S2 cells require ATP for the production of ~22-nucleotide sequences. The accumulation of lower-mobility products was not observed in these cases, although ~22-nucleotide sequences were routinely observe in ATP-depleted embryo extracts. The requirement of Dicer nuclease for ATP is an unusual property, and may indicate that unwinding of guide RNAs by the helicase domain is required for the enzyme to act catalytically (Bernstein, 2001).

For efficient induction of RNAi in C. elegans and in Drosophila, the initiating RNA must be double-stranded and must also be several hundred nucleotides in length. Similarly, Dicer is inactive against single-stranded RNAs regardless of length. The enzyme can digest both 200- and 500-nucleotide dsRNAs, but is significantly less active with shorter substrates. In contrast, Escherichia coli RNase III can digest to completion dsRNAs of either 35 or 22 nucleotides. This suggests that the substrate preferences of the Dicer enzyme may contribute to, but not wholly determine, the size dependence of RNAi (Bernstein, 2001).

These results indicate that the process of RNAi can be divided into at least two distinct steps. Initiation of RNAi would occur upon processing of a dsRNA by Dicer into ~22-nucleotide guide sequences, although the possibility that another Dicer-associated nuclease may participate in this process cannot be formally excluded. These guide RNAs would be incorporated into a distinct nuclease complex (RISC) that targets single-stranded mRNAs for degradation. An implication of this model is that the guide sequences are themselves derived directly from the dsRNA that triggers the response. In accord with this model, it has been shown that ³²P-labelled, exogenous dsRNAs that have been introduced into S2 cells by transfection are incorporated into the RISC enzyme as 22-nuclotide sequences (Bernstein, 2001).

With the identification of Dicer as a potential catalyst of the initiation step of RNAi, the biochemical basis of this unusual mechanism of gene regulation is now open to investigation. It is now important to determine whether the conserved family members from other organisms, particularly mammals, also have a function in dsRNA-mediated gene regulation (Bernstein, 2001).

Dicer-1-dependent Dacapo suppression acts downstream of Insulin receptor in regulating cell division of Drosophila germline stem cells

It is important to understand the regulation of stem cell division because defects in this process can cause altered tissue homeostasis or cancer. The cyclin-dependent kinase inhibitor Dacapo (Dap), a p21/p27 homolog, acts downstream of the microRNA (miRNA) pathway to regulate the cell cycle in Drosophila germline stem cells (GSCs). Tissue-extrinsic signals, including insulin, also regulate cell division of GSCs. Intrinsic and extrinsic regulators intersect in GSC division control; the Insulin receptor (InR) pathway regulates Dap levels through miRNAs, thereby controlling GSC division. Using GFP-dap 3'UTR sensors in vivo, this study shows that in GSCs the dap 3'UTR is responsive to Dicer-1, an RNA endonuclease III required for miRNA processing. Furthermore, the dap 3'UTR can be directly targeted by miR-7, miR-278 and miR-309 in luciferase assays. Consistent with this, miR-278 and miR-7 mutant GSCs are partially defective in GSC division and show abnormal cell cycle marker expression, respectively. These data suggest that the GSC cell cycle is regulated via the dap 3'UTR by multiple miRNAs. Furthermore, the GFP-dap 3'UTR sensors respond to InR but not to TGF-beta signaling, suggesting that InR signaling utilizes Dap for GSC cell cycle regulation. The miRNA-based Dap regulation may act downstream of InR signaling; Dcr-1 and Dap are required for nutrition-dependent cell cycle regulation in GSCs and reduction of dap partially rescues the cell cycle defect of InR-deficient GSCs. These data suggest that miRNA- and Dap-based cell cycle regulation in GSCs can be controlled by InR signaling (Yu, 2009).

Previous studies have shown that miRNAs may regulate Dap, thereby controlling the cell division of GSCs. This study shows that the dap 3'UTR directly responds to miRNA activities in GSCs. Using luciferase assays, miR-7, miR-278 and miR-309 were identified as miRNAs that can directly repress Dap through the dap 3'UTR in vitro. Although miR-278 and miR-7 play a role in regulating GSC division and cell cycle marker expression, respectively, neither of these mutants showed as dramatic a defect in the GSC cell cycle as Dcr-1-deficient GSCs. Thus, the dap 3'UTR may serve to integrate the effect of multiple miRNAs during cell cycle regulation. It remains possible that some miRNAs involved in this process remain to be identified. It was further shown that InR signaling controls the dap 3'UTR in GSCs. This led to an exploration of the interaction between InR signaling and miRNA/Dap cell cycle regulation. GSCs deficient for InR or Dcr-1 show similar cell cycle defects. Using starvation to control InR signaling, it was shown that both Dcr-1 and dap are required for proper InR signaling-dependent regulation of GSC division. Further, reduction of dap partially rescues the cell division defect of the InR mutant GSCs, suggesting that InR signaling regulates the cell cycle via Dap. These results suggest that miRNAs and Dap act downstream of InR signaling to regulate GSC division (Yu, 2009).

The data suggest that multiple miRNAs can regulate the 3'UTR of dap: miR-7, miR-278 and miR-309 can regulate the dap 3'UTR directly, whereas bantam and miR-8 may regulate it indirectly, or through cryptic MREs in the dap 3'UTR. Using GFP sensor assays, it was also shown that the dap 3'UTR may be directly regulated by miRNAs in the GSCs in vivo. However, which specific miRNAs control endogenous Dap levels in Drosophila GSCs remains unknown. Mammalian p21^cip1 has also been shown to be a direct target for specific miRNAs of the miR-106 family, including miR-290s and miR-372. Further, the mouse miR-290 family has recently been identified as regulating the G1-S transition. In addition, miR-221 and miR-222 have been shown to regulate p27^kip1, thereby promoting cell division in different mammalian cancer cell lines. Neither the miR-290 nor miR-220 family is conserved in Drosophila. Together, these results indicate that the CKIs (Dap) might be a common target for miRNAs in regulating the cell cycle in stem cells. However, the specific miRNAs that regulate the CKIs might vary between organisms (Yu, 2009).

This study reveals novel regulatory roles for miR-7 and miR-278 in the GSC cell cycle. miR-7 and miR-278 can directly target Dap. GSCs deficient for miR-278 show a mild but significant reduction in cell proliferation. Ectopic expression of miR-7 in follicle cells reduces the proportion of cells that stain positive for Dap. Furthermore, ablation of miR-7 in GSCs results in a perturbation of the frequency of CycE-positive GSCs. However, the cell division kinetics of miR-7 mutant GSCs is not reduced, by contrast with the dramatic reduction of cell division in Dcr-1-deficient GSCs. It is plausible that miR-7 and miR-278 act in concert with other miRNAs to regulate the level of Dap in GSCs and thereby contribute to cell cycle control in GSCs (Yu, 2009).

The interaction of multiple miRNAs with the dap 3'UTR might integrate information from multiple pathways. Further studies will reveal what regulates miR-7 and miR-278 expression in GSCs and which other miRNAs might act together in Dap regulation. It is known that miR-7 and the transcriptional repressor Yan mutually repress one another in the eye imaginal disk. In this model, Yan prevents transcription of miR-7 until Erk in the Egfr pathway downregulates Yan activity by phosphorylation, thereby permitting expression of miR-7. Conversely, miR-7 can repress the translation of Yan. Thus, a single pulse of Egfr signaling results in stable expression of miR-7 and repression of Yan. Whether similar regulation will be observed between miR-7 and the signaling pathways that regulate GSC division remains to be seen. It has been suggested that miR-7 might regulate downstream targets of Notch, such as Enhancer of split and Bearded. Thus, miR-7 may have a mild repressive effect on multiple targets in GSCs. Further experiments might illuminate this possibility (Yu, 2009).

miR-278, on the other hand, has been implicated in tissue growth and InR signaling. Overexpression of miR-278 promotes tissue growth in eye and wing imaginal discs. Deficiency of miR-278 leads to a reduced fat body, which is similar to the effect of impaired InR signaling in adipose tissue. Interestingly, miR-278 mutants have elevated insulin/Dilp production and a reduction of insulin sensitivity. Furthermore, miR-278 regulates expanded, which may modulate growth factor signaling including InR. Since InR signaling plays important roles in tissue growth and cell cycle control, it will be interesting to further test how miR-278 may regulate InR signaling, and whether InR signaling might regulate miR-278 in a feedback loop in GSCs (Yu, 2009).

Other miRNAs or miRNA-dependent mechanisms might also play roles in Drosophila GSCs. For example, the miRNA bantam is required for GSC maintenance. A recent study has shown that the Trim-NHL-containing protein Mei-P26, which belongs to the same family as Brain tumor (Brat), affects bantam levels and restricts cell growth and proliferation in the GSC lineage (Neumuller, 2008). Interestingly, most miRNAs are upregulated in mei-P26 mutant flies. By contrast, overexpression mei-P26 in bag of marbles (bam) mutants broadly reduces miRNA levels. This suggests that Mei-P26 regulates proliferation and maintenance of GSC lineages via miRNA levels. Since InR signaling cell-autonomously regulates GSC division but not maintenance, the possible interaction between Mei-P26 and InR signaling might be complex (Yu, 2009).

The systemic compensatory effect of insulin secretion in mammals with defective InR signaling is well documented. Insulin levels in mice with liver-specific InR (Insr - Mouse Genome Informatics) knockout are ~20-fold higher than those of control animals owing to the compensatory response of the pancreatic β-cells and impairment of insulin clearance by the liver. Knockout of the neuronal InR also leads to a mild hyperinsulinemia, indicating whole-body insulin resistance. Furthermore, the knockout of components in the InR signaling pathway, such as Akt2 and the regulatory and catalytic subunits of PI3 kinase, also leads to hyperinsulinemia and glucose intolerance. Therefore, a systemic decrease in InR signaling may lead to compensatory responses (Yu, 2009).

To understand the roles of InR signaling in the GSCs while avoiding any systemic compensatory effect the phenotypes of GSC clones were analyzed. Using a panel of cell cycle markers, it was found that InR mutant GSCs show cell cycle defects similar to those of Dcr-1 mutant GSCs: a reduction of cell division rate, an increased frequency of cells staining positive for Dap and CycE, and a decreased frequency of cells staining positive for CycB. Using GFP-dap 3'UTR sensors, it was shown that the dap 3'UTR responds to InR signaling in GSCs, suggesting that InR signaling can regulate Dap expression through the dap 3'UTR. This, together with genetic data indicating that InR/starvation-dependent cell cycle regulation requires Dcr-1 and dap, has led to the proposalthat InR signaling regulates the cell cycle through miRNAs that further regulate Dap levels. Since a reduction in dap only partially rescues the cell cycle defects of InR mutant GSCs, it is possible that InR signaling might also regulate GSC division by additional mechanisms (Yu, 2009).

InR signaling regulates the cell cycle through multiple mechanisms, mainly through the G1-S, but also partly through the G2-M, transition. Recent work has shown a delay in the G2-M transition in GSCs during C. elegans dauer formation. Starvation and InR deficiency may also affect the G2-M checkpoint in Drosophila GSCs (Hsu, 2008). This study has dissected one possible molecular pathway that InR signaling utilizes to regulate the Drosophila GSC G1-S transition and show that InR signaling can control the cell cycle through miRNA-based regulation of Dap (Yu, 2009).

Many studies have connected InR and CKIs to Tor (Target of rapamycin) or Foxo pathways downstream of InR signaling. In S. cerevisiae, the yeast homolog of p21/p27 is upregulated when Tor signaling is inhibited. Foxo, a transcription factor that can be repressed by InR signaling, is known to play important roles in nutrition-dependent cell cycle regulation by upregulating p21 and p27. In C. elegans, starvation causes L1 cell cycle arrest mediated by InR (daf-2) and Foxo (daf-16): InR represses the function of Foxo, thereby downregulating the CKI (cki-1) and upregulating the miRNA lin-4. This study has shown that a miRNA-based regulation of Dap can be coordinated by InR in Drosophila GSCs (Yu, 2009).

Insulin and insulin-like growth factors (Igf1 and Igf2) are known to play important roles in regulating metabolic and developmental processes in many stem cells. In mammals, Igf signaling is required by different stem cell types, including human and mouse ES cells for survival and self-renewal, neural stem cells for expediting the G1-S transition and cell cycle re-entry, and skeletal muscle satellite cells for promoting the G1-S transition via p27^kip1 downregulation. This study has dissected the molecular mechanism of the InR pathway in another adult stem cell type, tDrosophila GSCs, showing that InR signaling can regulate stem cell division through miRNA-based downregulation of the G1-S inhibitor Dap. Further studies will reveal whether miRNAs also mediate InR signaling in other stem cell types (Yu, 2009).

REGULATION

Targets of Activity

The 21-nucleotide small temporal RNA (stRNA) let-7 regulates developmental timing in Caenorhabditis elegans and probably in other bilateral animals. In vivo and in vitro evidence is presented that in Drosophila a developmentally regulated precursor RNA (see microRNA encoding gene let-7), related in sequence to C. elegans let-7, is cleaved by an RNA interference-like mechanism to produce mature let-7 stRNA. Targeted destruction in cultured human cells of the messenger RNA encoding the enzyme Dicer, which acts in the RNA interference pathway, leads to accumulation of the let-7 precursor. Thus, the RNA interference and stRNA pathways intersect. Both pathways require the RNA-processing enzyme Dicer to produce the active small-RNA component that represses gene expression (Hutvagner, 2001).

Two small temporal RNAs (stRNAs), lin-4 and let-7, regulate the timing of development in Caenorhabditis elegans. stRNAs encode no protein, but instead appear to block the productive translation of mRNA by binding sequences in the 3'-untranslated region of their target mRNAs. let-7 is present in most if not all bilaterally symmetric animals, including Drosophila melanogaster and humans. In Drosophila, let-7 first appears at the end of the third larval instar, accumulates to high levels in pupae, and persists in adult flies (Hutvagner, 2001).

The mechanism by which stRNAs are synthesized is unknown. The ~21-nucleotide (nt) let-7 RNA has been proposed to be cleaved from a larger precursor transcript. The generation of small RNAs from a longer, structured precursor -- double-stranded RNA (dsRNA) -- is an essential feature of the RNA interference (RNAi) pathway, raising the possibility that stRNAs are generated by mechanisms similar to the initial steps in RNAi and suggesting that enzymes such as the Drosophila protein Dicer might play a role in generating stRNAs (Hutvagner, 2001),

Examination of the developmental expression of let-7 in Drosophila revealed a candidate for a let-7 precursor RNA, let-7L (Pasquinelli, 2000). let-7L was detected at the end of the third larval instar and at the beginning of pupation, the same developmental stages where let-7 itself is first expressed. Consistent with the transcript being a let-7 precursor, the amount of let-7L RNA declines as let-7 accumulates. let-7L RNA is slightly shorter than a 76-nt RNA standard. Previous analysis of the genomic sequence flanking Drosophila let-7 led to the proposal that a 72-nt RNA hairpin might be a let-7 precursor (Pasquinelli, 2000; Hutvagner, 2001).

A let-7 homolog is also expressed in human tissues (Pasquinelli, 2000) and in cultured human HeLa cells, but not in Drosophila embryos or cultured Drosophila S2 cells. Primer extension analyses confirmed that the mature Drosophila let-7 RNA detected by Northern hybridization was bona fide let-7. Primer extension products corresponding to the 5' ends of mature let-7 RNAs were detected in total RNA from early and unstaged Drosophila pupae and from human HeLa cells. Primer extension analysis of total RNA from unstaged worms, as well as Northern hybridization experiments, indicated that worm let-7 is 1 nt longer than that in flies and humans. In early pupae, primer extension analysis also detected three longer extension products. The major (middle) product and the less abundant (lower) product comigrate with primer extension products templated by a synthetic 72-nt RNA corresponding to putative pre-let-7. This longer transcript from early pupae has the same 5' end as the 72-nt let-7 precursor and is therefore a good candidate for a let-7 precursor RNA (Pasquinelli, 2000; Hutvagner, 2001).

To determine if the let-7L RNA detected in vivo is, in fact, the direct precursor of mature let-7, processing of the proposed pre-let-7 stem-loop RNA into let-7 was tested in Drosophila embryo lysates, which contain no detectable let-7 RNA (Pasquinelli, 2000). These lysates recapitulate RNAi in vitro, prompting the question of whether the proposed precursor RNA is cleaved into mature let-7 by an RNAi-like mechanism. The 72-nt RNA was incubated with Drosophila embryo lysate for various times, then assayed for the production of let-7 by primer extension. As seen in vivo, mature let-7 RNA accumulates in the cell-free reaction. Thus, an RNA corresponding to the proposed let-7 precursor is converted to an RNA with precisely the same 5' ends as authentic let-7 by one or more factors in the Drosophila embryo lysate (Hutvagner, 2001).

Only let-7 RNA, not its complement, has been detected in vivo in worms, flies, and human tissues (Pasquinelli, 2000). Thus, it is expected that bona fide let-7 maturation in vitro would be asymmetric, yielding only let-7 and not small RNAs complementary to let-7, such as antisense let-7. In contrast, processing of long, dsRNA by the RNAi pathway is symmetric, yielding double-stranded 21- to 22-nt RNAs. Therefore, it was asked if processing of the proposed pre-let-7 RNA in vitro is symmetric or asymmetric, yielding let-7 but not its complement. Four pre-let-7 RNAs were prepared by in vitro transcription, each uniformly labeled with a different alpha-³²P-nucleotide [adenosine 5'-triphosphate (ATP), cytidine 5'-triphosphate, guanosine 5'-triphosphate, or uridine 5'-triphosphate] and incubated separately in an in vitro reaction. Since let-7 contains no cytosine, accurate in vitro processing of pre-let-7 should produce a 21- to 22-nt product for RNAs labeled at A, G, or U but not at C. A product of the appropriate size for let-7 was produced for pre-let-7 transcripts labeled at A, G, and U. No ³²P-labeled product accumulated from the ³²P-C-labeled pre-let-7 RNA. Although pre-let-7 RNA continued to disappear with incubation in the lysate, mature-let-7 production rapidly reached a plateau. Because single-stranded 21-nt RNAs are generally unstable in the embryo lysate, this likely reflects degradation of let-7 in the lysate, which may lack factors required for let-7 stabilization and function. Nonetheless, it is remarkable that let-7 RNA accumulates at all, because exogenous, single-stranded, 21-nt RNAs are degraded by the lysate within minutes (Hutvagner, 2001).

Next, the products of an in vitro reaction were analyzed by Northern hybridization using three different deoxyoligonucleotide probes. Probe 2 was entirely complementary to mature let-7. Probe 3 was complementary to the first 21 nt of the precursor and therefore only partially complementary to mature let-7. Control experiments showed that probe 3 detected mature let-7 substantially less well than probe 2, whereas probe 3 detected as well or better than probe 2 products derived from the precursor sequence that is 5' to the region encoding let-7. Finally, probe 4 was complementary to the side of the stem of the precursor opposite the portion encoding let-7. Thus, probe 4 should detect the products of symmetric processing of the precursor RNA. Control experiments demonstrated that probe 4 readily detected synthetic antisense let-7 RNA, but not let-7 itself. Northern hybridization experiments were quantified by determining the amount of each probe that hybridized to the region of the blot corresponding to the ~21-nt reaction product and, as a control for hybridization efficiency, the amount of hybridization of each probe to the unreacted precursor remaining at 3 hours, because the full-length precursor is perfectly complementary to all three probes. Probe 2, which is complementary to let-7, readily detected an RNA that accumulated with time. In contrast, probe 3 detected only weakly an RNA that accumulated over the course of the reaction, consistent with it detecting by partial hybridization mature let-7 but not reaction products derived from the region of the precursor 5' to the let-7 sequence. Most important, probe 4, which was designed to detect reaction products like antisense let-7, did not detect products that accumulated upon incubation of pre-let-7 in the lysate. These data strongly imply that symmetric processing products such as antisense let-7 are either not generated at all or are far less stable than let-7 in the in vitro reaction. Thus, the in vitro reaction displays the same specificity and asymmetry that characterize let-7 biogenesis in vivo (Hutvagner, 2001).

It remained possible that the mechanisms of cleavage in vitro and in vivo differ. To assess the type of ribonuclease (RNase) that might be responsible for pre-let-7 processing, both in vitro and in vivo, the 5' and 3' ends of both the let-7 generated by the in vitro processing reaction and the let-7 from pupae were analyzed. Treatment with periodate, followed by ß-elimination (of either RNA from the in vitro processing reaction or total pupal RNA) increased the apparent mobility of let-7 by nearly 2 nt, a change diagnostic of RNAs bearing 2',3'-terminal hydroxyl groups. Treatment with calf intestinal phosphatase (CIP) of in vitro-generated let-7 or pupal RNA decreased the apparent mobility of let-7 by 1 nt, consistent with the removal of a charged phosphate group. Furthermore, treatment of the CIP-treated RNA with polynucleotide kinase and ATP restored its original mobility, demonstrating that let-7 contains a monophosphate. Because let-7 contains 2'- and 3'-terminal hydroxyls, this single phosphate must be at its 5' end. Thus, let-7 produced by in vitro processing and let-7 isolated from pupae have the same terminal structure: a 5' monophosphate and 2'- and 3'-terminal hydroxyls. Notably, such termini are characteristic of the products of cleavage of dsRNA by RNase III (Hutvagner, 2001).

The small interfering RNAs (siRNAs) that mediate RNAi also bear a 5' monophosphate and 2'- and 3'-terminal hydroxyls. In Drosophila, siRNA duplexes are produced by the cleavage of long dsRNA by the enzyme Dicer (Bernstein, 2001). Cleavage by Dicer is thought to be catalyzed by its tandem RNase III domains. Only two types of RNase III enzymes are predicted to occur in Drosophila : Drosha (Filippov, 2000) and Dicer. Dicer is the only RNase III domain protein in the publicly available sequence of the Drosophila genome that contains an ATP-binding motif, the DEAD-box RNA helicase domain (Bernstein, 2001). Cleavage of dsRNA by Dicer is strictly ATP-dependent (Bernstein, 2001). Cleavage of pre-let-7 into mature let-7 in Drosophila embryo lysates also requires ATP. Taken together, the chemical structure of mature let-7 RNA in vitro and in vivo and the ATP dependence of pre-let-7 processing in vitro strongly implicate Dicer in let-7 maturation. However, it is noted that expression of Dicer protein in Drosophila larvae or pupae has not yet been demonstrated, although the RNAi pathway, which requires Dicer, functions in larvae and pupae (Hutvagner, 2001).

A more stringent test of a role for Dicer in pre-let-7 processing would be to assay let-7 production in flies lacking Dicer protein. However, mutant alleles of Dicer have yet to be identified in Drosophila . As an alternative approach, a recently reported sequence-specific method was used in which cultured mammalian cells were transfected with synthetic 21-nt siRNA duplexes to suppress gene expression. Because they are <30 base pairs long, the siRNA duplexes do not trigger the sequence-nonspecific responses that complicate standard dsRNA-induced interference in mammalian cells (Hutvagner, 2001).

This method was used to evaluate the role of the human ortholog of Dicer (Helicase-MOI) in let-7 biogenesis. Human Dicer was identified by its unique domain structure, comprising an NH₂-terminal DEXH-box ATP-dependent RNA helicase domain, PAZ domain, tandem RNase III motifs, and COOH-terminal dsRNA-binding domain, and by its sequence homology to Drosophila Dicer. HeLa cells were transfected with a single, synthetic siRNA duplex containing 19 nt of the coding sequence of human Dicer mRNA, beginning at position 183 relative to the start of translation. Three days after transfection, total RNA was prepared from the cells and analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) for Dicer and actin mRNA levels and by primer extension for the presence of let-7. The level of Dicer mRNA in the Dicer siRNA-treated cells was four- to six-fold lower than in the control samples, whereas actin mRNA levels were unchanged. Separate controls showed that ~70% to 80% of the cells were transfected. Thus, the observed decrease in Dicer mRNA levels demonstrates that the Dicer siRNA induced substantial degradation of Dicer mRNA in the fraction of the cells that were successfully transfected (Hutvagner, 2001).

Transfection of HeLa cells with the siRNA duplex corresponding to human Dicer, but not the control siRNA duplex, led to the accumulation of a longer let-7-containing RNA, let-7L. Primer extension analysis of RNA from cells transfected with the Dicer siRNA detected an RNA with a 5' end ~7 nt and ~11 to 12 nt upstream of the mature let-7 product. These products are consistent with the accumulation of the predicted human let-7 precursor RNA (Pasquinelli, 2000) and with a longer form of this precursor containing an extended stem. The mature human let-7 RNA was readily detected in control cells, but not in the cells transfected with the Dicer siRNA duplex, providing additional evidence for a role for Dicer in let-7 maturation. These findings, together with in vitro data, provide strong evidence that Dicer protein function is required for the maturation of let-7. Thus, the RNAi and stRNA pathways intersect; both require the RNA-processing enzyme Dicer to produce the active small-RNA component that represses gene expression. The two pathways must also diverge after the action of Dicer, because siRNA duplexes are generated from long, dsRNA direct mRNA cleavage, whereas the single-stranded stRNA let-7 represses mRNA translation (Hutvagner, 2001).

Recently, Grishok (2001) has shown that the Dicer homolog Dcr-1 is required for both lin-4 and let-7 function in C. elegans. Thus, Dicer is likely to have a broad role in the biogenesis of stRNAs and perhaps other small regulatory RNAs. Furthermore, mutations in the Arabidopsis homolog of Dicer, SIN-1/CARPEL FACTORY (SIN1/CAF), have dramatic developmental consequences (A. Ray, 1996; S. Ray, 1996; Jacobsen, 1999). Perhaps SIN1/CAF protein in plants, like Dicer in bilateral animals, processes structured RNA precursors into small RNAs that regulate development (Hutvagner, 2001).

Pre-let-7 is processed asymmetrically to yield only let-7. It is not yet known what structural or sequence features of pre-let-7 determine its asymmetric cleavage. RNase III enzymes cleave perfectly paired dsRNA on both strands, producing a pair of cuts, one on each strand, displaced by two nucleotides. For the R1.1 RNA hairpin of T7 bacteriophage, internal loops and bulges constrain the Escherichia coli RNase III dimer to cut only one strand of the stem. The proposed let-7 precursor contains such an internal loop at the site of 5' cleavage. It is possible that if the stem were uninterrupted by such distortions, a pair of 21- to 22-nt RNAs might be generated, rather than the single stRNA let-7. If so, it might be possible to design stem-loop RNA precursors that produce an siRNA duplex. The hope is that such an siRNA duplex, generated in vivo in a specific cell type or at a specific developmental stage, would be able to target an mRNA for destruction by the RNAi machinery, thereby extending the utility of RNAi to the study of mammalian development (Hutvagner, 2001).

Characterization of RISC (RNA-induced silencing complex) in Drosophila

In a diverse group of organisms that includes Caenorhabditis elegans, Drosophila, planaria, hydra, trypanosomes, fungi and plants, the introduction of double-stranded RNAs inhibits gene expression in a sequence-specific manner. These responses, called RNA interference or post-transcriptional gene silencing, may provide anti-viral defence, modulate transposition or regulate gene expression. A biochemical approach has been taken towards elucidating the mechanisms underlying this genetic phenomenon. 'Loss-of-function' phenotypes can be created in cultured Drosophila cells by transfection with specific double-stranded RNAs. This coincides with a marked reduction in the level of cognate cellular messenger RNAs. Extracts of transfected cells contain a nuclease activity (termed RISC for RNA-induced silencing complex) that specifically degrades exogenous transcripts homologous to transfected double-stranded RNA. This enzyme contains an essential RNA component. After partial purification, the sequence-specific nuclease co-fractionates with a discrete, approximately 25-nucleotide RNA species, which may confer specificity to the enzyme through homology to the substrate mRNAs (Hammond, 2000).

Although double-stranded RNAs (dsRNAs) can provoke gene silencing in numerous biological contexts including Drosophila, the mechanisms underlying this phenomenon have remained mostly unknown. It was therefore important to establish a biochemically tractable model in which such mechanisms could be investigated. Transient transfection of cultured, Drosophila S2 cells with a lacZ expression vector results in ß-galactosidase activity that is easily detectable by an in situ assay. This activity is greatly reduced by co-transfection with a dsRNA corresponding to the first 300 nucleotides of the lacZ sequence, whereas co-transfection with a control dsRNA (CD8) or with single-stranded RNAs of either sense or antisense orientation has little or no effect. This indicates that dsRNAs could interfere, in a sequence-specific fashion, with gene expression in cultured cells (Hammond, 2000).

To determine whether RNA interference (RNAi) could be used to target endogenous genes, S2 cells were transfected with a dsRNA corresponding to the first 540 nucleotides of Drosophila cyclin E, a gene that is essential for progression into S phase of the cell cycle. During log-phase growth, untreated S2 cells reside primarily in G2/ M. Transfection with lacZ dsRNA has no effect on cell-cycle distribution, but transfection with the cyclin E dsRNA causes a G1-phase cell-cycle arrest. The ability of cyclin E dsRNA to provoke this response is length-dependent. Double-stranded RNAs of 540 and 400 nucleotides are quite effective, whereas dsRNAs of 200 and 300 nucleotides are less potent. Double-stranded cyclin E RNAs of 50 or 100 nucleotides are inert in this assay, and transfection with a single-stranded, antisense cyclin E RNA has virtually no effect (Hammond, 2000).

One hallmark of RNAi is a reduction in the level of mRNAs that are homologous to the dsRNA. Cells transfected with the cyclin E dsRNA (bulk population) show diminished endogenous cyclin E mRNA as compared with control cells. Similarly, transfection of cells with dsRNAs homologous to fizzy, a component of the anaphase-promoting complex (APC) or cyclin A, a cyclin that acts in S, G2 and M, also causes reduction of their cognate mRNAs. The modest reduction in fizzy mRNA levels in cells transfected with cyclin A dsRNA probably results from arrest at a point in the division cycle at which fizzy transcription is low (Hammond, 2000).

These results indicate that RNAi may be a generally applicable method for probing gene function in cultured Drosophila cells. The decrease in mRNA levels observed upon transfection of specific dsRNAs into Drosophila cells could be explained by effects at transcriptional or post-transcriptional levels. Data from other systems have indicated that some elements of the dsRNA response may affect mRNAdirectly. Attempts were therefore made to develop a cell-free assay that reflected, at least in part, RNAi (Hammond, 2000).

S2 cells were transfected with dsRNAs corresponding to either cyclin E or lacZ. Cellular extracts were incubated with synthetic mRNAs of lacZ or cyclin E. Extracts prepared from cells transfected with the 540-nucleotide cyclin E dsRNA efficiently degraded the cyclin E transcript; however, the lacZ transcript was stable in these lysates. Conversely, lysates from cells transfected with the lacZ dsRNA degraded the lacZ transcript but left the cyclin E mRNA intact. These results indicate that RNAi ablates target mRNAs through the generation of a sequence-specific nuclease activity (Hammond, 2000).

This enzyme has been termed RISC (RNA-induced silencing complex). Although possible intermediates in the degradation process were occasionally observed, the absence of stable cleavage end-products indicates an exonuclease (perhaps coupled to an endo-nuclease). However, it is possible that the RNAi nuclease makes an initial endonucleolytic cut and that non-specific exonucleases in the extract complete the degradation process. In addition, the ability to create an extract that targets lacZ in vitro indicates that the presence of an endogenous gene is not required for the RNAi response (Hammond, 2000).

To examine the substrate requirements for the dsRNA-induced, sequence-specific nuclease activity, a variety of cyclin-E- derived transcripts was incubated with an extract derived from cells that had been transfected with the 540-nucleotide cyclin E dsRNA. Just as a length requirement was observed for the transfected dsRNA, the RNAi nuclease activity showed a dependence on the size of the RNA substrate. Both a 600-nucleotide transcript that extends slightly beyond the targeted region and an ~1-kilobase (kb) transcript that contains the entire coding sequence are completely destroyed by the extract. Surprisingly, shorter substrates are not degraded as efficiently. Reduced activity is observed against either a 300- or a 220-nucleotide transcript, and a 100-nucleotide transcript is resistant to nuclease in this assay. This was not due solely to position effects because ~100- nucleotide transcripts derived from other portions of the transfected dsRNA behave similarly. As expected, the nuclease activity (or activities) present in the extract can also recognize the antisense strand of the cyclin E mRNA. Again, substrates that contained a substantial portion of the targeted region are degraded efficiently whereas those that contained a shorter stretch of homologous sequence (~130 nucleotides) were recognized inefficiently. For both the sense and antisense strands, transcripts that had no homology with the transfected dsRNA were not degraded (Hammond, 2000).

Although the possibility that nuclease specificity could have migrated beyond the targeted region cannot be excluded, the resistance of transcripts that do not contain homology to the dsRNA is consistent with data from C. elegans. Double-stranded RNAs homologous to an upstream cistron have little or no effect on a linked downstream cistron, despite the fact that unprocessed, polycistronic mRNAs can be readily detected. Furthermore, the nuclease is inactive against a dsRNA identical to that used to provoke the RNAi response in vivo. In the in vitro system, neither a 5' cap nor a poly(A) tail is required, since such transcripts are degraded as efficiently as uncapped and non-polyadenylated RNAs (Hammond, 2000).

Gene silencing provoked by dsRNA is sequence specific. A plausible mechanism for determining specificity would be incorporation of nucleic-acid guide sequences into the complexes that accomplish silencing. In accord with this idea, pre-treatment of extracts with nuclease (micrococcal nuclease) abolishes the ability of these extracts to degrade cognate mRNAs. Activity can not be rescued by addition of non-specific RNAs such as yeast transfer RNA. Although micrococcal nuclease can degrade both DNA and RNA, treatment of the extract with DNAse I has no effect. Sequence-specific nuclease activity, however, does require protein. Together, these results support the possibility that the RNAi nuclease is a ribonucleoprotein, requiring both RNA and protein components. Biochemical fractionation is consistent with these components being associated in extract rather than being assembled on the target mRNA after its addition (Hammond, 2000).

In plants, the phenomenon of co-suppression has been associated with the existence of small (~25-nucleotide) RNAs that correspond to the gene that is being silenced. To address the possibility that a similar RNA might exist in Drosophila and guide the sequence-specific nuclease in the choice of substrate, the Drosophila activity was followed through several fractionation steps. Crude extracts contain both sequence-specific nuclease activity and abundant, heterogeneous RNAs homologous to the transfected dsRNA. The RNAi nuclease fractionates with ribosomes in a high-speed centrifugation step. Activity can be extracted by treatment with high salt, and ribosomes can be removed by an additional centrifugation step. Chromatography of soluble nuclease over an anion-exchange column results in a discrete peak of activity. This peak retains specificity since it is inactive against a heterologous mRNA. Active fractions also contain an RNA species of 25 nucleotides that is homologous to the cyclin E target. The band observed on Northern blots may represent a family of discrete RNAs because it could be detected with probes specific for both the sense and antisense cyclin E sequences and with probes derived from distinct segments of the dsRNA. At present, it cannot be determine whether the 25-nucleotide RNA is present in the nuclease complex in a double-stranded or single-stranded form (Hammond, 2000).

RNA interference allows an adaptive defence against both exogenous and endogenous dsRNAs, providing something akin to a dsRNA immune response. These and other data are consistent with a model in which dsRNAs present in a cell are converted, either through processing or replication, into small specificity determinants of discrete size in a manner analogous to antigen processing. The results suggest that the post-transcriptional component of dsRNA-dependent gene silencing is accomplished by a sequence-specific nuclease that incorporates these small RNAs as guides that target specific messages based upon sequence recognition. The identical size of putative specificity determinants in plants and animals predicts a conservation of both the mechanisms and the components of dsRNA-induced, post-transcriptional gene silencing in diverse organisms. In plants, dsRNAs provoke not only post-transcriptional gene silencing but also chromatin remodelling and transcriptional repression. It is now critical to determine whether conservation of gene-silencing mechanisms also exists at the transcriptional level and whether chromatin remodelling can be directed in a sequence-specific fashion by these same dsRNA-derived guide sequences (Hammond, 2000).

Short interfering RNAs (siRNAs) induce RNAi

Double-stranded (ds) RNA causes the specific degradation of homologous RNAs in a process called 'RNA interference (RNAi)'; this process is called 'posttranscriptional gene silencing (PTGS)' in plants. The duplex RNA becomes processed by Dicer or another RNase III-like enzyme to short dsRNA fragments of about 21-23 nucleotides (nt), which are incorporated in the RNA-induced silencing complex (RISC) that directs target-specific RNA degradation. Different synthetic dsRNA cassettes, consisting of two 5'-phosphorylated RNA strands of 22 nt each, can initiate RNAi in Drosophila embryos. The cassettes were active at similar quantities required to initiate RNAi by conventional dsRNA. Their sequence specificity was confirmed using synthetic dsRNA cassettes for two different genes, Notch and hedgehog; each time, only the relevant embryonic phenotype was observed. Introduction of point mutations had only a moderate effect on the silencing potential, indicating that the silencing machinery does not require perfect sequence identity. 5'-phosphorylated synthetic RNA was more active than its hydroxylated form. Substitution of either RNA strand by DNA strongly reduces activity. Synthetic cassettes of siRNA will provide a new tool to induce mutant phenotypes of genes with unknown function (Boutla, 2001).

The Notch gene was chosen for the first target, because it is ubiquitously expressed in the early embryo and loss of function produces a characteristic 'neurogenic' phenotype. The expressivity of the neurogenic phenotype can be used as a rough quantitative estimate of the severity of Notch function disruption. An in vitro-synthesized 985-bp dsRNA fragment of the Notch mRNA was injected into Drosophila precellular embryos at 5 µM, which is a typical concentration for RNAi, of which about 100 pl, equivalent to about 0.5 fmole, were actually transferred. The dsRNA induced a strong Notch phenotype with high penetrance, indicative of an almost complete inactivation of the Notch mRNA, both zygotic and maternal (Boutla, 2001).

Next, two Notch-specific RNAs of 22 nucleotides were synthesized. Selection of the sequence within the 985-bp cDNA fragment was based solely on structural considerations, to avoid self-dimerization or undesired intramolecular basepairing of each RNA molecule. In contrast with previous methods, a simple all-RNA cassette was used, without deoxynucleotides in the 3' protruding ends and without special considerations of which nucleotide would form the 3' end. However, a 5' phosphate was additionally introduced as an authentic RNase III product and it was compared with the nonphosphorylated RNA for its silencing potential. Both cassettes were adjusted to 100 µM and were used for injection. It should be noted that this is a 20-fold higher molar concentration than that of the dsRNA; however, in terms of absolute amount of RNA, it is less than half. The phosphorylated cassette was able to induce a strong Notch phenotype, exactly as observed after the injection of the long dsRNA. The nonphosphorylated cassette gave phenotypes with decreased penetrance, but the expressivity remained strong. At present, it cannot be distinguish whether the reduced efficiency is a general property or whether the phosphorylated 5' terminus simply provided protection against exonucleases. It is noteworthy that the phosphorylated cassette had a higher penetrance at a 10-fold dilution compared to the undiluted nonphosphorylated dsRNA cassette, but under these conditions, its expressivity was slightly lower (Boutla, 2001).

To test for the specificity and the general applicability of inducing RNAi, a second synthetic phosphorylated RNA cassette of the same general design was used, this time directed against the hedgehog (hh) gene. At a concentration of 100 µM, the hh dsRNA cassette induced a strong mutant phenotype in 88% of the 268 injected embryos. As for Notch, the strength of the observed phenotype suggested complete silencing of the hh gene (Boutla, 2001).

According to current models, the antisense RNA confers sequence specificity upon the RNAi-mediated RNA degradation process. In view of this, a test was made as to the extent one of the RNA strands of the Notch siRNA cassette could be substituted by DNA. The combination DNA sense/RNA antisense was the most promising, since it left the antisense RNA intact. However, substitution of either sense or antisense strand by DNA resulted in a dramatic drop in both penetrance and expressivity of the Notch phenotypes to levels comparable to those obtained with ordinary antisense RNA. A phosphorylated dsDNA cassette had an even weaker effect, inducing only a very mild Notch phenotype that had not been observed with any of the other samples tested (Boutla, 2001).

Subsequently, tests were performed to see whether the 3' protruding ends, as generated by RNase III, are required. It was reported that blunt-ended RNAs were less active in insect tissue culture, and it has been reported that a synthetic blunt-ended 26mer dsRNA is about 250-times less effective than an 81mer dsRNA, although a 26mer might be too large to act as a siRNA. In this study, after injection with a blunt-ended RNA cassette an increased number of viable embryos were obtained and, in accordance with this, a reduced expressivity. At 10-fold dilution, it became evident that this construct was less active than the proper siRNA. Thus, the protruding 3' ends are not mandatory to elicit RNAi; although, in this case, the difference is not as pronounced as reported earlier. A potential difference to the blunt-ended cassette used previously is the presence of the 5' phosphate in the construct used in this study (Boutla, 2001).

Tests were performed of several RNA cassettes that carried mutations. In the first example, a single nucleotide exchange was introduced that would be likely to interfere as much as possible with substrate binding. Therefore, the mutation was positioned centrally and was simultaneously introduced into the sense and the antisense strand, so that the RNA cassette remained double stranded. In previous reports, nonmatching nucleotides greatly impaired the silencing potential when introduced to the antisense strand of longer dsRNAs. Surprisingly, this synthetic cassette was also able to induce a strong Notch phenotype with high penetrance, indicating that a perfect match to the target RNA is not necessary to initiate the RNAi response. The 10-fold-diluted sample was still active, but penetrance and, in particular, expressivity were reduced. Next, each of these mutated sense and antisense RNAs was tested in combination with the wild-type sequence. Either of the combinations, characterized by a G:U pair or a mismatch, was highly active. As a third example, an RNA cassette with a double mutation in the antisense strand was paired with the nonmutated 22mer. Even this RNA construct, with its central bulge loop, had some silencing potential. However, both penetrance and expressivity dropped significantly compared with the single mutant. It will require a more detailed analysis to determine at what position and to what degree sequence deviations can be tolerated without loss of silencing function (Boutla, 2001).

Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila embryo lysate

Duplexes of 21-23 nucleotide (nt) RNAs are the sequence-specific mediators of RNA interference (RNAi) and post-transcriptional gene silencing (PTGS). Synthetic, short interfering RNAs (siRNAs) were examined in Drosophila embryo lysate for their requirements regarding length, structure, chemical composition and sequence in order to mediate efficient RNAi. Duplexes of 21 nt siRNAs with 2 nt 3' overhangs were the most efficient triggers of sequence-specific mRNA degradation. Substitution of one or both siRNA strands by 2'-deoxy or 2'-O-methyl oligonucleotides abolished RNAi, although multiple 2'-deoxynucleotide substitutions at the 3' end of siRNAs were tolerated. The target recognition process is highly sequence specific, but not all positions of a siRNA contribute equally to target recognition; mismatches in the center of the siRNA duplex prevent target RNA cleavage. The position of the cleavage site in the target RNA is defined by the 5' end of the guide siRNA rather than its 3' end. These results provide a rational basis for the design of siRNAs in future gene targeting experiments (Elbashir, 2001b).

The structural determinants of siRNA duplexes required to promote efficient target RNA degradation in D.melanogaster embryo lysate were systematically analyzed, thus providing rules for the design of most potent siRNA duplexes. A perfect siRNA duplex is able to silence gene expression with an efficiency comparable to a 500 bp dsRNA, given that comparable quantities of total RNA are used (Elbashir, 2001b).

Efficiently silencing siRNA duplexes are composed of 21 nt sense and 21 nt antisense siRNAs and must be selected to form a 19 bp double helix with 2 nt 3'-overhanging ends. 2'-deoxy substitutions of the 2 nt 3'-overhanging ribonucleotides do not affect RNAi, but help to reduce the costs of RNA synthesis and may enhance RNase resistance of siRNA duplexes. More extensive 2'-deoxy or 2'-O-methyl modifications reduce the ability of siRNAs to mediate RNAi, probably by interfering with protein association for siRNP assembly (Elbashir, 2001b).

Target recognition is a highly sequence-specific process, mediated by the siRNA complementary to the target. The 3'-most nucleotide of the guide siRNA does not contribute to the specificity of target recognition, while the penultimate nucleotide of the 3' overhang affects target RNA cleavage and a mismatch reduces RNAi 2- to 4-fold. The 5' end of the guide siRNA also appears more permissive for mismatched target RNA recognition when compared with the 3' end. Nucleotides in the center of the siRNA, located opposite the target RNA cleavage site, are important specificity determinants and even single nucleotide changes reduce RNAi to undetectable levels. This suggests that siRNA duplexes may be able to discriminate mutant or polymorphic alleles in gene targeting experiments, which may become an important feature for future therapeutic developments (Elbashir, 2001b).

Sense and antisense siRNAs, when associated with the protein components of the endonuclease complex or its commitment complex, were suggested to play distinct roles; the relative orientation of the siRNA duplex in this complex defines which strand can be used for target recognition (Elbashir, 2001a). Synthetic siRNA duplexes with an equal number of overhanging nucleotides have dyad symmetry with respect to the double-helical structure, but not with respect to sequence. The association of siRNA duplexes with the RNAi proteins in the D. melanogaster lysate leads to the formation of two asymmetric complexes. In such hypothetical complexes, the chiral environment is distinct for sense and antisense siRNA, hence their function. The prediction obviously does not apply to palindromic siRNA sequences or to RNAi proteins that could associate as homodimers. To minimize sequence effects that may affect the ratio of sense- and antisense-targeting siRNPs, using siRNA sequences with identical 3'-overhanging sequences is suggested. Adjusting the sequence of the overhang of the sense siRNA to that of the antisense 3' overhang is recommended because the sense siRNA does not have a target in typical knock-down experiments. Asymmetry in the reconstitution of sense- and antisense-cleaving siRNPs is partially responsible for the variation in RNAi efficiency observed for various 21 nt siRNA duplexes with 2 nt 3' overhangs used in this study. Alternatively, the nucleotide sequence at the target site and/or the accessibility of the target RNA structure may be responsible for the variation in efficiency observed for these siRNA duplexes. It should be noted that all siRNAs used in this study are derived from a short region of one gene. Thus, it is more likely that differences in siRNA efficiency are a consequence of the primary sequences of the siRNAs and the respective target sites, rather than the secondary or tertiary structure of the targeted RNA (Elbashir, 2001b).

In Drosophila, siRNA duplexes are produced in vitro and in vivo from long dsRNAs. About 45% of these short RNAs are precisely 21 nt long, 28% are 22 nt long and a few percent are shorter or longer RNAs (Elbashir, 2001a). This length distribution correlates with the finding that 21 nt siRNA duplexes are the most efficient mediators of mRNA degradation. In addition to the length, the paired structure and overhang are also important. This structural feature may explain why siRNA duplexes isolated from the dsRNA processing reaction under denaturing conditions are less potent for RNAi than longer dsRNAs that are processed to siRNAs during the targeting reaction. Presumably, denaturation followed by renaturation favours the formation of the thermodynamically more stable, blunt-ended, but less active, siRNA duplexes. Isolation of siRNAs under native conditions does not reduce siRNA activity (Elbashir, 2001b).

Production of siRNAs from long dsRNA requires the RNase III enzyme Dicer (Bernstein, 2001). Dicer is a bidentate RNase III, which also contains an ATP-dependent RNA helicase domain and a PAZ domain, presumably important for dsRNA unwinding and mediation of protein-protein interactions, respectively. Dicer is evolutionarily conserved in worms, flies, plants, fungi and mammals, and has a second cellular function important for the development of these organisms. At present, it is uncertain whether Dicer activity in species other than Drosophila produces siRNAs of predominantly 21 nt in length. The estimates of siRNA size vary in the literature between 21 and 25 nt (Elbashir, 2001b).

An RNA-dependent RNA polymerase activity amplifies short interfering RNAs

In posttranscriptional gene silencing (PTGS) and RNA interference (RNAi), 21-25 nucleotide RNA fragments are produced from the initiating dsRNA. Short interfering RNAs (siRNAs) mediate RNAi by an unknown mechanism. GFP and Pp-Luc siRNAs, isolated from a protein complex in Drosophila embryo extract, target mRNA degradation in vitro. Most importantly, these siRNAs, as well as a synthetic 21-nucleotide duplex GFP siRNA, serve as primers to transform the target mRNA into dsRNA. The nascent dsRNA is degraded to eliminate the incorporated target mRNA while generating new siRNAs in a cycle of dsRNA synthesis and degradation. Evidence is presented that mRNA-dependent siRNA incorporation to form dsRNA is carried out by an RNA-dependent RNA polymerase activity (RdRP) (Lipardi, 2001).

This study demonstrates the template-specific incorporation of the 21-25 nucleotide RNAs, or siRNAs, to generate dsRNA that is subsequently cleaved by RNase III activity into new siRNAs. In this way, mRNA is degraded through a cycle of 'degradative-PCR'. Evidence for RdRP activity in Drosophila extracts and it is suggested siRNA incorporation into dsRNA involves RdRP, the crucial step in the amplification of the target RNA for rapid degradation by RNase III-type activity. Although it cannot be excluded that siRNAs may be incorporated into dsRNA by a direct 'guide' mechanism not involving RdRP, such a process would not give the sufficient amplification of the double-stranded RNA target. This would be needed to trigger efficient RNAi with substoichiometric levels of the initiating double-stranded trigger RNA. Consistent with the genetic screens in other lower eukaryotes, the results suggest a role for RdRP in Drosophila RNAi as well (Lipardi, 2001).

The requirement for a dsRNA trigger as the effector for silencing can be partially explained by the nature of the dsRNA cleavage step required for siRNA production. Any factor that significantly alters the double-stranded nature of the dsRNA trigger, such as sequence divergence or chemical modification, affects silencing substantially. In the model proposed here, any changes in strand complementarity could presumably reduce the susceptibility of the triggering dsRNA to RNase III-type cleavage. The nature of the sense and antisense strands in the triggering dsRNA would also play a role in the efficacy of silencing since the RdRP amplification step would depend upon the production and functionality of the siRNAs. Both strands of a synthetic 21 nucleotide GFP duplex siRNA function as primers to give the expected RNA products when the appropriate GFP template strand is used (Lipardi, 2001).

The length of the siRNAs may be an important aspect of their function. Previous reports indicated that 29-36 nucleotide dsRNAs transcribed in vitro do not direct RNAi efficiently in Drosophila extracts, and that a 26 nucleotide dsRNA, also transcribed in vitro, when injected into worms, triggers lower than expected levels of RNA interference at 25°C and none at 16°C. However, chemically synthesized 21 and 22 nucleotide siRNAs can mediate targeted RNA cleavage in Drosophila embryo extracts and in Schneider cells. The siRNAs produced in Drosophila embryo extract by micrococcal nuclease and CIP treatment are essentially as efficient on a weight basis in RNAi as the full-length dsRNA from which they were derived, suggesting there is some optimal length for siRNAs in RNAi. The conservation in the size range for the small RNAs associated with silencing in all the species examined proposes that it may be closely correlated with primer function. This could be due to some unique property of primer activity in a protein complex that has yet to be identified (Lipardi, 2001).

siRNAs require a 3' hydroxyl group for function in RNAi. The authentic siRNAs, produced in Drosophila extracts by RNase III-related enzymes such as Dicer (Bernstein, 2001), have been chemically characterized and shown to have a 5' phosphate and a 3' hydroxyl group. The micrococcal nuclease generated siRNAs described in this study are functional in RNAi and can be incorporated into dsRNA after phosphatase treatment to remove the 3' phosphate group produced by the nuclease digestion. The 21 nucleotide synthetic siRNA primer also has a 3' hydroxyl group that would be required for incorporation into dsRNA by RdRP activity. Whether or not the 3' hydroxyl group is also used in a primer ligation step remains to be determined (Lipardi, 2001).

Cleavage of the GFP target RNA occurs after the synthetic GFP siRNA is incorporated into dsRNA. If cleavage occurred in the template RNA immediately upon binding to the synthetic GFP siRNA, no full-length GFP dsRNA would have been observed. Therefore, cleavage occurs in the nascent dsRNA in regions inside and outside the zone represented by the initial siRNA since the primers are extended to make dsRNA. The fact that the synthetic GFP siRNA is extended to the 5' end of the sense strand template would also restrict cleavage, in this instance, to the region upstream of the 3' terminus of the siRNA. Any region of the target RNA converted into duplex by a given siRNA would be subject to digestion by RNase III activity (Lipardi, 2001).

It has been proposed previously that there is no amplification of the trigger dsRNA in RNAi in C. elegans, based upon the effects of asymmetric strand substitutions in the input dsRNA. This study provides evidence that both single- and double-stranded RNAs can serve as templates for siRNA incorporation into dsRNA in Drosophila extract. However, the rapid degradation of dsRNA suggests that amplification of the trigger dsRNA is of limited value. The antisense siRNA strand would be the most important for the synthesis of new dsRNA from the mRNA template (Lipardi, 2001).

Genetic studies have identified several mutants in C. elegans, Neurospora crassa, and Arabidopsis thaliana that resist RNA interference. These include mutants that affect the initiation of silencing activity (rde-1 and rde-4 in C. elegans, qde-2 in Neurospora, and AGO-1 in Arabidopsis, mutants in the effectors of silencing (rde-2 and mut-7 in C. elegans, the latter related to RNase D), mutants in helicase (qde-3 in Neurospora, and SDE-3 in Arabidopsis, and mutants in RNA-dependent RNA polymerase (ego-1 in C. elegans, qde-1 in Neurospora, and SGS-2/SDE-1 in Arabidopsis). Although by sequence comparison an RdRP homolog in Drosophila has not yet been identified, the results presented in this paper suggest the presence of an RdRP gene (Lipardi, 2001 and references therein).

RdRP-dependent as well as -independent mechanisms may be involved in the generation of dsRNA up to the full-length of the target RNA, according to one of the following schemes: (1) a single siRNA primer would be extended from various positions along individual template strands by RdRP to generate dsRNAs; and (2) different siRNAs would associate along a single template RNA and be extended by RdRP to the adjacent siRNA primer. The extension products would be ligated to generate dsRNAs. This model would require RdRP activity as well as an RNA ligase step; and (3) dsRNAs would be formed by a primer 'guide' mechanism where they would align along the template for subsequent ligation. All these mechanisms could generate dsRNA of sufficient length to be cleaved by RNase III-type activity since this requires a minimum of 39 base pairs. The results favor the first and second models since RdRP activity would be required to amplify the target dsRNA sufficiently when substiochiometric amounts of the trigger dsRNA are involved in initiating RNAi. A primer 'guide' mechanism would require the involvement of an RNA ligase in order to generate larger dsRNAs, and genetic screens have not identified related genes as candidates essential for RNAi. As previously noted, RdRP genes have been shown to be involved in posttranscriptional gene silencing in three different lower eukaryotes. In addition, the 'guide' primer ligation model is not supported by observations using synthetic siRNAs. siRNAs generated from dsRNAs greater than 111 nucleotides in length are not well defined and are derived from several overlapping regions of different lengths (18-24 nucleotides) to make the siRNA population heterogeneous in composition. It is unlikely that a 'guide' mechanism could sort out the precise siRNAs for ligation along the target RNA to rapidly generate the full-length dsRNA. The most convincing evidence for the involvement of RdRP activity in Drosophila RNAi comes from results using the synthetic 21 nucleotide GFP duplex siRNA, where full-length GFP dsRNA is produced from a single primer with the same time course of synthesis and degradation as dsRNA produced using the micrococcal-nuclease-generated GFP siRNAs. The extension of both strands of the synthetic siRNA in a template-dependent manner to yield the expected dsRNA products would specifically require an RdRP. The role for helicase activity in RNAi, as shown for qde-3 in Neurospora and SDE-3 in Arabidopsis in the genetic screens, may be to unwind the primers or the dsRNA trigger, but this remains to be demonstrated (Lipardi, 2001).

Double-stranded RNA is processed into siRNA primers that convert the target mRNA into dsRNA for subsequent degradation and the formation of new siRNAs. Since the siRNA primers are double stranded, they should direct the degradation of either sense or antisense cognate target RNAs. This is exactly what is observed when either sense or antisense GFP mRNA is incubated in extract with GFP dsRNA. Therefore, dsRNA representing transcripts derived from opposite strands of a complementary template would be targeted simultaneously to effect silencing of more than a single gene in some instances. The siRNA primer model also suggests a single siRNA should target transcript degradation as long as the primer extended product is of sufficient length to be cleaved by RNase III activity, roughly 39 nucleotides. When the siRNA-21 is used in a silencing assay, GFP mRNA is selectively degraded, as predicted, since the extended primer produces dsRNA 44 bp long. Therefore, a single primer is sufficient to target mRNA silencing. This result also predicts that a primer producing the longest dsRNA product would be the most efficient since the second generation of siRNAs would represent more of the target RNA (Lipardi, 2001).

A Drosophila fragile X protein interacts with components of RNAi and ribosomal proteins

In Drosophila, Fmr1 binds to and represses the translation of an mRNA encoding of the microtuble-associated protein Futsch. A Fmr1-associated complex has been isolated that includes two ribosomal proteins, L5 and L11, along with 5S RNA. The Fmr1 complex also contains Argonaute2 (AGO2) and a Drosophila homolog of p68 RNA helicase (Dmp68). AGO2 is an essential component for the RNA-induced silencing complex (RISC), a sequence-specific nuclease complex that mediates RNA interference (RNAi) in Drosophila. Dmp68 is also required for efficient RNAi. Fmr1 is associated with Dicer, another essential component of the RNAi pathway, and microRNAs (miRNAs) in vivo, suggesting that Fmr1 is part of the RNAi-related apparatus. These findings suggest a model in which the RNAi and Frm1-mediated translational control pathways intersect in Drosophila. The findings also raise the possibility that defects in an RNAi-related machinery may cause human disease (Ishizuka, 2002).

The connection that has been established between Fmr1, components of RNAi, miRNAs, and the general translation machinery is of considerable significance because they provide intriguing clues and possible connections to the function of Fmr1 and the pathways with which it may intersect. Recent work in numerous organisms has shown that RNAi shares features with a developmental gene regulatory mechanism that involves miRNAs. For example, both the foreign dsRNAs that trigger RNAi and the endogenous miRNA precursors that function in development are processed into small RNAs by Dicer. Members of the Argonaute gene family are also involved in both the siRNA and miRNA pathways. In C. elegans, Dicer, the dsRNA-binding protein RDE-4, and a conserved DExH-box RNA helicase (DRH-1) are in a complex with RDE-1, an AGO2 ortholog. Furthermore, the human AGO2 ortholog, eIF2C2, is in a complex, the miRNP, that contains the DEAD-box RNA helicase Gem3. Therefore, Argonaute proteins appear to be in a complex that contains an RNA helicase(s), Dicer and small guide RNAs, and function in a variety of homology-dependent mechanisms that involve base-pairing between small guide RNAs and target mRNAs. The findings that Fmr1 interacts with AGO2, Dmp68, Dicer, miRNAs, and the general translation machinery, provide a means to link RNAi enzymes to translational control pathways, and are also consistent with the fact that the RISC nuclease fractionates with ribosomes (Ishizuka, 2002).

Processing of pre-microRNAs by the Dicer-1-Loquacious complex

microRNAs (miRNAs) are a large family of 21- to 22-nucleotide non-coding RNAs that interact with target mRNAs at specific sites to induce cleavage of the message or inhibit translation. miRNAs are excised in a stepwise process from primary miRNA (pri-miRNA) transcripts. The Drosha-Pasha/DGCR8 complex in the nucleus cleaves pri-miRNAs to release hairpin-shaped precursor miRNAs (pre-miRNAs). These pre-miRNAs are then exported to the cytoplasm and further processed by Dicer to mature miRNAs. Drosophila Dicer-1 interacts with Loquacious, a double-stranded RNA-binding domain protein. Depletion of Loquacious results in pre-miRNA accumulation in Drosophila S2 cells, as is the case for depletion of Dicer-1. Immuno-affinity purification experiments revealed that along with Dicer-1, Loquacious resides in a functional pre-miRNA processing complex, and stimulates and directs the specific pre-miRNA processing activity. Efficient miRNA-directed silencing of a reporter transgene, complete repression of white by a dsRNA trigger, and silencing of the endogenous Stellate locus by Suppressor of Stellate, all require Loqs. In loqs^f00791 mutant ovaries, germ-line stem cells are not appropriately maintained. Loqs associates with Dcr-1, the Drosophila RNase III enzyme that processes pre-miRNA into mature miRNA. Thus, every known Drosophila RNase-III endonuclease is paired with a dsRBD protein that facilitates its function in small RNA biogenesis. These results support a model in which Loquacious mediates miRNA biogenesis and, thereby, the expression of genes regulated by miRNAs (Forstemann, 2005; Saito, 2005).

It was asked if Loqs forms a complex in vivo with Dicer-1. For these studies, Dicer-1 tagged with the Flag epitope and Loqs tagged with the myc epitope were simultaneously expressed in S2 cells. Then Dicer-1 was immunoprecipitated with anti-Flag antibodies, and Loqs with anti-myc antibody and then the precipitates were analyzed by immunoblotting. In reciprocal assays, Dicer-1 and Loqs were found to co-precipitate. Consistent with these findings that Dicer-1 and Loqs form a complex in vivo, both proteins are localized predominantly in the cytoplasm of S2 cells (Saito, 2005).

It was further investigated whether Loqs can bind to Dicer-1 in vitro. Dicer-1 was produced by an in vitro translation system and used in binding assays with recombinant Loqs fused to glutathione S-transferase (GST). GST-Loqs interacts with Dicer-1 even in the presence of RNase A, whereas GST itself shows no detectable binding. These results demonstrate that the association of Loqs with Dicer-1 occurs both in vivo and in vitro, and that RNA molecules do not appear to mediate the association (Saito, 2005).

To examine the functional connection between the Dicer-1-Loqs complex and pre-miRNA processing, whether depletion of Dicer-1 or Loqs has any effect on the production of mature miRNA from the precursor was investigated. First whether cytoplasmic lysates of S2 cells are capable of processing synthetic Drosophila let-7 precursor RNA into functional mature let-7 was investigated. In this experiment, the synthetic let-7 precursor RNA was converted to mature let-7 in S2 cytoplasmic lysates, as is the case in embryo lysates. In an in vitro RNAi assay, target RNA harboring a sequence perfectly complementary to mature let-7 was cleaved efficiently within the let-7 complementary sequence, thus showing production of functional let-7 in S2 cell lysates. Cytoplasmic lysates from Dicer-1- or Loqs-depleted cells were then subjected to the pre-let-7 processing assay. Both Dicer-1 and Loqs depletion led to reductions of mature let-7 compared with controls, showing that both Dicer-1 and Loqs function in pre-miRNA processing (Saito, 2005).

Next, pre-miR-ban was used as a substrate for pre-miRNA processing assays. It has been shown that S2 cell extracts contain primary-miRNA processing activity that cleaves pri-miRNA into an approximately 60- to 70-bp pre-miRNA precursor. This processing is known to occur in the nucleus; thus pre-miR-ban was prepared by in vitro processing of pri-miR-ban incubated with S2 nuclear extracts. Uniformly labeled pre-miR-ban was then gel-purified and used as a substrate for analysis of pre-miRNA processing. Incubation of the pre-miRNA with S2 cytoplasmic extracts results in the appearance of a mature 21-nucleotide miR-ban. Then the requirement of Dicer-1 and Loqs in pre-miR-ban processing was examined. Incubation of pre-miRNA with Dicer-1- and Loqs-depleted S2 cytoplasmic extracts results in a marked reduction in mature miRNA levels. In contrast, depletion of Dicer-2 or R2D2 shows no measurable reduction of mature miRNA levels. Then the pre-miRNA processing activity of the purified complexes (both Flag-Dicer-1 and Flag-Loqs complexes) was assayed. That the Flag-Loqs complex contains Dicer-1 was confirmed by immunoblotting. Both Dicer-1 and Loqs complexes are capable of generating maturemiR-ban from pre-miR-ban. Several steps in the RNAi and miRNA pathways are known to require a divalent metal ion. In addition, it is well known that RNase III-type enzymes require divalent metals for cleavage. Flag-Dicer-1 complex was employed and the processing was performed in the presence of magnesium ions or EDTA in a buffer. No pre-miRNA processing activity is detected at 10 mM EDTA. These results demonstrate that the Dicer-1-Loqs complex converts pre-miRNAs into mature miRNAs in a divalent metal ion-dependent manner (Saito, 2005).

To further examine the requirement for Loqs in pre-miRNA processing, Flag-Dicer-1 complex was purified under a harsher condition (high salt), where Dicer-1 is stripped of most Loqs protein, and this Dicer-1 complex was used in pre-miRNA processing assays with or without supplement of recombinant GST-Loqs. Without any supplement, the Flag-Dicer-1 complex purified under the harsh condition showed less activity than that under mild condition. Then GST-Loqs was added in the assay mixture. The addition of GST-Loqs to the Dicer-1 complex stimulates the processing of pre-miRNA. GST-Loqs alone does not show any significant pre-miRNA processing activity. These results show that Loqs is required for stimulating the processing of pre-miRNAs. Interestingly, it was found that the Dicer-1 complex purified under the harsh condition displays considerable siRNA-generating activity on the long dsRNA substrate in vitro, although previous genetic studies have shown that Dicer-1 is not required for siRNA production. The addition of GST-Loqs inhibits this effect. Western blot analysis shows that the Dicer-1 complex used in this experiment does not contain appreciable Dicer-2. GST-Loqs alone shows no activity for generating siRNAs from long dsRNAs. These results suggested that Dicer-1, stripped of much of its bound Loqs, processes both dsRNA and pre-miRNA substrates, but re-addition of recombinant Loqs suppresses dsRNA processing activity and enhances pre-miRNA processing activity. These findings thus imply that much of the apparent substrate specificity of Dicer-1 in vivo results from its association with Loqs. Although very unlikely, it is, however, formally possible that the Dicer-1 immunoprecipitates may contain very small amounts of Dicer-2 protein that can catalyze long dsRNA cleavage, and that addition of a large amount of dsRBD-containing Loqs may block the activity of Dicer-2 in this experiment (Saito, 2005).

The presence of endogenous miRNA was examined in RNA preparations from Flag-Dicer-1 and Flag-Loqs complexes obtained from S2 cells using anti-Flag antibodies. The Dicer-1 complex contains both the pre- and mature form of miR-ban, and the complex seems to preferentially bind the precursor form of miR-ban. In contrast, the precursor form of miR-ban is barely detectable in the Loqs complex, though it contains mature miR-ban. However, EDTA treatment, which inhibits pre-miRNA processing activity, results in an accumulation of pre-miR-ban in the Loqs complex. This may suggest that part of Flag-tagged Loqs protein interacts with Dicer-1 or pre-miRNAs or both. Alternatively, Flag-Loqs complexes may rapidly process pre-miRNAs into mature miRNAs and, therefore, may only transiently interact with them. Nonetheless, these results suggest that Dicer-1-Loqs complexes associate with both pre- and mature miRNAs in vivo (Saito, 2005).

Argonaute protein AGO1 is required for stable production of mature miRNAs and associates with Dicer-1. Thus, attempts were made to ascertain if Loqs is also present in an AGO1-associated complex, and if so, if the AGO1 complex is capable of processing pre-miRNA in vitro. Flag-Loqs and AGO1 tagged with TAP were simultaneously expressed in S2 cells, and the AGO1-TAP complex was purified through immunoglobulin G (IgG) bead-binding. The IgG bound was then subjected to Western blot analysis using anti-Dicer-1, anti-AGO1, or anti-Flag (for Loqs detection) antibodies. Not only Dicer-1 but also Loqs was detected in the AGO1 complex. These results indicate that all three proteins are present in the same complex, although they cannot exclude the possibility that there is one complex that contains AGO1 and Dicer-1 but not Loqs, and another complex that contains AGO1 and Loqs but not Dicer-1. The pre-miRNA processing activity of the AGO1 complex was then examined. Pre-miR-ban was utilized as a substrate. The AGO1 complex is able to efficiently process pre-miR-ban into the mature form. In contrast, another Argonaute protein AGO2-associated complex shows no such activity, which is consistent with the finding that the AGO2-associated complex does not contain Dicer-1. Considered together, these results showed that Dicer-1 and Loqs form a functional complex that mediates the genesis of mature miRNAs from pre-miRNAs, and suggested that the resultant mature miRNAs are loaded onto an AGO1-associated complex, which probably is miRNA-associated RISC, through specific interaction of AGO1 with Dicer-1 and Loqs (Saito, 2005).

Normal microRNA maturation and germ-line stem cell maintenance requires Loquacious, a double-stranded RNA-binding domain protein

Reduction of R2D2 protein by RNAi destabilizes Dcr-2; conversely, RNAi of Dcr-2 renders R2D2 unstable. In contrast, RNAi of loqs in S2 cells reduced Dcr-1 protein levels by no more than 15%, suggesting that Loqs functions together with Dcr-1 in pre-miRNA processing, rather than that Loqs is simply needed to stabilize Dcr-1 protein. However, loqs^f00791 mutant ovaries, which lack detectable Loqs protein, contain 70% less Dcr-1 than wild-type. A role for Loqs in both Dcr-1 function and in Dcr-1 stability suggests that the two proteins physically interact, like R2D2 and Dcr-2. Therefore, tests were performed to see if Dcr-1 and Loqs are components of a common complex (Forstemann, 2005).

Myc-tagged versions for two protein isoforms of Loqs, Loqs PA and Loqs PB, were expressed in S2 cells, and the tagged proteins were immunoprecipitated with anti-myc monoclonal antibodies. The immunoprecipitated protein was analyzed by Western blotting using a polyclonal anti-Dcr-1 antibody. Dcr-1 protein co-immunoprecipitates with myc-tagged Loqs. When myc-tagged GFP was expressed in place of myc-tagged Loqs, no Dcr-1 protein was recovered in the anti-myc immunoprecipitate. Similarly, an affinity purified, polyclonal antibody directed against the N-terminus of endogenous Loqs protein also co-immunoprecipitated Dcr-1 protein. This interaction is resistant to treatment with RNase A. No co-immunoprecipitation of Dcr-2 with myc-tagged Loqs PB was detected under conditions where Dcr-1 was readily detected, but it cannot be excluded that Dcr-2 is a substoichiometric component of a complex that contains both Dcr-1 and Loqs (Forstemann, 2005).

When immunoprecipitated with anti-Dcr-1 antibody, both myc-tagged Loqs protein isoforms-PA and PB-associate with Dcr-1. Moreover, the antibody against endogenous Loqs protein detected two bands corresponding in size to Loqs PA and Loqs PB in the proteins immunoprecipitated with the anti-Dcr-1 antibody. Loqs PB comprises only approximately 22% of the total Loqs protein in S2 cells, but corresponds to approximately 95% of the Loqs associated with Dcr-1. Loqs PA, which is expressed at comparable levels in S2 cells, accounts for most of the remaining Loqs associated with Dcr-1. In contrast, the putative Loqs PC protein comprises the majority of S2 cell Loqs, but is not recovered in the Dcr-1 immunoprecipitate. Intriguingly, Loqs PA and PB contain a third dsRBD that Loqs PC lacks; perhaps this third dsRBD is required for the association of Loqs with Dcr-1 (Forstemann, 2005).

The immunoprecipitated Dcr-1-Loqs complexes accurately convert pre-miRNA to mature miRNA. Pre-miRNA processing by the immunoprecipitates is efficient and accurate when the anti-Dcr-1 antibody was used, and also when anti-myc antibody and myc-tagged Loqs was used. Thus, Dcr-1 and Loqs co-associate in a complex capable of converting pre-miRNA into mature miRNA. The data also demonstrate that an N-terminal tandem myc tag does not perturb Loqs function in pre-miRNA cleavage (Forstemann, 2005).

Next, the size of the pre-miRNA processing complex was estimated by gel filtration chromatography. Pre-miRNA processing activity chromatographs as a broad approximately 525-kDa peak that overlaps the peaks of both Dcr-1 and Loqs proteins. Dcr-1 protein chromatographs as an approximately 480-kDa complex that overlaps the peak of Loqs PB, which chromatographs as an approximately 630-kDa complex. The Loqs PB isoform accounts for most of the Dcr-1-associated Loqs in S2 cells. The apparent size of the Dcr-1 complex suggests that it is either associated with proteins in addition to Loqs or that the complex has an elongated shape that increases its apparent molecular weight. Pre-miRNA processing activity, Loqs, and Dcr-1 were all well resolved from the approximately 230-kDa peak of Dcr-2, which corresponds to the Dcr-2/R2D2 heterodimer. Although the peaks of Loqs and Dcr-1 do not co-migrate, Dcr-1 is stably associated with Loqs after gel filtration: Dcr-1 and Loqs reciprocally co-immunoprecipitates from the pooled peak Dcr-1 fractions. Loqs was not detected in the Dcr-2 peak by this method. Loqs PC, which does not associate with Dcr-1 in immunoprecipitation, chromatographs as a 58-kDa protein, suggesting that it is a free monomeric protein (Forstemann, 2005).

Dicer-1 and R3D1-L catalyze microRNA maturation in Drosophila

In Drosophila melanogaster, Dicer-2/R2D2 and Dicer-1 generate small interfering RNA (siRNA) and microRNA (miRNA), respectively. A novel dsRNA-binding protein, R3D1-L, forms a stable complex with Dicer-1 in vitro and in vivo. While depletion of R3D1-L by RNAi causes accumulation of precursor miRNA (pre-miRNA) in S2 cells, recombinant R3D1-L enhances miRNA production by Dicer-1 in vitro. Furthermore, R3D1 deficiency causes miRNA-generating defect and severe sterility in male and female flies. Therefore, R3D1-L functions in concert with Dicer-1 in miRNA biogenesis and is required for reproductive development in Drosophila (Jiang, 2005).

Based on studies of Dicer-2/R2D2 in the siRNA pathway, it was hypothesized that Dicer-1 also functions in concert with an unknown dsRNA-binding protein in the miRNA pathway. Through a bioinformatics approach, an open reading frame (ORF, CG6866) was identified in the fly genome that showed considerable homology with R2D2 and RDE-4, an R2D2 homolog in C. elegans. Furthermore, PSI-Blast ranked this ORF as the best hit among R2D2-like proteins in FlyBase, and vice versa. This protein was named R3D1 because it contained three putative dsRNA-binding domains (R3) and was later found to associate with Dicer-1 (D1). The R3D1 gene encodes two alternatively spliced proteins, R3D1-L (long; 465 amino acids) and R3D1-S (short; 419 amino acids) (Jiang, 2005).

To test physical association of endogenous Dicer-1 and R3D1, coimmunoprecipitation (co-IP) experiments were performed by using anti-Dicer-1 or anti-R3D1 antibodies in the cytosolic (S100) extracts of S2 cells. R3D1-L (~55 kDa) was present in the IPs of anti-Dicer-1 but not anti-Dicer-2 antibodies. The presence of R3D1-S (~50 kDa) was not detected probably because it was absent or masked by the Immunoglobin (IgG) heavy chain. Reciprocal IP using anti-R3D1 antibody brought down Dicer-1 but not Dicer-2, whereas anti-R2D2 antibody only brought down Dicer-2. In addition, both Dicer-1 and R3D1 interacted with AGO1, a critical component of miRISC. These studies indicate that endogenous R3D1-L specifically associates with Dicer-1 and AGO1, which are key components of the initiation and effector complexes of the miRNA pathway (Jiang, 2005).

To study miRNA biogenesis, the miRNA-generating enzyme was purified from S2 cell extracts by biochemical fractionation. A single peak of activity was observed on all columns tested and was followed throughout the purification. Western blots were performed to detect the presence of Dicer-1, R3D1, Dicer-2, and R2D2 among individual fractions following each step of purification. Both Dicer-1 and R3D1-L showed perfect correlation with the miRNA-generating activity after every chromatography step. This was not the case for R3D1-S, nor for Dicer-2/R2D2, which has been shown to generate siRNA in S2 cells. Therefore, these results indicate that Dicer-1/R3D1-L and Dicer-2/R2D2 represent distinct initiation complexes of the miRNA and siRNA pathways in Drosophila cells (Jiang, 2005).

To determine if R3D1-L is required for miRNA biogenesis in vivo, Dicer-1, R3D1, or both were depleted in S2 cells by RNAi followed by Northern blotting to measure the levels of pre-bantam and bantam miRNA. Surprisingly, only R3D1-L (~55 kDa), but not R3D1-S (~50 kDa), protein was efficiently knocked down by treatment of R3D1 dsRNA. The R3D1 (~1.1 kb) dsRNA should efficiently target both R3D1-L and R3D1-S mRNA, which differ by 138 nt. It was likely that R3D1-S comigrated with a cross-reacting protein on the Western blot. Depletion of AGO1, a key component of miRISC, results in a specific reduction of bantam miRNA in S2 cells. In contrast, knocking down Dicer-1 causes accumulation of pre-bantam but no reduction in bantam. Targeting R3D1 produces a similar phenotype as Dicer-1 depletion. When Dicer-1 and R3D1 were both targeted, there was a greater accumulation of pre-bantam and a modest reduction in bantam. Since RNAi is transient and rarely a complete knockout, the lack of significant bantam reduction is probably because the remaining Dicer-1 is sufficient to maintain the level of miRNA production. Consistent with these results, the miRNA-generating activity was reduced by approximately twofold in Dicer-1- or R3D1-L-depleted cells and by ~3.5-fold in cells of double RNAi treatment. Thus, like Dicer-1, R3D1 is required for miRNA maturation in S2 cells (Jiang, 2005).

Genetic and biochemical studies have suggested that Dicer-1 and Dicer-2 may possess different biochemical activities. It is also possible that associated proteins, such as R3D1-L and R2D2, can help define the functional specificity for Dicer-1 and Dicer-2. To distinguish the two possibilities, polyhistidine-tagged Dicer-1 or Dicer-1/R3D1-L and Dicer-2 or Dicer-2/R2D2 recombinant proteins were expressed by using an insect cell expression system. These recombinant proteins were highly purified by Ni-NTA columns followed by SP-Sepharose and Q-Sepharose chromatography (Jiang, 2005).

Despite sharing extensive sequence homology, Dicer-1 and Dicer-2 display different substrate specificities. Dicer-1 demonstrates striking pre-miRNA processing activity, whereas miRNA generation is not detected for Dicer-2 at these concentrations. In contrast, Dicer-2 is much more active than Dicer-1 in processing long dsRNA into siRNA. In addition, Dicer-1 and Dicer-2 had different ATP requirements. Like human Dicer, Dicer-1 or Dicer-1/R3D1-L generates miRNA or siRNA in an ATP-independent manner, whereas Dicer-2 or Dicer-2/R2D2 required ATP hydrolysis for efficient siRNA production. Taken together, these reconstitution studies establish that Drosophila Dicer-1 and Dicer-2 are functionally distinct enzymes with different substrate specificities and ATP requirements (Jiang, 2005).

Recombinant Dicer-1 and R3D1-L formed a stable complex and cofractionated on multiple columns. Purified recombinant Dicer-1/R3D1-L complex is at least fivefold more active than Dicer-1 alone when measured in the pre-miRNA-processing assay. Consistent with this, addition of purified R3D1-L to Dicer-1 greatly enhances its miRNA-generating activity in a dose-dependent manner. Purified R3D1-S has a similar role but to a lesser degree. To compare the substrate affinity of Dicer-1 and Dicer-1/R3D1-L, gel-shift experiments were performed in the absence of Mg²⁺; this treatment blocks cleavage of pre-miRNA by Dicer-1. Addition of R3D1-L to Dicer-1 greatly enhances its affinity for pre-miRNA in a dose-dependent manner. These studies suggest that R3D1-L can enhance Dicer-1's miRNA-generating activity by increasing its substrate affinity (Jiang, 2005).

To study the physiological function of R3D1 in flies, a piggyBac (PB) fly strain was obtained in which the piggyBac transposon was inserted in the vicinity of the R3D1 gene. By cloning and sequencing the flanking sequences, it was found that the PB-element was inserted within the first exon and 221 nt upstream of the translational start codon of the R3D1 gene. The levels of R3D1-L and R3D1-S mRNA were much reduced in the homozygous flies when compared with wild type or heterozygotes by semiquantitative RT-PCR. However, corresponding reductions in R3D1 proteins could not be verified by Western blots due to masking by cross-reacting proteins. Nevertheless, this suggests that the PB-insertion creates a hypomorphic mutant allele of the R3D1 gene by attenuating its transcription (Jiang, 2005).

To examine if miRNA biogenesis is defective in the r3d1^PB mutant flies, the levels of pre-miR277 were compared in wild-type, heterozygous, and homozygous adult flies. As shown by Northern blots, there was significant accumulation of pre-miR277 in both male and female homozygotes. Consistent with this, there was an approximately sixfold reduction in the miRNA-generating activity in the whole fly extracts of r3d1^PB/r3d1^PB mutant females. The lack of reduction in mature miR277 can be explained by the fact that r3d1^PB is a partial loss-of-function allele. In addition, these data suggest that miRNA production may not be the rate-limiting step in the Drosophila miRNA pathway. Importantly, the miRNA-generating defect in the mutant lysates can be rescued by addition of recombinant R3D1-L, but not R3D1-S. Together, these results indicate that r3d1^PB mutant flies are defective for miRNA biogenesis (Jiang, 2005). It was suspected that r3d1 mutant flies might display developmental phenotypes because miRNAs play essential roles in animal development. Since r3d1^PB mutants survived to adulthood, it was decided to examine their fertility by setting up crosses between r3d1^PB homozygous males or females and their wild-type counterparts. Interestingly, while r3d1^PB mutant females are completely sterile, the males are ~60%-70% sterile when compared with the control crosses between heterozygotes and wild-type flies. To further analyze this phenotype, the testes and ovaries were dissected and examined from r3d1^PB mutant flies. Although mutant testes appeared normal, mutant ovaries contained a few maturing egg chambers and a shriveled germarium with few healthy germline stem cells. This is a classic 'germ cell loss' phenotype because a few egg chambers can develop from primordial germ cells when the adult ovary first forms. However, the mutant ovary did not sustain continuous egg chamber production since germline stem cells could not be properly maintained. These results indicate that R3D1 is required for normal reproductive development in male and female flies and suggest that specific miRNAs may play essential roles in maintaining germline stem cells in the fly ovary. It is suspected that R3D1 deficiency, and hence the miRNA biogenesis defect, is far more severe in mutant testes and ovaries than other parts of the fly body (Jiang, 2005).

miRNA and siRNA can be viewed as two parallel branches of the RNAi pathway. The biochemical studies demonstrate that Dicer-1/R3D1-L and Dicer-2/R2D2 are used as distinct initiation complexes for the miRNA and siRNA pathways, respectively, in Drosophila cells. The same concept can also be applied to species containing a single Dicer, such as C. elegans and humans. In C. elegans, DCR-1/RDE-4 functions as the initiation complex for the siRNA pathway. However, RDE-4 is not required for the miRNA pathway. It is likely that DCR-1 functions in concert with another dsRNA-binding protein in the miRNA pathway (Jiang, 2005).

By reconstitution, this study has established that Drosophila Dicer-1 and Dicer-2 enzymes are functional distinct enzymes with different ATP requirements and substrate specificities. Like Dicer-1, human Dicer generates miRNA or siRNA in an ATP-independent manner. Like Dicer-2, the C. elegans DCR-1 requires ATP hydrolysis for efficient siRNA production. While Dicer-1 is more suited for processing pre-miRNA, Dicer-2 favors long dsRNA as its ideal substrate. Thus, it will be important to identify the sequence and structural features that determine the evolutionary and functional differences between Dicer-1 and Dicer-2 (Jiang, 2005).

The Dicer-2/R2D2 complex not only generates siRNA, but also binds siRNA and facilitates siRNA loading onto the siRISC complex. It is likely that the Dicer-1/R3D1-L complex plays a similar role in facilitating miRNA loading onto the miRISC complex. Consistent with this hypothesis, recombinant Dicer-1/R3D1-L complex efficiently binds to the synthetic miRNA/miR* duplex. Since the majority of the miRNA/miR* duplexes have different stability at the two ends, this thermodynamic asymmetry is believed to cause preferential loading of miRNA onto miRISC and destruction of the miR* strand. It is reasonable to speculate that the Dicer-1/R3D1-L complex also functions as a sensor for the asymmetry of nascent miRNA/miR* duplex and helps to select the miRNA strand as the guide RNA for miRISC. Since R3D1-L interacts with both Dicer-1 and AGO1, it may play a similar role as R2D2 by bridging the initiation and effector steps of the miRNA pathway (Jiang, 2005).

While Dicer-2/R2D2 cleaves long dsRNA into siRNA, Drosha/Pasha (DGCR8 in human) and Dicer-1/R3D1-L catalyze sequential steps of miRNA biogenesis, processing of pri-miRNA into pre-miRNA and of pre-miRNA into miRNA, respectively. Although R2D2 does not regulate siRNA production, it facilitates the role of Dicer-2 in loading siRNA onto siRISC. While Pasha^fly/DGCR8^human is essential for Drosha to process pri-miRNA, R3D1-L greatly enhances miRNA generation by Dicer-1. Taken together, these studies indicate that all known RNase III enzymes (Drosha, Dicer-1, and Dicer-2) are paired with specific dsRNA-binding proteins (Pasha, R3D1-L, and R2D2) in catalyzing small RNA biogenesis and/or function in Drosophila. It remains uncertain if the same pattern will repeat in other species (Jiang, 2005).

Short interfering RNA strand selection is independent of dsRNA processing polarity during RNAi in Drosophila

Short interfering RNAs (siRNAs) guide mRNA cleavage during RNA interference (RNAi). Only one siRNA strand assembles into the RNA-induced silencing complex (RISC), with preference given to the strand whose 5' terminus has lower base-pairing stability. In Drosophila, Dcr-2/R2D2 processes siRNAs from longer double-stranded RNAs (dsRNAs) and also nucleates RISC assembly, suggesting that nascent siRNAs could remain bound to Dcr-2/R2D2. In vitro, Dcr-2/R2D2 senses base-pairing asymmetry of synthetic siRNAs and dictates strand selection by asymmetric binding to the duplex ends. During dsRNA processing, Dicer (Dcr) liberates siRNAs from dsRNA ends in a manner dictated by asymmetric enzyme-substrate interactions. Because Dcr-2/R2D2 is unlikely to sense base-pairing asymmetry of an siRNA that is embedded within a precursor, it is not clear whether processed siRNAs strictly follow the thermodynamic asymmetry rules or whether processing polarity can affect strand selection. This study used a Drosophila in vitro system in which defined siRNAs with known asymmetry can be generated from longer dsRNA precursors. These dsRNAs permit processing specifically from either the 5' or the 3' end of the thermodynamically favored strand of the incipient siRNA. Combined dsRNA-processing/mRNA-cleavage assays indicate that siRNA strand selection is independent of dsRNA processing polarity during Drosophila RISC assembly in vitro (Preall, 2006).

In Drosophila, Dcr enzymes are required for RISC assembly as well as dsRNA processing, suggesting that the two phases of RNAi might be functionally coupled in a manner that affects siRNA strand selection. However, these experiments indicate that Drosophila RISC assembly and siRNA strand selection are not significantly influenced by the dsRNA processing step and that the thermodynamic asymmetry rules apply equally well with processed and unprocessed siRNAs in this system. This suggests that Drosophila Dcr enzymes do not channel newly generated siRNAs directly into RISC, but rather release the siRNAs into solution (or onto another factor) before they enter the RISC assembly pathway (Preall, 2006).

Several observations have suggested that thermodynamic asymmetry governs strand selection for processed RNAi triggers. MicroRNAs (miRNAs) are diced from stem-loop precursors, and in most instances only one strand of the processed miRNA duplex is stably incorporated into RISC. The mature strand can be present at either the 5' or the 3' end of the stem-loop, but either way, the selected strand is generally compatible with the thermodynamic asymmetry guidelines. In addition, artificial dsRNAs introduced into plant cells give rise to a stable set of siRNAs that adhere to the asymmetry rules. Similar results have been reported with natural dsRNAs in plants. However, interpretation of these results is difficult because the Dcr processing polarities were not defined, and it is also not clear whether small RNA stability is always a suitable surrogate measure of RISC assembly. Furthermore, plant cells (unlike insect and mammalian cells) export siRNAs into the vasculature to enable systemic RNAi, and therefore the plant dsRNA processing machinery may have specifically evolved the propensity to release newly processed siRNAs. Thus the applicability of the plant analyses to insects and mammals has not been clear (Preall, 2006).

While this work was in progress, Rose (2005) characterized modified ~27 nt duplexes that force a defined Dcr processing polarity and give rise to specific, predictable 21 nt siRNAs. Experiments with these Dcr substrate RNAs revealed that hDcr processing polarity can in fact influence siRNA strand selection in transfected human cells, although it does not completely supercede thermodynamic asymmetry. The reasons for the discrepancy between the results and those of Rose are not clear, although one possibility is that different Dcr enzymes may vary in their tendencies to remain associated with newly generated siRNAs. It is noteworthy that Drosophila Dcr-2 (which is primarily devoted to the siRNA pathway) appears to lack the canonical PAZ domain that normally provides Dcr with a binding pocket for 2 nt 3' overhangs. A PAZ domain is present in hDcr, and mutational analyses indicate that the hDcr PAZ domain assists with dsRNA binding and processing when a 2 nt 3' overhang is present. The apparent lack of a PAZ domain in Dcr-2 may compromise its ability to remain bound to newly cleaved siRNA. It is curious that Drosophila Dcrs are required for RISC assembly but do not appear to couple dsRNA processing to siRNA strand selection, whereas mammalian Dcrs are not required for RISC assembly but apparently do couple dsRNA processing to siRNA strand selection (Preall, 2006).

Finally, it remains to be determined whether Dcr enzymes associate with long dsRNA processing substrates and siRNA RISC-assembly substrates in the same way. This issue is undoubtedly important for understanding the functional relationship between Dcr's roles in the initiator and effector phases of RNAi. Crystal structures of E. coli RNase III, an ancestor of eukaryotic Dcrs, are likely to be informative. The structural data, and models derived from them, depict a protein that can engage dsRNAs in a dynamic fashion. A single dsRBD on each subunit of the RNase III homodimer is tethered to the endonuclease domain by a flexible linker that can rotate roughly 90° around the catalytic core. Thus, there are likely to be at least two binding modes for dsRNA in complex with an RNase III enzyme: one in which the dsRBD braces the RNA helix from either side as it is channeled into the catalytic cleft, and another where the dsRBD holds the dsRNA above and orthogonal to the active site. It is possible that Dcr enzymes also exhibit alternate dsRNA binding modes depending on whether they are actively processing dsRNA or channeling siRNA into RISC. Interconversion between these two conformations may require at least transient release of the siRNA product. Additional dynamic dsRNA/protein interactions during dsRNA processing and RISC assembly presumably involve the dsRNA binding proteins Loquacious/R3D1, R2D2, and TRBP, which associate with Dcr-1, Dcr-2, and hDcr, respectively. Further functional analysis of Dcr's PAZ, RNase III, and dsRNA binding domains, aided by recent advances in the structural biology of Dcr, will be necessary to understand Dcr's roles in the transition between the initiation and effector phases of RNAi (Preall, 2006).

The mirtron pathway generates microRNA-class regulatory RNAs in Drosophila

The canonical microRNA (miRNA) pathway converts primary hairpin precursor transcripts into 22 nucleotide regulatory RNAs via consecutive cleavages by two RNase III enzymes, Drosha and Dicer. This study characterizes Drosophila small RNAs that derive from short intronic hairpins termed 'mirtrons.' Their nuclear biogenesis appears to bypass Drosha cleavage, which is essential for miRNA biogenesis. Instead, mirtron hairpins are defined by the action of the splicing machinery and lariat-debranching enzyme, which yield pre-miRNA-like hairpins. The mirtron pathway merges with the canonical miRNA pathway during hairpin export by Exportin-5, and both types of hairpins are subsequently processed by Dicer-1/loqs. This generates small RNAs that can repress perfectly matched and seed-matched targets, and evidence is provided that they function, at least in part, via the RNA-induced silencing complex effector Ago1. These findings reveal that mirtrons are an alternate source of miRNA-type regulatory RNAs (Okamura, 2007).

This study has characterized a class of intronic hairpins, termed mirtrons, that generate ~22 nt regulatory RNAs in Drosophila. The biogenesis of mirtrons is distinct from that of canonical miRNAs. Although alternate mechanisms are not excluded, the data points to a mechanism in which mirtron maturation bypasses cleavage by the pre-miRNA-generating enzyme Drosha but is instead initiated by splicing and intron lariat debranching. This differs explicitly from the processing of canonical intronic miRNA genes, whose cleavage by Drosha occurs prior to host intron splicing. However, the mirtron pathway merges with the canonical miRNA pathway to generate active regulatory RNAs, since debranched mirtrons are productive substrates of Exportin-5 and the Dicer-1/loqs system, yielding small RNAs that can repress target transcripts. This study showed specifically that mirtron-derived small RNAs can associate with Ago1 and require Ago1 to regulate seed-matched targets (Okamura, 2007).

The functional similarity between mirtrons and miRNA precursors is bolstered by the observation that miR-10-3p and the small-RNA product of a mirtron hairpin in Vha-SFD are extensively related across their 5' halves, are derived from the same (right-hand) hairpin arm, are the most abundant products of their respective hairpins, and have the same seed (positions 2-8, AAAUUCG). The small-RNA products of mirtrons are catagorized as a novel subclass of miRNAs (Okamura, 2007).

Fourteen mirtron loci were identified from a high-throughput sequencing effort that confidently identified 133 canonical miRNA genes; thus, mirtrons constitute a considerable fraction of total miRNA genes in Drosophila. In contrast, while a majority of canonical miRNA genes are well-conserved among the sequenced Drosophilids, most mirtrons arose recently during evolution. Since newly evolved miRNAs are thought to have fewer targets than highly conserved miRNAs, the regulatory networks involving mirtrons may be proportionally smaller than those mediated by canonical miRNAs. Still, the findings that both 'old' and 'young' mirtrons (1) produce miRNAs that associate with Ago1, (2) can actively repress minimally paired seed targets, and (3) display patterns of divergence on microevolutionary scales that indicate their incorporation into endogenous regulatory networks together suggest that mirtrons exert appreciable effects on biological networks. Indeed, the relative ease with which mirtrons have been born and/or lost raises the intriguing possibility that the changing mirtronic content of Drosophila genomes has contributed to fly speciation (Okamura, 2007).

The existence of mirtrons has implications for the interpretation of miRNA genetics. It is now recognized that the Dicer mutant condition does not solely reflect the loss of miRNAs, since Dicer has additional roles in chromatin dynamics and/or processing of exogenous or other endogenous dsRNA, depending on the organism. Drosha mutant cells do not accurately reflect the loss of miRNAs either; since Drosha processes other ncRNAs, including rRNAs. It has been suggested that DGCR8/Pasha mutant cells more purely reflect a 'miRNA null' state. This may not be the case either, because the mirtron pathway generates a subclass of miRNAs via a nuclear pathway that is largely, if not completely, distinct from the microprocessor. Therefore, caution should be exercised when using processing-enzyme mutants to assess the contribution of small RNAs to a given biological process (Okamura, 2007).

The data demonstrate that the Drosophila mirtron pathway merges the splicing/debranching pathway with the dicing pathway to generate functional miRNAs. Since the key parts of this hybrid small-RNA pathway are deeply conserved mechanisms for RNA processing, it seems plausible that mirtrons may exist outside of Drosophila. Since debranched introns are normally quite labile; however, it is hypothesize that critical to the operation of the mirtron pathway is a dedicated mechanism to hand-off debranched introns to the hairpin export machinery. Having such a mechanism in place may prove key to the existence of mirtrons in other species (Okamura, 2007).

Drosophila R2D2 mediates follicle formation in somatic tissues through interactions with Dicer-1

The miRNA pathway has been shown to regulate developmentally important genes. Dicer-1 is required to cleave endogenously encoded microRNA (miRNA) precursors into mature miRNAs that regulate endogenous gene expression. RNA interference (RNAi) is a gene silencing mechanism triggered by double-stranded RNA (dsRNA) that protects organisms from parasitic nucleic acids. In Drosophila, Dicer-2 cleaves dsRNA into 21 base-pair small interfering RNA (siRNA) that are loaded into RISC (RNA induced silencing complex) that in turn cleaves mRNAs homologous to the siRNAs. Dicer-2 co-purifies with R2D2, a low-molecular weight protein that loads siRNA onto Ago-2 in RISC. Loss of R2D2 results in defective RNAi. However, unlike mutants in other RNAi components like Dicer-2 or Ago-2, r2d2¹ mutants have striking developmental defects. r2d2¹ mutants have reduced female fertility, producing less than 1/10 the normal number of progeny. These escapers have normal morphology. R2D2 functions in the ovary, specifically in the somatic tissues giving rise to the stalk and other follicle cells critical for establishing the cellular architecture of the oocyte. Most interestingly, the female fertility defects are dramatically enhanced when one copy of the dcr-1 gene is missing and Dicer-1 protein co-immunoprecipitates with R2D2 antisera. These data show that r2d2¹ mutants have reduced viability and defective female fertility that stems from abnormal follicle cell function, and Dicer-1 impacts this process. It is concluded that R2D2 functions beyond its role in RNA interference to include ovarian development in Drosophila (Kalidas, 2008).

R2D2 has a well characterized role with Dicer-2 and Ago-2 in RNA interference. R2D2 forms a stable heterodimeric complex with Dicer-2 in vivo and in vitro. R2D2 does not affect the enzymatic activity of Dicer-2, but instead orchestrates the transfer of siRNAs produced by Dicer-2 to Ago-2. Dicer-2 and Ago-2 physically interact in the same complex with R2D2 during RNA interference. dcr-2 mutants are defective for RNAi but have normal fertility. ago-2 mutants are also viable and fertile, but have been recently reported to have defects in nuclear divisions and migration in early embryonic development, with a reduction in the number of pole cells that give ultimately give rise to the germline. However, these subtle developmental defects in ago-2 mutants are compensated for during development so they have little effect on overall fertility, and were not reported in the initial characterization of the null mutants. By contrast, r2d2¹ mutants have striking fertility defects. These abnormalities are not observed in dcr-2 and have much higher penetrance and are qualitatively different from ago-2 mutants, suggesting there is a Dicer-2 and Ago-2-independent role for R2D2 in vivo. The genetic interaction observed with Dicer-1 provides the first insights into this role (Kalidas, 2008).

The hatching frequency of r2d2¹ null mutant embryos was approximately 14% of that of wild type embryos. This drop in hatching is due in part to defective fertilization. However, fertilized r2d2¹ mutant embryos generally failed to develop, showing no signs of nuclear divisions and were usually arrested before the blastoderm stage. Viability was improved in embryos fertilized by wild type sperm, indicating R2D2 is important both maternally and zygotically. Therefore, these data indicates that R2D2 contributes to viability early in development and embryogenesis, in addition to its established role in RNA interference (Kalidas, 2008).

r2d2¹ mutants have defective ovaries and are partially sterile. The ovaries from these mutants show a range of phenotypic defects, all of which are completely rescued by introduction of an r2d2 transgene into the mutant background, clearly demonstrating loss of R2D2 produces these defects in oogenesis. Clonal analysis reveals a requirement for R2D2 in the somatic follicle cells, but the data indicates there is not a germline requirement for R2D2. Removal of R2D2 specifically from somatic cells results in the typical ovarian architecture defects including loss of stalks observed in r2d2¹, while loss of R2D2 in germ cells did not affect follicle morphology. The observed follicle cell phenotypes, while broader in scope, are similar to those previously described for defective polar cell specification and formation. Once specified, polar cells are known to secrete a variety of signals that pattern the follicular epithelium. The robust expression of R2D2 protein in the stalk cells will focus future efforts on candidate signaling pathways expressed in these cells (Kalidas, 2008).

Several genes related to RNAi components have defects in ovarian development. These include piwi, aubergine and Spindle-E. However, the phenotypes associated with these other mutants are quite different from found here for r2d2¹. piwi is an Argonaut homologue involved in stem cell maintenance, co-suppression, heterochromatin formation, and transposon silencing. However piwi, while expressed in both somatic and germline cells, is required in the terminal filament for stem cell maintenance but is also needed for cell division in the germline. Aubergine and spindle-E are also involved in heterochromatin formation and aubergine plays a role in suppression of stellate required for male fertility. However, aubergine and spindle-E are required in the germline. Most recently Loquacious was reported to have a role in germline stem cell maintenance and stellate suppression. R2D2 appears unique among the RNAi-related components identified to date because it is required in the follicle tissue and not in the germline, and has no effect on male fertility. Therefore, the function of R2D2 in female fertility appears to be distinct from these other RNAi related proteins (Kalidas, 2008).

The genetic and physical interaction between Dicer-1 and R2D2 implies these gene products are operating in the same pathway to orchestrate female follicle cell patterning. Dicer-1 mutants are lethal, likely due to loss of mature miRNAs and subsequent regulation of endogenous gene function. r2d2¹ flies can survive to adulthood and are morphologically normal, so R2D2 is unlikely to have a significant role in processing most miRNAs. One possibility is that Dicer-1 and R2D2 are partnered in the ovary to process one or a small number of miRNAs in the follicle cells. Dicer-1 has three splicing variants, so specific splicing variants of Dicer-1 could potentially partner with R2D2. Alternatively, R2D2 and Dicer-1 may perform an unknown function independent of miRNA processing. Screens to identify suppressor mutations of r2d2¹ with increased fertility will prove valuable in providing future insights into R2D2-dependent follicle formation (Kalidas, 2008).

Processing of Drosophila endo-siRNAs depends on a specific Loquacious isoform

Drosophila expresses three classes of small RNAs, which are classified according to their mechanisms of biogenesis. MicroRNAs are ~22-23 nucleotides (nt), ubiquitously expressed small RNAs that are sequentially processed from hairpin-like precursors by Drosha/Pasha and Dcr-1/Loquacious complexes. MicroRNAs usually associate with AGO1 and regulate the expression of protein-coding genes. Piwi-interacting RNAs (piRNAs) of ~24-28 nt associate with Piwi-family proteins and can arise from single-stranded precursors. piRNAs function in transposon silencing and are mainly restricted to gonadal tissues. Endo-siRNAs are found in both germline and somatic tissues. These ~21-nt RNAs are produced by a distinct Dicer, Dcr-2, and do not depend on Drosha/Pasha complexes. They predominantly bind to AGO2 and target both mobile elements and protein-coding genes. Surprisingly, a subset of endo-siRNAs strongly depend for their production on the dsRNAbinding protein Loquacious (Loqs), thought generally to be a partner for Dcr-1 and a cofactor for miRNA biogenesis. EndosiRNA production depends on a specific Loqs isoform, Loqs-PD, which is distinct from the one, Loqs-PB, required for the production of microRNAs. Paralleling their roles in the biogenesis of distinct small RNA classes, Loqs-PD and Loqs-PB bind to different Dicer proteins, with Dcr-1/Loqs-PB complexes and Dcr-2/Loqs-PD complexes driving microRNA and endo-siRNA biogenesis, respectively (Zhou, 2009).

Dicer partner proteins tune the length of mature miRNAs in flies and mammals

Drosophila Dicer-1 produces microRNAs (miRNAs) from pre-miRNA, whereas Dicer-2 generates small interfering RNAs (siRNAs) from long dsRNA. Alternative splicing of the loquacious (loqs) mRNA generates three distinct Dicer partner proteins. To understand the function of each, flies were constructed expressing Loqs-PA, Loqs-PB, or Loqs-PD. Loqs-PD promotes both endo- and exo-siRNA production by Dicer-2. Loqs-PA or Loqs-PB is required for viability, but the proteins are not fully redundant: a specific subset of miRNAs requires Loqs-PB. Surprisingly, Loqs-PB tunes where Dicer-1 cleaves pre-miR-307a, generating a longer miRNA isoform with a distinct seed sequence and target specificity. The longer form of miR-307a represses glycerol kinase and taranis mRNA expression. The mammalian Dicer-partner TRBP, a Loqs-PB homolog, similarly tunes where Dicer cleaves pre-miR-132. Thus, Dicer-binding partner proteins change the choice of cleavage site by Dicer, producing miRNAs with target specificities different from those made by Dicer alone or Dicer bound to alternative protein partners (Fukunaga, 2012).

The simplest interpretation of these data that Loqs-PA and Loqs-PB both decrease the KM of Dcr-1 for pre-miRNA substrates is that Loqs partner proteins increase the affinity of Dcr-1 for some pre-miRNA substrates. For other pre-miRNAs, Loqs-PB can also increase Dcr-1 enzyme turnover. Thus preventing Dcr-2 from processing pre-miRNAs (R2D2), promoting the processing of 'foreign' dsRNA and of esiRNAs and other endo-siRNA precursors (Loqs-PD), enhancing the efficiency of pre-miRNA processing by Dcr-1 (Loqs-PA and PB), and tuning cleavage site choice by Dcr-1 for a small number of pre-miRNAs (Loqs-PB) (Fukunaga, 2012).

The last two functions are surprising because the number of pre-miRNAs that require Loqs-PB and not Loqs-PA for the efficient production of their miRNAs is small and even fewer require Loqs-PB to produce the correct isomir. Nonetheless, the results suggest that the loss of GSCs in flies lacking Loqs- PB reflects the reduced abundance of specific miRNAs such as miR-79, miR-283, miR-305, miR-311, and miR-318 or perhaps the production of the wrong isomirs for miR-307a, miR-87, or miR-316. Among these, miR-318 is the most abundant miRNA in ovaries (140,000 ppm in w1118 ovaries but only 8 ppm in heads). Future experiments using Loqs-independent versions of pre-miR-318 and other miRNAs should help test this idea (Fukunaga, 2012).

The amino acid sequence of Loqs-PB suggests it is slightly more related to PACT (28% identity; 34% similarity) than to TRBP (24% identity; 33% similarity). Nonetheless, the data suggest that the functional homolog of Loqs-PB in mammals is TRBP. Like Loqs-PA and Loqs-PB, TRBP may act generally to enhance the binding of Dicer to pre-miRNA. Such a role for TRBP is consistent with earlier observations that the two cleavage reactions catalyzed by mammalian Dicer are less well coordinated in the absence of TRBP. But like Loqs-PB, TRBP also helps Dicer produce specific isoforms of a one or more miRNAs. This novel function of Dicer partner proteins may be widely conserved, enabling plants and animals to effectively and accurately dice difficult but important pre-miRNA substrates. Perhaps the Drosha-binding partner Pasha in flies or DGCR8 in mammals similarly tunes pri-miRNA cleavage site choice by Drosha (Fukunaga, 2012).

The stem, but not the loop, of pre-miR-307a enables Loqs-PB to influence where Dcr-1 cleaves. One possible explanation for how Loqs-PB and TRBP change where Dcr-1 and Dicer cleave is that the mismatches and internal loop causes the stems of pre-miR-307a, pre-miR-87, and mammalian pre-miR-132 to be longer than the corresponding A-form helix; binding of Loqs-PB or TRBP might then 'shrink' the stems. In contrast, Loqs-PB binding may extend of the stem of premiR-316. Because Loqs-PB does not change where Dcr-2 cleaves pre-miR-307a, Loqs-PB likely acts only when bound to Dcr-1 (Fukunaga, 2012).

The data suggest that the effect of Loqs-PB is biologically relevant. The long miR-307a isomir predominates in wild-type flies, and miR-307^a23-mer but not miR-307^a21-mer can repress the Gk and tara mRNAs in vivo. Why has evolution failed to select for easier-to-dice variants of pre-miR-307a and premiR- 87 in flies and pre-miR-132 in mice, so that a specific partner is no longer needed to ensure production of the 'right' isomir? The persistence of the miR-307^a21-mer in wild-type flies, for example, suggests that most, if not all, abundant isomirs have distinct biological functions and that the optimal function of miRNAs like miR-307a, miR-87, and miR-132 requires a defined ratio of isomirs (Fukunaga, 2012).

Although this study failed to detect altered regulation of predicted miR-307a^21-mer targets in ovaries from flies lacking Loqs-PB, miR-307a^21-mer may have functions in other tissues or times in development. In flies, mice, and humans, the relative abundance of miRNA isomirs generated by different Dicer cleavage sites, including 50 isomirs with distinct seed sequences, varies among tissues and developmental stages. Perhaps the abundance of Loqs-PB versus Loqs-PA or TRBP versus PACT is regulated across development and differentiation to ensure the correct relative abundance of isomirs from various pre-miRNAs, much as the ratios of alternatively spliced mRNAs are regulated in different tissues and cell types. The loqsKO flies rescued with transgenes producing individual Loqs isoforms should facilitate the testing of this idea in vivo (Fukunaga, 2012).

Drosophila oncogene Gas41 is RNAi modulator that intersects heterochromatin and siRNA pathway

Glioma amplified sequence41 (Gas41) is a highly conserved putative transcription factor that is frequently abundant in human gliomas. Gas41 shows oncogenic activity by promoting cell growth and viability. This study shows Gas41 is required for proper functioning of RNAi machinery in the nuclei, though three basic structural domains of RNAi components PAZ, PIWI and dsRNA binding are absent in the structural sequences. Variations of structural domains are highly conversed among prokaryotes and eukaryotes. Gas41 interacts with cytological RNase III enzyme Dicer1 both biochemically and genetically. However, Drosophila Gas41 functions as chromatin remodeler and interacts with different heterochromatin markers and repeat induced transgene silencing by modulating PEV. This study also shows that transcriptional inactive Gas41 mutant interferes the functional assembly of heterochromatin associated proteins, H3K9me2 and HP1 in developing embryos. A reduction of heterochromatic markers is accompanied with mini-w promoter sequence in Gas41 mutants. These findings suggest that, Drosophila Gas41 guides the repeat associated gene silencing, and Dicer1 interaction thereby depicting a new role of the Gas41. It is a critical RNAi component. In Drosophila, Gas41 plays a dual role. In one hand, it seems to participate with Dicer 1 in the RNAi pathway and alternatively also participate in repeat-induced gene silencing by accumulating heterochromatin proteins at the mw array promoters. Therefore, it proposes an intriguing and seemingly paradoxical new finding in RNA technology in the process of heterochromatin gene silencing (Gandhi, 2014).

DEVELOPMENTAL BIOLOGY

Dicer mRNA is expressed in embryos, in S2 cells and in adult flies, which is consistent with the presence of functional RNAi machinery in all of these contexts (Bernstein, 2001).

Localization of Dicer in oocytes

A hallmark of germline cells across the animal kingdom is the presence of perinuclear, electron-dense granules called nuage. In many species examined, Vasa, a DEAD-box RNA helicase, is found in these morphologically distinct particles. Despite its evolutionary conservation, the function of nuage remains obscure. A null allele of maelstrom (mael) has been characterized. Maelstrom protein is localized to nuage in a Vasa-dependent manner. By phenotypic characterization, maelstrom has been defined as a spindle-class gene that affects Vasa modification. In a nuclear transport assay, it has been determined that Maelstrom shuttles between the nucleus and cytoplasm, which may indicate a nuclear origin for nuage components. Interestingly, Maelstrom, but not Vasa, depends on two genes involved in RNAi phenomena for its nuage localization: aubergine and spindle-E (spn-E). Furthermore, maelstrom mutant ovaries show mislocalization of two proteins involved in the microRNA and/or RNAi pathways, Dicer and Argonaute2, suggesting a potential connection between nuage and the microRNA-pathway (Findley, 2003).

The dissociation of Maelstrom from nuage particles in aubergine and spn-E backgrounds was intriguing in light of their requirement in RNAi in Drosophila spermatogenesis and late oogenesis. Importantly, proteins (or homologs) of RNAi pathway components also act in micro RNA (miRNA) processing. Since miRNAs have been shown to regulate RNA translation, it is conceivable that miRNAs are assembled in RNP particles formed in nuage. In this setting, nuage could represent a step in the generation of specificity in translational control in the germline. To explore this potential relationship between nuage and RNAi/miRNA processing pathways, the localization of additional RNAi components was examined in wild-type and maelstrom ovaries. Argonaute1 and Argonaute2 are RDE1/AGO1 homologs required for RNAi in Drosophila. Dicer is the core RNase of RNAi in Drosophila; it is also required for production of the small RNA effectors of the RNAi and miRNA pathways in C. elegans. In vertebrate cell lines, Dicer is primarily cytoplasmic. In wild-type Drosophila ovarioles, Dicer and AGO1 appear uniform and cytoplasmic in nurse cell cytoplasm; AGO2 appears cytoplasmic but relatively more granular. In maelstrom ovaries, AGO1 distribution is relatively unperturbed. However, AGO2 and Dicer are both dramatically mislocalized in maelstrom ovarioles. Beginning around stage 3, Dicer aggregates in discrete, often perinuclear foci in nurse cells. AGO2 is observed in perinuclear regions of nurse cells, which, by contrast, can colocalize with Vasa in nuage (Findley, 2003).

EFFECTS OF MUTATION

To determine whether the Dicer enzyme is involved in RNAi in vivo, Dicer activity was depleted from S2 cells and the effect on dsRNA-induced gene silencing was tested. Transfection of S2 cells with a mixture of dsRNAs homologous to the two Drosophila Dicer genes (Dicer-1 and CG6493/Dicer-2) resulted in a roughly 6-7-fold reduction of Dicer activity either in whole-cell lysates or in Dicer immuno-precipitates. Transfection with a control dsRNA had no effect. Qualitatively similar results were seen if Dicer mRNA was examined by Northern blotting. Depletion of Dicer substantially compromises the ability of cells to silence an exogenous, green fluorescent protein transgene by RNAi. These results indicate that Dicer may be involved in RNAi in vivo. The lack of complete inhibition of silencing may result from an incomplete suppression of Dicer or may indicate that in vivo guide RNAs may be produced by more than one mechanism (Bernstein, 2001).

Distinct roles for Drosophila Dicer-1 and Dicer-2 in the siRNA/miRNA silencing pathways

RNA silencing phenomena, either the regulation of mRNA translation or regulation of mRNA degradation, intersect at the ribonuclease Dicer. In animals, the double-stranded RNA-specific endonuclease Dicer produces two classes of functionally distinct, tiny RNAs: microRNAs (miRNAs) and small interfering RNAs (siRNAs). miRNAs regulate mRNA translation, whereas siRNAs direct RNA destruction via the RNA interference (RNAi) pathway. siRNAs and miRNAs then direct a RNA-induced silencing complex (RISC) to cleave mRNA or block its translation (RNAi). Mutations have been characterized in the Drosophila dicer-1 and dicer-2 genes. Mutation in dicer-1 blocks processing of micro RNA precursors, whereas dicer-2 mutants are defective for processing siRNA precursors. It has been recently found that Drosophila Dicer-1 and Dicer-2 are also components of siRNA-dependent RISC (siRISC). Dicer-1 and Dicer-2 are required for siRNA-directed mRNA cleavage, though the RNase III activity of Dicer-2 is not required. Dicer-1 and Dicer-2 facilitate distinct steps in the assembly of siRISC. However, Dicer-1 (but not Dicer-2) is essential for miRISC-directed translation repression. Thus, siRISCs and miRISCs are different with respect to Dicers in Drosophila (Lee, 2004).

An important though not absolute role has been established for Dcr-2 in Drosophila RNAi. Since another Dicer (Dcr-1) is present in Drosophila, it is possible that it has a redundant function with Dcr-2. The dcr-1 gene is located at 94C4 on the third chromosome, and a mutation was found that mapped by linkage and complementation analysis to that region. The coding sequence of dcr-1 in the mutant contained a premature stop codon such that the truncated product lacks the PAZ and RNase III domains. Moreover, dcr-1 mRNA is not detectable in the mutant as determined by RT-PCR and Northern blot analysis, suggesting that the transcript is unstable when truncated Dcr-1 protein is produced. This transcript null dcr-1^Q1147X mutant exhibited an eye color phenotype when assayed in a GMR-wIR genetic background. The mutant appeared to partially suppress silencing by GMR-wIR, with patches of dark orange eye color. In addition, the eye was half its normal size, the organization of ommatidial facets was disrupted, and sensory bristles were missing over the eye surface. Other bristles, which flank the eye surface, were sometimes absent or exhibited hyperplasia (Lee, 2004).

Despite an effect on white gene silencing, the dcr-1 mutant had normal levels of wIR siRNAs. This observation is consistent with Dcr-2 processing the great majority of wIR dsRNA. It is further consistent with a central role for Dicer helicase activity in dsRNA processing, since Dcr-1 lacks a DExH-box helicase domain. If the dcr-1 mutant has normal dsRNA processing, why is it partially disrupted for gene silencing? To answer this, clones of homozygous dcr-1 mutant germ cells were generated in heterozygous females, and then dcr-1 mutant eggs were injected with either dsRNA or siRNA complementary to bicoid transcripts. Loss of bicoid mRNA was measured as a consequence. dcr-1 mutant eggs exhibit an impaired RNAi response to dsRNA and siRNA. Six-fold more bicoid mRNA was present in dcr-1 mutant eggs compared to wild-type eggs after either dsRNA or siRNA treatment. This result indicates that Dcr-1 acts downstream of siRNA production in the RNAi pathway. Dcr-1 plays an important though not absolute role in siRNA-dependent RNAi. Since Dcr-2 is also required downstream of siRNAs, these data suggest that Dcr-1 and Dcr-2 function might be partially redundant in some downstream activity (Lee, 2004).

Dcr-1 is essential to generate mature miRNAs. This role was demonstrated by analyzing miRNA levels in dcr-1 mutant eggs. No mature miRNAs belonging to the miR-2 group were detected in dcr-1 mutant eggs. Thus, Dcr-1 is critical for miRNA production whereas Dcr-2 is required primarily for siRNA production (Lee, 2004).

Although Dcr-1 and Dcr-2 preferentially produce different types of small RNAs, both are required for efficient siRNA-dependent mRNA degradation. Does this dual requirement extend to the miRNA pathway as well? To test this possibility, a genetic assay for miRNA-dependent gene silencing was used in dcr-1 and dcr-2 mutants. Several classes of motifs are present in the 3'UTR regions of the E(spl) and Bearded genes. The 3'UTR motifs are complementary to a variety of miRNAs, and they mediate posttranscriptional repression of gene expression. A series of reporter transgenes was constructed that mimics this posttranscriptional repression. The reporter genes contain a constitutive promoter from armadillo, lacZ coding sequence, and the 3'UTR from the Bearded or E(spl)m8 gene. When the reporter contains a wild-type Bearded 3'UTR, its expression in the developing eye disc is very weak. It is somewhat more strongly expressed in the eye disc posterior to the morphogenetic furrow and is equally weak in the anterior eye disc and antennal disc. When the reporter contains a Bearded 3'UTR with its three B motifs mutated, expression is ubiquitously strong in the eye and antennal discs, confirming that the B motifs mediate a silencing effect on gene expression (Lee, 2004).

Expression of a wild-type reporter gene was examined in clones of mutant dcr-2 cells that were generated in the developing eye disc. Clones expressed the reporter at levels indistinguishable from wild-type tissue, indicating that Dcr-2 is not required for this gene silencing mechanism. In contrast, expression of a wild-type reporter gene in clones of mutant dcr-1 cells was much stronger than in wild-type tissue. The derepressive effect of the dcr-1 mutation requires intact B motifs in the Bearded 3'UTR, since mutant clones did not affect expression of a reporter gene with mutated B motifs. These results argue that dcr-1 but not dcr-2 is necessary for posttranscriptional gene silencing that is mediated by a miRNA mechanism. This conclusion is also validated by other mutant phenotypes associated with each gene. Loss of dcr-1 has profound effects on Drosophila development within both somatic- and germ-lineages, whereas loss of dcr-2 appears to have little or no effect on development (Lee, 2004).

The dcr-1 mutant clones exhibited an interesting pattern of reporter expression. Clones in the antennal disc and eye disc, anterior to the morphogenetic furrow, exhibited little or no derepression of the reporter gene. Typically, only a few mutant cells in each clone had high levels of reporter gene expression. No overt cell differentiation occurs in this region of the eye disc. In contrast, almost all eye disc clones posterior to the furrow, where cell differentiation actively occurs, exhibited extensive derepression. Many, if not most, mutant cells in a clone exhibited this behavior. A boundary of reporter gene expression within a clone could be detected if the clone was bisected by the furrow. This boundary coincided with the morphogenetic furrow. Three interpretations seem possible. (1) Different sets of miRNAs repress the reporter in different regions of the eye disc, one set of which requires Dcr-1 and one set of which does not. However, all of these miRNAs would have to act through the 3'UTR binding sites, since a mutated reporter is constitutively derepressed. Thus, this interpretation is not favored. (2) Dcr-1 might not be essential in anterior disc cells because Dcr-2 or another factor substitutes if Dcr-1 is missing. (3) Anterior disc cells may contain miRNAs that were originally generated in dcr-1⁺ progenitor cells, and may therefore not require dcr-1. Passage of the morphogenetic furrow may trigger miRNA turnover, resulting in renewed dependence on dcr-1 posterior to the furrow (Lee, 2004).

The RNAi pathway can be divided into discrete biochemical steps: dsRNA processing, maintenance of siRNA 5' phosphate termini, siRNA loading into siRISC, and siRISC-based cleavage of target mRNA. It was confirmed that Dcr-2 but not Dcr-1 is required for dsRNA processing by incubating radiolabeled dsRNA substrate in lysates made from mutant embryos and monitoring siRNA formation. Labeled 21-23 nucleotide RNAs were readily detectable from reactions with wild-type and dcr-1 lysates, but were greatly reduced in reactions with dcr-2 lysate (Lee, 2004).

The genetic experiments suggested a role for Dcr-1 and Dcr-2 downstream of dsRNA processing. Therefore, lysates made from mutant embryos were assayed for steps downstream of siRNA production. siRNAs require 5' phosphate termini for proper association with RISC, and the 5' phosphates are maintained by a kinase that recognizes siRNAs (Nykanen, 2001). Both wild-type and dcr-2 mutant lysates efficiently converted synthetic siRNAs bearing 5' hydroxyl groups into 5'-phosphorylated forms, indicating that the mutant lysates have normal end-maintenance activity (Lee, 2004).

Nykanen (2001) reported that siRNAs are incorporated into a 350 kDa complex when incubated with embryo lysate. The complex is converted to active siRISC using ATP hydrolysis. Radiolabeled siRNA was incubated with wild-type or dcr-2 mutant embryo lysates in the presence of ATP, and then the products were size-fractionated by gel filtration chromatography. siRNA was predominantly associated with a 350 kDa complex in wild-type lysate. However, most of the siRNA in the dcr-2 mutant lysate fractionated as if unbound by proteins, indicating that Dcr-2 is essential for siRNA entry into functional complexes. Thus, dcr-2 lysates should be defective for siRNA-directed mRNA cleavage. SiRNA complementary to a radiolabeled target mRNA was coincubated with embryo lysate. Incubation in wild-type lysate produced a truncated mRNA whose length was consistent with it being the 5' cleavage product. Incubation in dcr-2 lysate generated 5- to 15-fold less cleavage product. This indicates that Dcr-2 is required for mRNA target cleavage by a siRNA, and is consistent with the in vivo observations (Lee, 2004).

To examine siRISC formation in dcr-1 mutant lysate, native gel electrophoresis was used. Gel filtration chromatography requires large lysate volumes, which could not be obtained from dcr-1 embryos. Pham (2004) has developed a gel electrophoretic method to characterize siRNA complexes. The R1 gel complex corresponds to Dcr-2 and R2D2 proteins bound to labeled siRNA. R2 complex appears to be an intermediate that links R1 to a third complex, R3. The R3 complex corresponds to a siRISC that is competent to cleave cognate mRNA (Pham, 2004). To address the role of Dcr-1 in complex formation, complexes were sought in a dcr-1 mutant lysate. R1 complex was detected, but its mobility was slightly shifted and more heterogeneous. No complex with comparable mobility to R2 was detected. The data indicate that proper formation of the R2 intermediate from the R1 precursor complex is dependent upon Dcr-1 (Lee, 2004).

Bacterial RNase III is an antiparallel dimer containing a deep cleft within the catalytic domain. At each end of the cleft lies a symmetric cluster of acidic residues that are conserved among RNase III enzymes. Some of the residues at each end coordinate a divalent metal ion (Mg²⁺) that is essential for the nucleophilic attack on the RNA phosphodiester bonds at each active site. The metal ion makes a bidentate interaction (inner-sphere and outer-sphere) with an invariant glutamate residue. In one model of catalysis, the nucleophile is used twice, thereby cleaving both strands. In another model, based on a crystal structure, each active site uses two separate clusters of residues to cleave the two phosphodiesters of the RNA helix. One cluster involves the metal ion, and the other cluster acts independent of the metal ion (Lee, 2004 and references therein).

Dicers contain two catalytic domains, which based on the structure of bacterial RNase III, might fold into a pseudodimer structure or might associate within a dimeric holoenzyme. In either scenario, the catalytic repeats contain many of the invariant acidic residues implicated in RNase III catalysis. Moreover, like bacterial RNase III, Dicers cleave dsRNA to produce fragments with 3' overhangs, and with 5'-phosphate and 3'-hydroxy termini in a reaction that requires a divalent metal ion. On this basis, the chemistry of phosphodiester hydrolysis is likely to be similar. Accordingly, certain invariant residues were mutated within Dcr-2 that ware predicted to specifically disrupt phosphodiester hydrolysis. E1371 and E1617 in the first and second RNase III repeats, respectively, are homologous to the E residue that extensively interacts with Mg²⁺ in the bacterial holoenzyme. An E->K mutant in E. coli RNase III fails to cleave dsRNA, but still binds the dsRNA substrate. To elucidate the functions of the homologous residues in Dcr-2, single and double E1371K and E1617K substitution mutants of dcr-2 were generated and the mutant genes were transformed into a Drosophila strain null for dcr-2. Their ability to silence white expression in a GMR-wIR background was tested. The single mutants gave barely detectable silencing activity, while the double mutant gave no detectable silencing. Thus, E1371 and E1617 are essential for Dcr-2 activity in vivo (Lee, 2004).

Two other conserved acidic residues in bacterial RNase III form an interdomain bridge near each metal binding site, but do not coordinate the metal. Interestingly, all known Dicers have acidic residues in homologous positions of the first domain repeat, but have nonacidic residues in the homologous positions of the second domain repeat. To test the functionality of the residues in the first domain, an E1210V or E1237A substitution was introduced into the dcr-2 gene. The same substitutions at the homologous positions of E. coli RNase III abolish activity. However, both dcr-2 point mutants fully rescued the dcr-2 null phenotype, indicating that E1210 and E1237 are not critical for Dcr-2 activity. These results are consistent with the notion that the nonacidic partner residues in the second repeat normally render these clusters nonfunctional (Lee, 2004).

These genetic and biochemical analyses support the idea that siRISC activity is dependent on Dcr-1 and Dcr-2. Pham (2004) has observed both Dcr-1 and Dcr-2 proteins in siRISC that is competent for target cleavage. At least three functions that Dicer could play in siRISC can be imagined. (1) Dicer may stably associate with siRISC after having passed an siRNA molecule to other RISC factors, but has no further role in siRISC activity. This is unlikely since siRNAs can UV-crosslink to Dcr-1 and Dcr-2 in assembled siRISC. (2) Dicer may use its dsRNA binding activity to retain double-stranded siRNA or a siRNA/mRNA duplex within siRISC. (3) The RNase III domain of Dicer may be responsible for RNA cleavage by siRISC. To test this latter possibility, the E1371K and E1617K variants of Dcr-2 were examined for siRISC activity (Lee, 2004).

Lysates were prepared from mutant embryos in which dcr-2^E1371K, dcr-2^E1617K, or dcr-2^{E1371K E1617K} genes were expressed in place of the endogenous dcr-2 gene. Lysates were incubated with siRNA duplexes and a labeled mRNA substrate, and siRNA-directed cleavage of the substrate was monitored by 5' product formation. All three mutants exhibited normal mRNA cleavage activity in vitro. To demonstrate that the mutant proteins are nevertheless defective for RNase III activity, they were tested for dsRNA processing. Neither dcr-2^E1371K nor dcr-2^E1617K lysates were able to support dsRNA cleavage to form siRNAs. These data indicate that siRISC activity is unaffected when Dcr-2 RNase III activity is specifically impaired (Lee, 2004).

Role of Dicer in mRNA stability: Involvement of microRNA in AU-rich element-mediated mRNA instability

AU-rich elements (AREs) in the 3' untranslated region (UTR) of unstable mRNAs dictate their degradation. An RNAi-based screen performed in Drosophila S2 cells has revealed that Dicer1, Argonaute1 (Ago1) and Ago2, components involved in microRNA (miRNA) processing and function, are required for the rapid decay of mRNA containing AREs of tumor necrosis factor-alpha. The requirement for Dicer in the instability of ARE-containing mRNA (ARE-RNA) was confirmed in HeLa cells. miR16, a human miRNA containing an UAAAUAUU sequence that is complementary to the ARE sequence, is required for ARE-RNA turnover. The role of miR16 in ARE-RNA decay is sequence-specific and requires the ARE binding protein tristetraprolin (TTP). TTP does not directly bind to miR16 but interacts through association with Ago/eiF2C family members to complex with miR16 and assists in the targeting of ARE. miRNA targeting of ARE, therefore, appears to be an essential step in ARE-mediated mRNA degradation (Jing, 2005).

The ARE motif (AUUUA) is the most studied cis-acting element responsible for rapid turnover of unstable mRNAs in mammalian cells. In the quest for a genetic system that allows a comprehensive search for components involved in ARE-mediated decay of mRNA, Drosophila S2 cells were examined and it was found that the decay of ARE-containing RNA in S2 cells is regulated in a manner similar to that in mammalian cells. Inhibition of gene expression by RNAi is much easier and more cost effectively conducted in Drosophila S2 cells compared to mammalian cells: this allowed for an investigation of a large number of genes for their involvement in ARE-mediated RNA decay. Surprisingly, knockdown of Drosophila Dicer1 gene expression leads to stabilizing an ARE-RNA reporter. Further studies revealed that Drosophila Ago1 and Ago2 are required for ARE-mediated RNA degradation, suggesting involvement of the miRNA system. It was then confirmed that human Dicer is required in ARE-RNA degradation in HeLa cells, which implies that this underlying mechanism is conserved in the mammalian cells. Given the involvement of Dicer in HeLa cells, it was reasoned that miRNA(s) are involved in ARE-mediated RNA decay and a search was conducted for miRNAs that possess a complementary sequence to the canonical AUUUA sequence of ARE. miR16 is a potential candidate due to the presence of the sequence UAAAUAUU, and it was shown that downmodulation and overexpression of miR16 increases or decreases, respectively, the stability of a RNA reporter containing ARE of TNF or Cox2, but not uPAR. Furthermore, it was determined that the regulation of ARE^TNF-RNA decay by miR16 is sequence specific. Just as with Dicer, a function of Ago family members in ARE^TNF-RNA degradation is likely to be the processing of miR16. However, the interaction with the ARE binding protein TTP indicates that Ago/eiF2C family members also play a crucial role in the targeting of miR16 to ARE. These data demonstrate the involvement of miR16 in controlling ARE-RNA turnover and suggest that cooperation of miRNA and ARE binding proteins is essential in the recognition of ARE and in triggering mRNA degradation (Jing, 2005).

Studies have shown that the ability of miRNA to target mRNA is directed by the pairing of miRNA to mRNA. The ARE-complementary sequence in miR16 is indeed required for miR16 function in destabilizing ARE-RNA. However, pairing with no more than an eight-base ARE-sequence may not be sufficient for miR16 to target ARE-RNA. In addition, the pairing of miR16 to ARE is not in the 5′ region of miRNA, which is believed to be more critical in causing gene repression than the 3′ region. It is speculated, then, that TTP is a factor that assists miR16 targeting to ARE sequences due to its ability to interact with the ARE and RISC complex. This explains why miR16-mediated ARE-RNA instability requires TTP. In addition, the requirement of miR16 in TTP-mediated destabilization of ARE-RNA suggests that targeting of miR16 to ARE is a necessary step for RNA degradation (Jing, 2005).

ARE sequences from different mRNA can vary dramatically, with some containing multiple AU-rich elements that allow for simultaneous interaction with more than one miRNA. This could influence the ability of miRNA to promote RNA degradation because of the potential synergistic effect of miR16 to bind to multiple sites. This synergism has been demonstrated in a study that shows the addition of multiple binding sites of CXCR4 siRNA into 3′UTR of a reporter results in more translation inhibition than expected when summing up the individual effects of each binding site. The number of pairs that miR16 can form with different ARE sequences varies from five to eight, and the strength of interaction between miR16 and different AREs in a given mRNA may also vary. The number of miRNAs targeted to an mRNA and the strength of the interaction may both contribute to the quantitative control of mRNA turnover or translation. Perhaps since no more than six pairs can form between miR16 and ARE of uPAR and since uPAR has only one AUUUA motif in the 3′UTR, miR16 does not have a significant effect on the stability of mRNA containing uPAR 3′UTR (Jing, 2005).

miR16 is conserved in mammals. Although a homolog of miR16 has not been found in Drosophila, miR289 contains UAAAUAUUUA, and four other known Drosophila miRNAs contain a UAAAU sequence. Among them, at least miR277, miR289, and miR304 are expressed in S2 cells. 2′-O-methyl oligonucleotides were used to test for Drosophila miRNA that could be involved in ARE-RNA degradation in S2 cells. The anti-miR289 oligo significantly stabilizes mRNA containing TNF-α ARE, while the other four oligos have no or very modest effects on the stability of ARE^TNF-RNA. miR289 has a similar effect on the stability of ARE^IL-6-RNA and ARE^IL-8-RNA. Sequence comparisons showed that miR289 partially complements with ARE, but not the other regions of these 3′UTRs. Thus, miR289 is likely to be a miRNA that has a role in regulating ARE-RNA in S2 cells (Jing, 2005).

Though the association of miR16 with ARE-RNA in the presence of TTP and S-100 in vitro has been demonstrated, the exact mechanism of miRNA targeting of ARE and regulation of RNA degradation remains undetermined. Because of the similarity between siRNA and miRNA in regulating gene expression, miR16-mediated ARE-RNA degradation could be similar to siRNA-mediated mRNA decay. It is theoretically possible that the targeting of ARE with miRNA leads to mRNA cleavage at the targeting site since RISC has been shown to be an RNA endonuclease in vitro. However, translational suppression caused by miRNA or imperfect pairing of siRNA suggests that endonuclease activity is not always associated with RISC. Since ARE-RNA degradation is believed to be initiated by deadenylation and subsequent targeting by the exosome pathway, and since endocleaved ARE-RNA was not detected in the experimental system that was used, it is believed that the RISC involved in ARE-RNA decay is not associated with endonuclease activity. At the present, it is not clear if RISC can execute an exonuclease function, although an exonuclease, Tudor-SN, has been found in the RISC complex. TTP has been shown to bind to extended ARE sequences by virtue of its zinc finger and associates with components of exosomes; this study shows that TTP is also associated with eiF2C/Ago family members. A recent study reported that an exosome associated DexH box helicase facilitates ARE-RNA deadenylation and decay in mammalian cells. Interestingly, a C. elegens homolog of this DexH box protein has been shown to interact with a protein complex containing Dicer, RDE-1, and RDE-4. It appears that ARE binding proteins, miRNA, deadenylase, and exosomes cooperate with each other in regulating mRNA degradation. A model is favored in which TTP binds to an ARE and transiently interacts with the RISCs that scan mRNA. When a RISC containing miR16 encounters TTP, it stays with ARE and TTP due to base complementarity between miR16 and ARE. It is conceivable that RISC, in conjunction with TTP, serves to recruit proteins for deadenylation and/or exosomes for mRNA degradation (Jing, 2005).

Hundreds of miRNAs have been identified, but the targets of most miRNAs are unknown. Since perfectly or nearly perfectly paired sequences can only be found for a few miRNAs, computational as well as experimental approaches have been developed to identify potential miRNA targets that do not contain perfect complementary sequences. Although these approaches have been shown to be very useful, ARE was not identified as the target of miR16 through currently available computer programs. The current data suggest that additional factors, such as sequence-specific RNA binding proteins, needs to be considered in studying the function of miRNA. As in the case of miR16, many miRNAs may require specific proteins in binding to their mRNA targets. The role of many miRNAs may need to be studied, not only in the context of miRNA-mRNA interaction, but also the interaction of miRNA complexes with other proteins (Jing, 2005).

Stem cell division is regulated by the microRNA pathway: Effect of dicer mutation on oogenesis

One of the key characteristics of stem cells is their capacity to divide for extended periods of time in an environment where most of the cells are quiescent. Therefore, a critical question in stem cell biology is how stem cells escape cell division stop signals. This study reports the necessity of the microRNA (miRNA) pathway for proper control of germline stem cell (GSC) division in Drosophila. Analysis of GSCs mutant for dicer-1 (dcr-1), the double-stranded RNaseIII essential for miRNA biogenesis, has revealed a marked reduction in the rate of germline cyst production. These dcr-1 mutant GSCs exhibit normal identity but are defective in cell cycle control. On the basis of cell cycle markers and genetic interactions, it is concluded that dcr-1 mutant GSCs are delayed in the G1 to S transition, which is dependent on the cyclin-dependent kinase inhibitor Dacapo, suggesting that miRNAs are required for stem cells to bypass the normal G1/S checkpoint. Hence, the miRNA pathway might be part of a mechanism that makes stem cells insensitive to environmental signals which normally stop the cell cycle at the G1/S transition (Hatfield, 2005).

MicroRNAs and short interfering RNAs (siRNAs), processed by the type III double-stranded RNase Dicer, function in an RNA-based mechanism of gene silencing. Most characterized miRNAs from animals repress gene expression by blocking the translation of complementary messenger RNAs into protein; they interact with their targets by imperfect base-pairing to mRNA sequences within the 3' untranslated region (3' UTR). Experimental evidence has suggested that small RNAs regulate stem cell character in plants and animals. Moreover, some miRNAs are differentially expressed in stem cells, suggesting a specialized role in stem cell regulation. However, the molecular mechanisms underlying stem cell control by miRNAs are not understood (Hatfield, 2005).

To determine the role of miRNAs in the control of stem cell biology, processing of all miRNAs in stem cells was specifically eliminated. The Drosophila genome contains two Dicer isozymes: Dicer-1 and Dicer-2. Dicer-1 (Dcr-1) is essential for processing miRNAs, whereas Dicer-2 (Dcr-2) is required for siRNAs; loss of Dcr-1 completely disrupts the miRNA pathway and only has a weak effect on the siRNA pathway. Using Drosophila GSCs as a model system, Dcr-1 activity was impaired with two dcr-1 alleles: dcr-1 ^d102 and a null dcr-1 ^Q1147X. Drosophila oogenesis depends on the presence of self-renewing GSCs in the adult ovary, as has recently been reported in a mammalian system. The continuous division of GSCs generates an array of progressively developed egg chambers in wild-type ovarioles (Hatfield, 2005).

Analysis of dcr-1 mutant clones in the Drosophila ovary 12 days after clone induction has revealed a marked depletion of developing egg chambers. In contrast, dcr-2 null mutant GSCs produced a normal progression of egg chambers. These data suggest that Dcr-1 is required for efficient germline production. Although dcr-1 mutants show reduced numbers of gametes, most developing gametes appear morphologically normal (although they exhibit polarity defects. Therefore potential problems in GSC maintenance, identity and division were analyzed. Clonal experiments revealed that the percentage of germaria with clonal stem cells at different time points after clone induction was similar in the dcr-1 ^Q1147X mutant and the wild-type control, suggesting that the loss of cysts in dcr-1 mutants is not primarily due to problems in the maintenance of GSCs (Hatfield, 2005).

To determine whether reduced cyst production in dcr-1 germaria is due to altered GSC fate, the identity of the dcr-1 mutant GSCs were analyzed. Female GSCs are identified by their location and the expression patterns of three markers: the presence of Adducin, a protein present in the spectrosome; the presence of phosphorylated Mad protein (P-Mad), indicating TGF-beta, and the absence of Bam, repressed by the TGF-beta. Thedcr-1 ^Q1147X GSCs showed normal spectrosome morphology and position, and normal TGF-beta pathway activity. Furthermore, as with wild-type GSCs, dcr-1 ^Q1147X GSCs did not stain positively for the Bam protein. From these analyses, it is concluded that decreased cyst production from dcr-1 ^Q1147X GSCs does not result from either a loss of GSCs or a change in their identity (Hatfield, 2005).

The frequency of cell division in dcr-1 ^Q1147X GSCs was impaired. Examination of individual germaria containing a single heterozygous GSC and a single dcr-1 ^Q1147X mutant GSC revealed that GSCs lacking Dcr-1 activity produced cysts at a frequency that was reduced to 18% of normal levels (41% for dcr-1 ^d102). In contrast, the frequency of division was not significantly reduced for GSCs that were homozygous for the dcr-2 mutation or for the isogenized parental chromosome from which the dcr-1 mutant alleles were generated. Thus, Dcr-1 is required cell autonomously in GSCs for the cell divisions that produce developing cystoblasts (no obvious defect in cyst division was observed (Hatfield, 2005).

To determine whether the reduced cyst formation reflected a block in the normal cell cycle programme, the distribution of cell cycle stages was analyzed in mutant dcr-1 ^Q1147X GSCs by staining mosaic germaria with antibodies against different cell cycle markers. An increase in the number of dcr-1 mutant GSCs staining positive for Cyclin E (CycE) was observed using two independent dcr-1 alleles. In contrast, GSCs that are homozygous for dcr-2 or the parental chromosome express CycE with frequencies similar to those of wild-type GSCs. Furthermore, pulse labelling of ovaries with the nucleotide analogue 5-bromodeoxyuridine (BrdU) revealed that the number of dcr-1 ^Q1147X mutant GSCs in S phase was reduced. Similarly, the number of dcr-1 ^Q1147X mutant GSCs staining positive for Cyclin A (CycA), Cyclin B (CycB) and the mitotic marker Phosphohistone-3 (PH3) was reduced. These data indicate that perturbation of the miRNA pathway by mutant dcr-1 in GSCs delays the cell cycle at the G1/S transition (Hatfield, 2005).

Whether loss of Dcr-1 function has similar consequences on the cell cycle in the GSCs of male flies was tested. Each male testis contains approximately ten GSCs surrounding a somatic structure called the hub. Similar to female GSCs, the number of male GSCs staining positive for CycE was increased in dcr-1 mutants. These data show that Dcr-1 also functions in the male GSC niche, and suggest that Dcr-1 has a conserved role in GSC division (Hatfield, 2005).

To test the possibility that the miRNA pathway might be a general cell cycle regulator, other cell types were tested to determine whether the G1/S delay and reduced cell division frequency would also be observed in other mitotically dividing dcr-1 mutant cells. dcr-1 ^Q1147X clones in imaginal discs revealed that the number of CycE-positive cells was not increased in mutant cells. The number of dcr-1 ^Q1147X mutant cells in imaginal discs was approximately equal to the number of marked wild-type cells that descended from a common parent cell, indicating that the frequency of cell division in imaginal disc cells is not reduced in a dcr-1 mutant. dcr-1 ^Q1147X dividing germline cysts express CycE at a frequency comparable to that of wild-type dividing cysts, suggesting that the mitotic cystoblast cell divisions are not affected in dcr-1 mutants. Therefore, the reduction in cell division frequency observed in the dcr-1 mutant germ line is specific to the GSC division. Together, these data suggest that the miRNA pathway has a specific role in regulating stem cell division (Hatfield, 2005).

The potential cause for the G1/S arrest was explored by examining the expression of Dacapo (Dap; a homologue of the p21/p27 family of cyclin-dependent kinase (CDK) inhibitors) in dcr-1 ^Q1147X mutant GSCs. The transition between the G1 and S phases of the cell cycle is negatively regulated by Dap. Dap protein traps the CycE/CDK2 complex in a stable but inactive form, and elevated levels of Dap lead to cell cycle arrest at the G1/S phase transition with prolonged expression of CycE protein. Notably, the number of Dap-positive GSCs increased in the dcr-1 mutant GSC population (Hatfield, 2005).

To determine whether Dap mediates the effect of dcr-1 on the GSC cell cycle, the level of Dap was reduced by 50% in dcr-1 ^Q1147X mutant GSCs and a partial rescue in cyst production was observed. Furthermore, the number of GSCs staining positive for CycE was reduced to normal levels, demonstrating that the CycE defect observed in dcr-1 mutant GSCs is dependent on Dap. Consistent with this, overexpression of a Dap transgene results in some germaria resembling dcr-1 germline mutants: the germaria are small, containing a few cysts, and had a high number of CycE-positive GSCs. The fact that reduction of Dap levels led to a normal GSC CycE profile, but partial rescue of cyst generation, suggests that Dcr-1 might also regulate later cyst development (Hatfield, 2005).

These data suggest that miRNAs act on stem cell division by reducing the levels of Dap. How is this regulation achieved? It was found that expression of a Dap transgene containing the Dap promoter and essentially all of the endogenous gene except some of the 3' UTR is similar in dcr-1 mutant and wild-type GSCs. These data suggest that the effect of Dcr-1 on Dap regulation in GSCs is at a post-transcriptional level and might involve the 3' UTR region that is missing in the dap-5gm transgene (Hatfield, 2005).

It is proposed that miRNAs are required for GSCs to transit the G1/S checkpoint by repressing directly or indirectly the G1/S inhibitor Dap. Because Dap is a key component of the G1/S transition, it is a plausible target for machinery that assures continuous cell division in a microenvironment in which most of the cells are quiescent. It is proposed that while the TGF-beta pathway -- which can upregulate p21/p27 -- is active in GSCs, miRNAs downregulate Dap to assure the continuous cell division essential for stem cells. This downregulation might be direct, because the Dap 3' UTR contains several predicted miRNA-binding sites. A Dap transgene lacking these sites shows no response to Dcr-1 levels, suggesting that the potential binding sites are responsive to Dicer-1. However, it is also possible that the Dap misregulation in dcr-1 mutant GSCs might be due to a secondary effect of Dcr-1 loss. The finding that miRNAs are required for stem cell division suggests that miRNAs might be part of a mechanism that makes stem cells insensitive to environmental signals that normally stop the cell cycle. Because miRNAs are a novel class of genes involved in human tumorigenesis, it is tempting to speculate that miRNAs could have a similar role in cancer cells (Hatfield, 2005).

Targets of microRNA regulation in the Drosophila oocyte proteome

MicroRNAs (miRNAs) are a class of small RNAs that silence gene expression. In animal cells, miRNAs bind to the 3' untranslated regions of specific mRNAs and inhibit their translation. Although some targets of a handful of miRNAs are known, the number and identities of mRNA targets in the genome are uncertain, as are the developmental functions of miRNA regulation. To identify the global range of miRNA-regulated genes during oocyte maturation of Drosophila, the proteome from wild-type oocytes was compared with the proteome from oocytes lacking the dicer-1 gene, which is essential for biogenesis of miRNAs. Most identified proteins appeared to be subject to translation inhibition. Their transcripts contained putative binding sites in the 3' untranslated region for a subset of miRNAs, based on computer modeling. The fraction of genes subject to direct and indirect repression by miRNAs during oocyte maturation appears to be small (4%), and the genes tend to share a common functional relationship in protein biogenesis and turnover. The preponderance of genes that control global protein abundance suggests this process is under tight control by miRNAs at the onset of fertilization (Nakahara, 2005).

At the completion of Drosophila oocyte maturation, the number of genes negatively regulated by miRNAs appears to be limited. This study detected 41 of 1,003 proteins that are down-regulated by miRNA production, representing a maximum 4% of genes that might be directly inhibited. Although this value is close to the genome-wide 7% level predicted by sequence comparisons, it is not certain that 4% represents a typical fraction of genes regulated by miRNAs in a cell. First, this estimate reflects only the 1,000 most-abundant proteins at this stage of development. Many less-abundant proteins are not detected by Difference Gel Electrophoresis, and so the fraction of those that are regulated is not known. For example, dcr-1 mutant oocytes exhibit defective translation of Oskar and Gurken proteins, leading to a mild ventralization phenotype. However, these two proteins are too rare to have been detected by the proteomic analysis. Second, it is not known whether the relative fraction of target genes varies during development. Possibly, fewer genes are regulated by miRNAs during oogenesis. It is known that RNAi is activated during oocyte maturation, and that the short interfering RNA pathway depends upon translation of target mRNAs. It is possible that there are fewer miRNA targets in mature oocytes due to dependence on different translation control mechanisms (Nakahara, 2005).

Why are these particular gene products repressed by miRNAs during oocyte maturation? In most animal species, translation serves as the main mechanism to regulate gene expression during oocyte maturation and early embryogenesis. Indeed, oocyte maturation and early embryogenesis proceed without transcription of nuclear RNA, including rRNA. In parallel, no ribosomal proteins are synthesized de novo, and consequently no new ribosomes are produced. In Drosophila, ribosomal protein-mRNA levels are constant throughout oogenesis and embryogenesis, but their translation drops as oogenesis ends and embryogenesis begins. Translation of ribosomal protein-mRNAs then rises in conjunction with the onset of rRNA transcription in the embryo. It was found that the accumulation of ribosomal protein-S2 is inhibited by miRNAs during oocyte maturation, suggesting that the translation block exerted on Drosophila ribosomal proteins is partially mediated by the miRNA pathway. The status of other ribosomal-proteins is not known, because most are basic in charge and would not be detected in the 2D gels used in the analysis (Nakahara, 2005).

A lack of new ribosome production during oocyte maturation and early embryogenesis would also eliminate the need to synthesize other factors involved in protein biogenesis, reflecting a coordinated effort to balance various steps along the biogenesis pathway. This study indicates that the synthesis of several chaperones and other biogenic factors is specifically inhibited during oocyte maturation, possibly for those reasons. Another cellular process that appears to be attenuated by miRNAs at this developmental stage is protein turnover. Why is proteolysis dampened? Possibly, reduced proteolysis allows preexisting ribosomes to remain abundant during the period when they cannot be replenished. Another possibility is that reduced proteolysis links the rate of protein biogenesis to the rate of protein turnover, thereby maintaining steady-state protein levels. Finally, lowered protein turnover during this highly dynamic stage of development would allow for rapid and global accumulation of new proteins necessary for early embryogenesis (Nakahara, 2005).

The role of PIWI and the miRNA machinery in Drosophila germline determination

The germ plasm has long been demonstrated to be necessary and sufficient for germline determination, with translational regulation playing a key role in the process. Beyond this, little is known about molecular activities underlying germline determination. This study reports the function of Drosophila Piwi, Dicer-1, and dFMRP (Fragile X Mental Retardation Protein) in germline determination. Piwi is a maternal component of the polar granule, a germ-plasm-specific organelle essential for germline specification. Depleting maternal PIWI does not affect Osk or Vasa expression or abdominal patterning but leads to failure in pole-plasm maintenance and primordial-germ-cell (PGC) formation, whereas doubling and tripling the maternal piwi dose increases Osk and Vasa levels correspondingly and doubles and triples the number of PGCs, respectively. Moreover, Piwi forms a complex with dFMRP and Dicer-1, but not with Dicer-2, in polar-granule-enriched fractions. Depleting Dicer-1, but not Dicer-2, also leads to a severe pole-plasm defect and a reduced PGC number. These effects are also seen, albeit to a lesser extent, for dFMRP, another component of the miRISC complex. Because Dicer-1 is required for the miRNA pathway and Dicer-2 is required for the siRNA pathway yet neither is required for the rasiRNA pathway, the data implicate a crucial role of the Piwi-mediated miRNA pathway in regulating the levels of Osk, Vasa, and possibly other genes involved in germline determination in Drosophila (Megosh, 2006).

It has been nearly a century since the discovery of germ plasm and its function in germline fate determination in diverse organisms. In recent decades, the components and assembly of the polar granule in Drosophila and its equivalent in C. elegans have been effectively explored. Translational regulation has also been implicated in pole plasm for abdominal patterning and germline determination. In addition, germ cell-less (gcl) and mitochondrial large-subunit ribosomal RNAs (mtlr RNAs) have been shown to be required for germline determination. However, the biochemical activities of these molecules remain largely unknown. This study identified Piwi and likely the miRNA machinery as a germ-plasm regulatory activity that is involved in germline fate determination (Megosh, 2006).

Germ-plasm assembly occurs in a stepwise fashion. Step 1 involves the transport of polar granule materials to the posterior end of the oocyte during oogenesis, a process that involves a microtubule-based transport system as well as genes such as cappuccino and staufen. Step 2 is the assembly of polar-granule components at the posterior end, a process that is almost concurrent with the transport and that is completed by stage 12 of oogenesis. A critical component for the assembly is Osk, which determines the pole-cell number in a dose-dependent manner and has the ability to recruit Vasa and Tud as well as to induce pole-cell formation at ectopic sites within the embryo. Three lines of data suggest that Piwi is downstream of Osk, Tud, and Vasa in the assembly process: (1) Osk, Tud, and Vasa appear to assemble normally into the pole plasm in Piwi-depleted developing oocytes; (2) Piwi cannot recruit Osk or Vasa ectopically to the anterior pole, yet Osk can recruit Piwi to the anterior pole; (3) Osk, Tud, and Vasa all have both germline determination and posterior-patterning functions, but Piwi does not appear to have a detectable function in patterning (Megosh, 2006).

Although the assembly of polar-granule components occurs in a hierarchical fashion, there is growing evidence for interactions between polar-granule components beyond what is required for assembly. For example, a regulatory relationship between nanos and tudor has been reported. In nanos mutant embryos, both Tudor levels and the number of pole cells increase. Other experiments suggest that the presence of mtlrRNA in the polar granules is required for stabilization of the polar-granule components Vasa, Gcl, nos mRNA, and pgc mRNA. The regulatory function reported in this study for Piwi toward Osk, Vasa, and Nos further supports the interplay and interdependency among pole-plasm components. A previous study implicates osk as a rate-limiting factor for all aspects of pole-plasm function. The results suggest that Piwi, likely working through the miRNA pathway, is also a limiting factor for germ-cell formation. This function of Piwi is likely achieved via regulation of the levels of Osk, Tud, and Vasa, and possibly that of other polar-granule components, in a dose-dependent fashion (Megosh, 2006).

The regulation of Piwi toward the expression of Osk, Tud, Vasa, and Nos appears to be dispensable; Piwi-deficient oocytes and early embryos do not display detectable defects in their expression of Osk, Tud, Vasa, and Nos. This redundancy is likely due to an overlapping function of Piwi with other proteins involved in the RNAi pathway and/or colocalized in nuage during oogenesis; such proteins might include Maelstrom, Armitage, and Aubergine. Among these proteins, Aubergine, a close homolog of Piwi, is a known polar-granule component in early embryos. It regulates the translation of Osk during oogenesis and is required for both pole-cell formation and posterior patterning during embryogenesis (Megosh, 2006).

It is intriguing that Piwi regulates Osk and Vasa expression yet does not display a posterior-patterning phenotype. This function is different from that of Aubergine, so it is possible that Piwi and Aubergine each have their own regulatory targets in addition to Osk and Vasa. The Piwi targets may be specifically involved in maintaining polar-granule localization and may not be subject to Aubergine regulation, whereas Aubergine targets might be involved in both germline determination and posterior patterning. In support of this possibility, it has recently been shown that the generation of certain rasiRNAs shows varying dependencies on Piwi and Aubergine. The regulation of Piwi toward its specific target genes may be activated during oocyte maturation, similar to the oocyte maturation-dependent activation of RNAi as observed for aubergine and spindle-E. Thus, Piwi is not required for Osk and Vasa localization during oogenesis but is required for maintaining their localization during embryogenesis. An alternative hypothesis is that Piwi, like Aubergine, also regulates patterning genes but that this function is redundant. This hypothesis, however, does not explain the fact that neither ectopic expression nor overexpression of Piwi causes a detectable defect in posterior patterning (Megosh, 2006).

Given the association of Piwi with Dcr-1 and dFMRP, the Piwi-mediated regulation is likely via the miRNA but not the siRNA mechanism, which is Dcr-2-dependent, or the rasiRNA mechanism, which does not depend on either Dcr-1 or Dcr-2. This hypothesis is further supported by the similar phenotypes observed in embryos depleted of Piwi, Dcr-1, and dFMRP but not Dcr-2. It is possible that Piwi might bind to novel small RNAs to achieve this function, given recent findings that mammalian Piwi subfamily proteins bind to Piwi-interacting RNAs (piRNAs). If so, these novel RNAs must function in a Dcr-1-dependent pathway in the cytoplasm given Piwi's localization to the cytoplasm in early pole cells. The function of the Piwi/DCR-1-mediated miRNA or novel small-RNA pathway in germline specification is very similar to that of other germ-cell regulators, such as gcl and mtlr RNAs, in that these genes are required for pole-cell formation but not for abdominal segmentation. However, unlike embryos from the gcl-bcd females, embryos from the piwi-bcd females exhibit no cell-cycle delays in the anterior nuclei and no significant changes in the morphology of anterior nuclei. Furthermore, GCL mediates a transcriptional repression mechanism [72]. Thus, the effect of the Piwi-miRNA mechanism on pole-cell formation may be distinct from the gcl-mediated mechanism (Megosh, 2006).

It is important to note that the Piwi-mediated miRNA pathway positively regulates the expression of Osk and Vasa, in contrast to the known translational repression role of the miRNA pathway. In support of this observation, the Piwi ortholog in the mouse, MIWI, also appears to positively regulate gene expression, likely by enhancing mRNA stability and translation. Alternatively, it is possible that Piwi regulates an unidentified intermediate protein whose function is to repress the expression of Osk and Vasa (Megosh, 2006).

piwi is essential for the self-renewal of adult germline stem cells in Drosophila. Recent studies have demonstrated that the miRNA pathway is involved in division and self-renewal of adult germline stem cells in the Drosophila ovary. This study further connects Piwi and the miRNA pathway and reveals their crucial role in germline fate determination during embryogenesis. These observations suggest that the germline and stem cells may share a common miRNA-mediated mechanism in defining their fates. Given the high degree of conservation of the miRNA machinery during evolution, this pathway may function in diverse organisms in determining the germline and stem cell fates (Megosh, 2006).

I>Dcr-1 maintains Drosophila ovarian stem cells

MicroRNAs (miRNAs) regulate gene expression by controlling the turnover, translation, or both of specific mRNAs. In Drosophila, Dicer-1 (Dcr-1) is essential for generating mature miRNAs from their corresponding precursors. Because miRNAs are known to modulate developmental events, such as cell fate determination and maintenance in many species, whether a lack of Dcr-1 would affect the maintenance of stem cells (germline stem cells, GSCs; somatic stem cells, SSCs) in the Drosophila ovary was investigated by specifically removing its function from the stem cells. The results show that dcr-1 mutant GSCs cannot be maintained and are lost rapidly from the niche without discernable features of cell death, indicating that Dcr-1 controls GSC self-renewal but not survival. bag of marbles (bam), the gene that encodes an important differentiating factor in the Drosophila germline, however, is not upregulated in dcr-1 mutant GSCs, and its removal does not slow down dcr-1 mutant GSC loss, suggesting that Dcr-1 controls GSC self-renewal by repressing a Bam-independent differentiation pathway. Furthermore, Dcr-1 is also essential for the maintenance of SSCs in the Drosophila ovary. These data suggest that miRNAs produced by Dcr-1 are required for maintaining two types of stem cells in the Drosophila ovary (Zin, 2007).

This study has demonstrated that Dcr-1 is required for the maintenance of GSCs and SSCs in the Drosophila ovary. Because Dcr-1 is an essential component of the miRNA pathway in Drosophila, it is further proposed that miRNAs processed by Dcr-1 are essential for controlling self-renewal of GSCs and SSCs. Consistent with this idea, the Dcr-1 partner, Loqs, has also shown to be required for GSC maintenance. Because Dcr-1 is intrinsically required for controlling GSC self-renewal, Loqs functions intrinsically in GSCs for controlling GSC self-renewal. Without one or more miRNAs generated by Dcr-1, ovarian GSCs and SSCs undergo premature differentiation that leads to the depletion of these stem cells in their corresponding niches. This study has also provided insight into how Dcr-1 controls GSC self-renewal. The miRNA pathway controls GSC self-renewal by repressing a bam-independent differentiation pathway. Furthermore, the miRNA pathway is required for the development and growth of later-differentiated germ cells, although it is dispensable for cyst division and oocyte specification. Similarly, the miRNA pathway is also required for follicle-cell proliferation and growth. To further understand how the miRNA pathway controls GSC and SSC self-renewal, it is essential to identify miRNAs and study their functions in GSC and SSCs. Because Dcr-1 contributes to the maintenance of Drosophila ovarian GSCs and SSCs tested so far, it is tempting to speculate that miRNAs may have a general role in maintaining different types of stem cells. It will be of great interest to test this hypothesis and to elucidate the underlying mechanisms as to how miRNAs contribute to the stem cell self-renewal and proliferation (Zin, 2007).

MicroRNA processing pathway regulates olfactory neuron morphogenesis

The microRNA (miRNA) processing pathway produces miRNAs as posttranscriptional regulators of gene expression. The nuclear RNase III Drosha catalyzes the first processing step together with the dsRNA binding protein DGCR8/Pasha generating pre-miRNAs. The next cleavage employs the cytoplasmic RNase III Dicer producing miRNA duplexes. Finally, Argonautes are recruited with miRNAs into an RNA-induced silencing complex for mRNA recognition. This study identified two members of the miRNA pathway, Pasha and Dicer-1, in a forward genetic screen for mutations that disrupt wiring specificity of Drosophila olfactory projection neurons (PNs). The olfactory system is built as discrete map of highly stereotyped neuronal connections. Each PN targets dendrites to a specific glomerulus in the antennal lobe and projects axons stereotypically into higher brain centers. In selected PN classes, pasha and Dicer-1 mutants cause specific PN dendritic mistargeting in the antennal lobe and altered axonal terminations in higher brain centers. Furthermore, Pasha and Dicer-1 act cell autonomously in postmitotic neurons to regulate dendrite and axon targeting during development. However, Argonaute-1 and Argonaute-2 are dispensable for PN morphogenesis. These findings suggest a role for the miRNA processing pathway in establishing wiring specificity in the nervous system (Berdnik, 2008).

To identify genes that are essential for dendrite targeting in Drosophila olfactory projection neurons (PNs), a MARCM-based mosaic forward genetic screen was performed by using novel piggyBac transposon insertions. The insertions LL03660 and LL06357, integrated in pasha and Dicer-1, respectively, were uncovered. Both alleles are homozygous lethal, likely to be null, and referred to as pasha^−/− and Dicer-1^−/− mutants throughout this study. The pasha^−/− allele is an insertion in the 5'UTR, resulting in undetectable Pasha protein in homozygous mutant neurons. The Dicer-1^−/− allele is an insertion in the coding region resulting in a truncated 740 amino acid protein lacking the RNase III, PAZ, and dsRNA binding domains (Berdnik, 2008).

The MARCM technique allows visualization and manipulation of PNs in neuroblast and single-cell clones in an otherwise heterozygous animal. Gal4-GH146 was used to label PNs from three neuroblast lineages, anterodorsal (ad), lateral (l), and ventral (v) PNs. Wild-type (WT) adPNs, lPNs, and vPNs target stereotyped sets of glomeruli in neuroblast clones. pasha^−/− PNs show two dendrite morphogenesis defects for all neuroblast clones. First, the dendritic density in most glomeruli is drastically reduced. Second, dendritic branches spill into incorrect glomerular classes. Very similar PN dendritic defects were observed in Dicer-1^−/− MARCM clones (Berdnik, 2008).

It was confirmed that the transposon insertions in pasha and Dicer-1 are the cause for the mutant phenotype with two further experiments. First, precise excision of both transposons fully revert PN morphogenesis defects. Second, expression of UAS-pasha-HA or UAS-Dicer-1 transgenes fully rescued pasha or Dicer-1 mutant PN phenotypes, respectively, in MARCM experiments. Because Gal4-GH146 is expressed only in postmitotic neurons, these experiments also demonstrate that Pasha and Dicer-1 act in postmitotic neurons to regulate dendrite morphogenesis (Berdnik, 2008).

As expected, in all rescue experiments, Pasha-HA localizes to the nucleus and Dicer-1 is enriched in the cytoplasm of PNs. Endogenous Pasha protein is found ubiquitously in all cell nuclei in the brain center at 18 hr after puparium formation (APF), when PN dendrites organize the proto-antennal lobe prior to olfactory receptor neuron (ORN) axon entry. Moreover, Pasha is undetectable in pasha^−/− adPNs and DL1 single neurons (Berdnik, 2008).

To study dendrite targeting with a better resolution, single-cell MARCM clones were examined. WT DL1 single-cell clones (hereafter referred to as DL1 single neurons) always target a posterior, dorsolateral glomerulus and fill the glomerulus with dendritic branches. In pasha^−/− PNs, 17/25 DL1 single neurons show stereotyped mistargeting defects: dendrites innervate DL1 more sparsely and also mistarget to several additional glomeruli (VA7m, VC2, VA6, DL2d, and DL5), all of which are partially innervated. 8/25 DL1 single neurons spill their dendrites medially to adjacent glomeruli, mostly D and DL5. Again, Dicer-1 single mutant neurons exhibit similar PN dendrite mistargeting although to a lower frequency. Similar stereotyped mistargeting pattern as in pasha mutants occur in 19/35 DL1 single neurons mutant for Dicer-1, 7/35 single neurons show medially spilled dendrites and 9/35 target normally. The variation of DL1 phenotypes could be caused by perdurance of WT protein in single-cell mutant clones, which might affect Dicer-1 more than Pasha. The stereotyped DL1 targeting defect was not found in more than 1400 other piggyBac insertions screened, supporting the specificity of the mutant phenotype for the miRNA processing pathway (Berdnik, 2008).

MARCM expression of UAS-Pasha-HA in pasha^−/− or UAS-Dicer-1 in Dicer-1^−/− DL1 single neurons fully rescued dendrite targeting, as is the case of neuroblast clones. These experiments demonstrate that Pasha and Dicer-1 act cell autonomously in postmitotic neurons to regulate DL1 dendrite targeting (Berdnik, 2008).

To expand the studies of dendrite targeting to other specific PN classes, Gal4-Mz19 was used to label fewer neurons in neuroblast clones. This Gal4 line labels ~6 adPNs that innervate VA1d and DC3 (posterior to VA1d) in WT. In 21/21 pasha^−/− adPNs, VA1d/DC3 is sparsely innervated and dendrites are incorrectly targeted to variable glomeruli such as DA1, VA2, and VM7. 23/25 Dicer-1^−/− PNs show similar medial mistargeting phenotypes albeit to a milder extent, innervating less distant glomeruli. Similarly, the dendritic density is reduced and incorrect glomeruli are innervated, as in GH146 MARCM experiments. Gal4-Mz19 is also expressed in ~7 lPNs innervating the dorsolateral DA1 glomuerlus in WT. DA1 PN targeting is much less affected in pasha and Dicer-1 mutants. 4/5 pasha mutant and 7/9 Dicer-1 mutant lPNs target normally to DA1 with WT dendrite densities, whereas 1/5 and 2/9 lPNs exhibit additional partial innervation of the adjacent DL3 glomerulus, respectively. Thus, Pasha and Dicer-1 are not required equally in all PN classes, suggesting that potential miRNAs might selectively regulate the targeting of specific classes of PNs (Berdnik, 2008).

In addition to dendrite mistargeting, axon defects were also observed in pasha and Dicer-1 mutants. WT DL1 axons project into the lateral horn (LH) via the mushroom body calyx (MBC) where they form several collateral branches. After entering the LH, DL1 axons always form one characteristic dorsal branch whereas the main branch terminates at the lateral edge of the LH. In pasha and Dicer-1 mutant DL1 single neurons, axons extend along the normal pathway, form collaterals in the MBC, and always reach the LH. However, more than half of the mutant DL1 axons do not reach the lateral edge but stop within the LH. The dorsal branch in the LH is either absent or reduced in length. Adding one copy of a UAS-pasha-HA transgene in pasha or UAS-Dicer-1 in Dicer-1 mutant DL1 single neurons rescued all axon phenotypes: the main branch fully extends to the lateral edge of the LH and the dorsal branch is indistinguishable from WT. Thus, Pasha and Dicer-1 cell autonomously regulate PN axon-terminal elaboration (Berdnik, 2008).

To determine whether the PN dendrite targeting errors are a result of initial mistargeting or failure to maintain stable synaptic connections later, developmental studies were performed. At 18 hr APF, when ORN axons have not yet entered the proto-antennal lobe, WT adPN, lPN, and vPN dendrites have already occupied a large area of the proto-antennal lobe . DL1 single neurons already target their dendrites in the area of the future DL1 glomerulus. In pasha^−/− PNs, dendritic elaboration within the proto-antennal lobe is extremely reduced in all neuroblast or DL1 single-cell clones at 18 hr APF. At 50 hr APF, glomeruli become first visible. In WT adPNs, lPNs, and DL1 single neurons, the same stereotyped innervation patterns as in adults are already evident even though the antennal lobe is smaller in its overall size. Dendrites of pasha^−/− PNs are reduced in density and spill into lineage-inappropriate glomeruli. Moreover, stereotyped mistargeting of DL1 single neurons is already evident in 4/4 pasha^−/− PNs at 50 hr APF (Berdnik, 2008).

These data, in combination with the observation that pasha mutant PN dendrite phenotypes do not vary in brains of 3- and 10-day-old adults indicate that Pasha regulates dendrite elaboration and correct targeting early during development (Berdnik, 2008).

Dicer functions in small RNA maturation across species. Dicer mutants are defective for both transcript destruction and translational repression, suggesting that Dicer is required for the siRNA (small interfering RNA) and miRNA maturation pathway. However, the Drosophila genome contains two Dicer genes, Dicer-1 and Dicer-2, that share similar protein domains but are different in their functions. Dicer-1 and Dicer-2 are both required for siRNA-dependent mRNA cleavage, with Dicer-2 acting in siRNA processing and Dicer-1 acting downstream of siRNA production. However, Dicer-1, but not Dicer-2, is essential for miRNA-induced silencing during translational repression (Berdnik, 2008).

To test whether the siRNA processing pathway is required for PN targeting, use was made of Dicer-2^L811fsX mutants that lack the two RNase III domains essential for dsRNA processing. It was found that Dicer-2^L811fsX mutant PNs exhibit normal dendrite and axon targeting, suggesting that Dicer-2 is dispensable and the siRNA pathway is not required for PN targeting (Berdnik, 2008).

Next it was asked whether Dicer-2 could compensate for Dicer-1's function in PN targeting because their protein domain organization is highly similar. UAS-Dicer-2 was expressed in Dicer-1^−/− PNs to test whether PN mistargeting phenotypes could be rescued as is the case for UAS-Dicer-1 expression. No alteration was seen in the Dicer1^−/− dendrite mistargeting phenotypes in DL1 PNs, adPNs, or lPNs. This observation suggests that Dicer-2 cannot replace Dicer-1's function during PN targeting. It is proposed that Dicer-1-dependent PN targeting defects are caused by the absence of one or several miRNA(s), because Dicer-1, but not Dicer-2, is essential for miRNA-directed translation repression and mRNA turnover (Berdnik, 2008).

Many distinct mechanisms have been described for miRNA-mediated gene silencing. However, for all these, the RNA-induced silencing complex (RISC) containing the Argonaute (AGO) proteins as core components is required. AGO members can be divided into two groups, the ubiquitously expressed AGO and the reproductive cell-specific Piwi subfamily. The AGO subclass containing AGO1 and AGO2 in Drosophila is involved in small RNA loading into the RISC. Both miRNAs and siRNAs act as components of RISCs but use different silencing mechanisms. miRNAs typically contain several mismatches when paired with target mRNAs, causing mostly translational repression, whereas siRNAs are perfectly paired with target mRNAs leading to their degradation. AGO2 is described as a multiple-turnover RNA-directed RNA endonuclease acting in mRNA cleavage, whereas AGO1 functions in translational repression but also plays a role in efficient mRNA degradation. However, mRNAs targeted by almost perfectly paired miRNAs can also be degraded via AGO2. Thus, AGO1 is typically necessary for stable miRNA maturation and is essential for viability, whereas AGO2 is an essential component of the siRNA-directed RNA interference response (Berdnik, 2008).

To determine which AGO member is involved in PN targeting, MARCM clones of the strong loss-of-function allele AGO1^k08121 and the AGO2⁴¹⁴ null allele were examined. Surprisingly, normal PN dendrite and axon targeting were observed in AGO1^k08121 and AGO2⁴¹⁴ adPNs, and DL1 single neurons as dendrites elaborate in the single dorsolateral DL1 glomerulus like in WT. To test whether AGO1 and AGO2 could act in a redundant manner, PN clones were generated homozygous mutant for AGO1 in an AGO2 homozygous mutant background. 7/7 adPNs and 9/9 DL1 PNs exhibit normal targeting. In addition, axon-terminal arborization is normal in AGO1/AGO2 mutant DL1 cells (Berdnik, 2008).

There are several explanations for this surprising result. First, the AGO1^k08121 allele may not be null. Second, perdurance of AGO1 protein from parental cells is capable of compensating for the loss of the AGO1 gene in homozygous mutant clones. AGO1^k08121 mutants have drastically reduced mRNA levels, AGO1 is absent in homozygous AGO1^k08121 embryo lysates, and AGO1^k08121 has been shown to disrupt stable miRNA maturation. AGO1^k08121 mutant wing disc clones miRNA function is disrupted as in pasha^−/− and Dicer-1^−/− clones as shown by using a bantam sensor transgene. Because of these facts and given that WT AGO1 mRNA or protein would be heavily diluted at least in neuroblast clones, the above two explanations imply that a very small amount of AGO1 would be sufficient for PN dendrite targeting. Third, perhaps one or more members of the Piwi subfamily thought to be expressed and function predominantly in the germline could compensate for the loss of AGO1/AGO2 in PNs. However, normal PN morphogenesis was observed in mutants for piwi¹ and aubergine^LL06590, and both are Piwi subfamily members. Lastly, PN dendrite targeting may utilize a novel miRNA-processing mechanism that is Dicer-1 dependent but AGO independent (Berdnik, 2008).

MicroRNA-mediated posttranslational regulation of gene expression has been documented in an increasing number of biological processes. Many miRNAs are developmentally regulated and show tissue-specific expression. In the nervous system, miRNAs have been shown to play roles during neurogenesis, specification of neuronal fate, neuronal morphogenesis, synaptogenesis, and neurodegeneration. This study has demonstrated a new function of the miRNA-processing pathway in regulating wiring specificity of the olfactory circuit (Berdnik, 2008).

The results support the model that one or more miRNA(s) are essential for regulating expression of genes that in turn regulate PN dendrite targeting and axon-terminal elaboration in identified neurons during development. Candidate target genes could be transcription factors that regulate wiring specificity in postmitotic neurons, cell-surface receptors for dendrite targeting, or their regulators. Expression or protein levels of such genes are essential for PN dendrite targeting. However, each miRNA is predicted to target hundreds of mRNAs and several miRNAs can regulate one mRNA, adding much more complexity to their regulatory function. Indeed, 7 miRNAs with available null mutants (out of 152 miRNAs predicted in the Drosophila genome were tested; none of them exhibit PN targeting defects. In flies, techniques that would allow the injection of individual or pools of mature miRNAs to rescue the neural phenotypes in pasha or Dicer-1 mutants, or mimic these phenotypes by injecting 'target protectors' that interfere with miRNA-mRNA interactions as in zebrafish, are currently not available. Therefore, it remains to be a future challenge to identify the miRNA(s), and ultimately their targets, for PN target selection. Looking for mutants with similar phenotypes as pasha and Dicer-1 in forward genetic screens or candidate gene approaches may help to identify specific miRNA and their targets (Berdnik, 2008).

Control of antagonistic components of the Hedgehog signaling pathway by microRNAs in Drosophila

Hedgehog (Hh) signaling is critical for many developmental processes and for the genesis of diverse cancers. Hh signaling comprises a series of negative regulatory steps, from Hh reception to gene transcription output. Stability of antagonistic regulatory proteins, including the coreceptor Smoothened (Smo), the kinesin-like Costal-2 (Cos2), and the kinase Fused (Fu), is affected by Hh signaling activation. This study shows that the level of these three proteins is also regulated by a microRNA cluster. Indeed, the overexpression of this cluster and resulting microRNA regulation of the 3'-UTRs of smo, cos2, and fu mRNA decreases the levels of the three proteins and activates the pathway. Further, the loss of the microRNA cluster or of Dicer function modifies the 3'-UTR regulation of smo and cos2 mRNA, confirming that the mRNAs encoding the different Hh components are physiological targets of microRNAs. Nevertheless, an absence of neither the microRNA cluster nor of Dicer activity creates an hh-like phenotype, possibly due to dose compensation between the different antagonistic targets. This study reveals that a single signaling pathway can be targeted at multiple levels by the same microRNAs (Friggi-Grelin, 2009).

cos2, fu, and smo mRNA can be regulated by a cluster of microRNAs, including miR-12 and miR-283, in Drosophila wing disc. The overexpression of this cluster decreases the levels of Smo, Cos2, and Fu proteins and activates the Hh pathway, as evidenced by the induction of dpp expression in the wing imaginal discs and by the adult wing outgrowth. The experiments presented in this study with the 3'-UTR sensors of smo, fu, or cos2 are in favor of a direct binding. To constitute a real proof of a direct effect, further experiments as direct biochemical binding assay or compensatory mutation between the 3'-UTR and the miRNAs will be necessary to perform (Friggi-Grelin, 2009).

Programs that have been created to genomewide predictions of Drosophila miRNA targets provide lists of presumptive miR-12, and miR-283 regulated genes. In addition to the current in vivo validations, miR-12 binding sites are predicted on the 3'-UTR of ci and no sites were found on the 3'-UTR of the Su(fu) gene. No decrease was observed in either of these two proteins in the microRNA cluster overexpressing clones. It is interesting to note that Su(fu) mRNA, encoding another negative regulator of Hedgehog signaling, has been shown to be targeted by miR-214 in zebrafish. Absence of miR-214 results in the reduction of muscle cell types, the specification of which is dependent on Hh pathway activity. Nevertheless, the current study shows that in Drosophila wing discs an absence of microRNA does not modify the Hh pathway, raising the question of what the role of microRNAs in Drosophila Hh pathway regulation is (Friggi-Grelin, 2009).

Could the microRNAs overexpression phenotype that was identified be artifactual and simply the result of forced overexpression of the microRNA cluster in a tissue in which it should be silent? It is thought that the answer is no, because Northern blot analysis and the increase of miR-sensor in the dcr-1 mutant clones showed that the microRNA cluster is indeed expressed in this tissue. This suggests that the cluster likely has a role in this tissue in which it is normally present. Is the microRNA cluster regulation of the cos2 and smo 3'-UTRs physiological? It is thought so, because an absence of either the microRNA cluster or of Dicer in the wing imaginal disc induces an increase in the Cos2- and Smo-sensor lines. This signifies that the microRNAs expressed from the cluster regulate the cos2 and smo 3'-UTRs and thus display some functionality in the disc during larval development. Altogether, these data clearly show that an artifactual situation in which the microRNA cluster is expressed in a tissue in which it should not be present has not been created. The miRs overexpression was also tested on embryonic patterning but it did not lead to any phenotype, suggesting that the miR cluster regulation on the Hh pathway is specific to larval tissues (Friggi-Grelin, 2009).

As miR-12 and miR-283, and likely redundant miRs, are present in every cell of the wing disc, one possibility is that their normal roles are to dampen down the levels of Hh pathway components, particularly Cos2 and Smo, to prevent the accidental activation or downregulation of the pathway. Indeed, expressing both the microRNA cluster and its targets in the same tissue could provide a means of 'buffering stochastic fluctuations' in mRNA levels or in protein translation rates within the Hh signaling pathway, as has been proposed for other processes (Friggi-Grelin, 2009).

The data possibly indicate that miRNAs are able to regulate two antagonistic components of the pathway, Cos2 and Smo. It has been shown that the stability of these two proteins is 'interdependent': an increased level of Cos2 in the wing imaginal disc lowers the level of Smo, and, in the opposite direction, increased Smo decreases the level of Cos2. It is proposed that the interregulation of Cos2/Smo levels is independent of their relative activities because Cos2 effect on Smo levels is observed in posterior cells in which Cos2 activity is strongly inhibited by the constitutive activation of the pathway. Therefore, eliminating the miRNA-mediated inhibition of Cos2 and Smo in Delta3miR or dcr-1 mutant cells likely initially increased the levels of both proteins, but then the resulting higher levels of each protein presumably downregulated the other; the net variation of Cos2 and Smo levels would therefore be null. This hypothesis is favored because the independent Smo- and Cos2-sensor lines, which are unaffected by this Cos2/Smo interregulation, showed increased levels of GFP staining in Delta3miR and dcr-1 mutant animals. This suggests that the levels of both Cos2 and Smo are increased in the mutant animals but, because of the downregulation of each protein by the other, no ultimate alterations in the levels of the proteins are observed. If so, an Hh phenotype would not be expected to be seen in the miR mutant (Friggi-Grelin, 2009).

The screen created a situation in which the expression of the microRNA cluster is deregulated, ultimately destabilizing Cos2 protein levels and thereby activating Ci and Hh target gene expression. Importantly, a similar situation might be encountered during tumoral development. Aberrant Hh signaling activity is known to trigger the development of diverse cancers. While several of these tumors have been linked to mutations in Hh signaling components, not all of them have, leaving open the possibility that they are caused by other factors such as microRNA misexpression. Interestingly, more than half of the known human microRNA genes are located near chromosomal breakpoints associated with cancer, and in some documented cases the microRNAs are amplified, leading to overexpression. Some upregulated microRNAs are possibly able to bind mRNAs encoding negative regulators of Hh signaling, such as Su(fu) or Ptc, and could thus induce the misactivation of the Hh pathway, as is observed in some cancers. Therefore, a fine analysis of microRNA expression levels and the levels of known Hh components should be considered in studies of Hh pathway-related cancers (Friggi-Grelin, 2009).

What does this study add to the current knowledge about miRNA regulation? The study shows that a cluster of three microRNAs can target several antagonistic components of the same pathway in vivo. This is novel and unexpected. This raises the question of how to interpret the miRNA expression signatures observed in human tumors. Indeed, as stated above, it has been proposed that miRNAs are differentially expressed in human cancers and contribute to cancer development. The working hypothesis in the cancer/miRNAs field is that key cancer genes are regulated by aberrant expression of miRNAs. The identification of a specific miRNA:mRNA interactor pair is generally accepted as being of biological importance when the mRNA encodes a tumor suppressor or an oncogene whose expression is modified in the tumor. This study shows indirectly that this is an oversimplified view, because identifying an oncogene or tumor suppressor as a target of a miRNA may not provide a full explanation for tumor development if the same miRNA hits other antagonistic components of the same pathway that nullify the effect of the identified miRNA:mRNA interactor pair (Friggi-Grelin, 2009).

A Drosophila genetic screen yields allelic series of core microRNA biogenesis factors and reveals post-developmental roles for microRNAs

Canonical animal microRNAs (miRNAs) are ~22-nt regulatory RNAs generated by stepwise cleavage of primary hairpin transcripts by the Drosha and Dicer RNase III enzymes. A genetic screen was performed using an miRNA-repressed reporter in the Drosophila eye, and the first reported alleles were recovered of fly drosha, an allelic series of its dsRBD partner pasha, and novel alleles of dicer-1. Analysis of drosha mutants provided direct confirmation that mirtrons are independent of this nuclease, as inferred earlier from pasha knockouts. These mutants were further used to demonstrate in vivo cross-regulation of Drosha and Pasha in the intact animal, confirming remarkable conservation of a homeostatic mechanism that aligns their respective levels. Although the loss of core miRNA pathway components is universally lethal in animals, hypomorphic alleles were unexpectedly recovered that gave adult escapers with overtly normal development. However, the mutant photoreceptor neurons exhibited reduced synaptic transmission, without accompanying defects in neuronal development or maintenance. These findings indicate that synaptic function is especially sensitive to optimal miRNA pathway function. These allelic series of miRNA pathway mutants should find broad usage in studies of miRNA biogenesis and biology in the Drosophila system (Smibert, 2011).

This study describes a forward genetic screen for factors involved in miRNA biogenesis or function and validate its utility by characterizing a series of core miRNA pathway mutants. These were used to to investigate Microprocessor cross-regulation in vivo, as well as to study post-developmental roles of miRNAs in neural function. In particular, this study provides the first loss-of-function analysis of Drosophila drosha. As expected, a strong block was observed in canonical miRNA biogenesis in the drosha-null mutant, which accumulated primary miRNA transcripts and was depleted of mature miRNAs, similar to pasha mutants. In addition mirtron biogenesis was found to be unaffected by drosha mutation, providing direct evidence that these splicing-derived miRNAs are completely independent of the Drosha nuclease. Animals null for drosha and pasha are generally similar with respect to all phenotypes examined, supporting the obligate nature of these core protein partners within the Microprocessor complex. The screen generated allelic series for the key Microprocessor components drosha and pasha. These allowed assessment of the sensitivities of development versus function in cells with mildly reduced miRNA levels (Smibert, 2011).

Compared with full knockouts that give only null states, forward genetic screening can yield allelic series of varying phenotypic severity, which can uncover interesting aspects of pathway and gene function. It is noted that the hypomorphic drosha and pasha mutants have very different effects in different assays, as highlighted by the differences in derepression of both an endogenous miRNA target and a transgenic sensor for miRNA activity between weak drosha and pasha alleles. This is the case despite the fact that these mutants have similar effects on total mature miRNA levels as measured by Northern blot. The reason for this phenotypic discrepancy is not yet clear but suggests the possibilities that miRNAs are differentially sensitive to availability of the core biogenesis machinery or that specific biological processes are especially sensitive to optimal miRNA biogenesis. Indeed, electrophysiological evidence of the latter is provided, and both of these hypotheses represent compelling future directions for analysis of these and other mutants that may emerge from genetic screening (Smibert, 2011).

The theme of miRNA pathway autoregulation has emerged at multiple levels in animals and in plants. An in vivo demonstration of the reciprocal regulation of the core Microprocessor components reveals that this mechanism is conserved and occurs within the intact animal. The role of Pasha within the Microprocessor to position Drosha catalytic centers is clearly of crucial importance to miRNA biogenesis. Perhaps the instability of Drosha in the absence of Pasha is a biological safeguard to prevent inappropriate cleavage of transcripts by Drosha in the absence of Pasha. Likewise, the capacity of Drosha to cleave pasha transcripts may also limit Drosha levels by restricting the amount of Pasha for it to associate with (Smibert, 2011).

The studies to date focused on mutants of known miRNA pathway components. This has been a productive effort, as indicated by the first reported Drosophila alleles of drosha, the first allelic series of pasha in any organism, and novel alleles of dicer-1. The screening recovered other suppressor mutations that do not map to known pathway components, as well as enhancer mutations that are dependent on the endogenous 3' UTR of the white gene (w-miR) trigger. It is anticipated that the cloning and characterization of these mutations may provide additional insights into the miRNA biogenesis machinery or the mechanism of miRNA-mediated silencing (Smibert, 2011).

miRNAs in whole organisms have to date mostly been studied for their roles in development. This is at least in part due to the early developmental defects that potentially mask later functional defects. Yet, miRNAs have long been viewed as a potentially key component of neural function and fine-tuning due to their regulatory potential. One tantalizing property of miRNAs is their ability to regulate gene expression locally. In neurons, where a synapse may be a great distance from the nucleus, this could provide a means for rapid post-transcriptional regulation of targets. How this may be regulated in a signal-dependent manner is a topic of ongoing study. This study presents novel hypomorphic mutants that mildly affect miRNA levels and cause synapse function defects without affecting development (Smibert, 2011).

The requirement of neurons for precise miRNA activity is emphasized by the specific synaptic transmission defects in hypomorphic miRNA pathway mutants. In weak alleles of either pasha or drosha, only a mild reduction was observed in miRNA biogenesis. While these animals quickly succumb just before or after adult eclosion, they exhibit grossly normal development of all external structures. Using whole eye clone analysis, normal specification and projection of mutant photoreceptors was document, but substantial synaptic transmission defects wee obtained that were very similar in drosha, pasha, and dicer-1 mutants. The lack of deterioration of this phenotype with age both argues for a specific synaptic transmission defect and also that the reduction in miRNA levels in these photoreceptor neurons does not affect their viability or general health (Smibert, 2011).

The commonality of these phenotypes among the different mutants indicates underlying defective biogenesis of one or more canonical miRNAs, as opposed to mirtrons or other noncanonical species. It is conceivable that the synaptic transmission defect is caused by the cumulative effect of mildly reducing all photoreceptor miRNAs. However, the specificity of this phenotype and its critical dependence on optimal miRNA pathway function may imply that there is some aspect of synaptic transmission machinery that is especially sensitive to a more limited set of miRNAs. As the number of mutant strains for Drosophila miRNA loci steadily increases, it will be productive to screen them using ERGs on mutant eyes. An alternative approach may be to test miRNA sponges expressed presynaptically, perhaps in a candidate screen of head-expressed miRNAs (Smibert, 2011).

Since loss of single alleles is typically well tolerated at the organismal level, dose-sensitive loci are of particular relevance to human disease. DGCR8 is one of about 20 genes within the 22q11.2 locus deleted in patients with diGeorge syndrome, for which heterozygosity results in cognitive and behavioral impairments in humans. In a mouse model of diGeorge syndrome bearing the 22q11.2 microdeletion, heterozygosity of dgcr8 contributes to a reduction in brain miRNAs. More recently, specific heterozygosity of dgcr8 was shown to cause subtle but detectable defects in neural developmental and physiology, providing more direct evidence of dgcr8 haploinsufficiency. In the Drosophila system, cells heterozygous for pasha[KO] have less Pasha protein than homozygous wild-type cells. Accordingly, the levels of the GFP-ban sensor indicated that it was repressed more strongly in homozygous wild-type cells than in neighboring pasha[KO] heterozygous cells. Therefore, despite the operation of feedback mechanisms that regulate Microprocessor levels, pasha exhibits functional haploinsufficiency. Altogether, these studies reaffirm that subtle alterations in miRNA biogenesis can lead to detectable organismal phenotypes, helping to explain the lengths to which animal evolution has gone to maintain Microprocessor homeostasis. Reciprocally, these studies define Drosophila as a suitable system for future exploration of the unique sensitivity of neuronal function to miRNA activity (Smibert, 2011).

Drosophila Argonaute 1 and its miRNA biogenesis partners are required for oocyte formation and germline cell division

Argonaute 1 (Ago1) is a member of the Argonaute/PIWI protein family involved in small RNA-mediated gene regulation. In Drosophila, Ago1 plays a specific role in microRNA (miRNA) biogenesis and function. Previous studies have demonstrated that Ago1 regulates the fate of germline stem cells. However, the function of Ago1 in other aspects of oogenesis is still elusive. This study reports the function of Ago1 in developing egg chambers. Ago1 protein was found to be enriched in the oocytes and is also highly expressed in the cytoplasm of follicle cells. Clonal analysis of multiple ago1 mutant alleles shows that many mutant egg chambers contain only 8 nurse cells without an oocyte; this phenotype is phenocopied in dicer-1, pasha and drosha mutants. These results suggest that Ago1 and its miRNA biogenesis partners play a role in oocyte determination and germline cell division in Drosophila (Azzam, 2012).

Drosophila Ago1 forms a complex with mature miRNAs and acts to repress mRNAs. However, the spatial distribution of Ago1 during development has not been well characterized. The protein trap lines from the Carnegie Protein Trap library provide a powerful way to characterize the spatial and temporal distribution of trapped genes. The distribution of Ago1 in the cytoplasm has been described and shown to be localized in small puncta in the egg chamber. The findings using two independent assays for Ago1 localization have shown that Ago1 is enriched in the oocyte and mutant analysis has revealed a role in oocyte formation and germline cell division (Azzam, 2012).

Nurse cells supply nutrition for oocyte growth. The germline cell division defect described in this study has been previously observed in a cyclin-E mutant where 30% of the egg chambers have 8 cells, but the egg chamber still manages to develop an oocyte. Other studies have also described 8 cell egg chambers when String is over expressed as well as in a tribbles mutant. Both String overexpression and the tribbles mutant have 8 cells per egg chamber, but only a proportion fail to develop an oocyte. This defect occurs in the germarium while the cyst cells are undergoing mitosis. In the wild-type situation, the cystoblast divides four times to produce 16 cyst-cells. In the absence of ago1, some of the cystoblasts undergo only three divisions, producing 8-cell cysts. However, the ago1 mutant ovarioles with this phenotype still express Cyclin E, suggesting that mitosis is still occurring although perhaps at a slower rate. Combined with the oocyte formation defect, the resulting egg chambers only have 8 nurse cells and lack an oocyte. The cyst cell division in the germarium is not well understood. One potential explanation for the observed phenotype is that when Ago1, and presumably miRNA mediated gene regulation, are lost, the signal to stop dividing occurs early. Another possibility is because the egg chamber grows more slowly, the oocyte reaches region 2A before it manages to divide 4 times, thus receiving a premature signal to stop dividing, or being prematurely enclosed by the migrating follicle cells. The smaller germarium of ago1 mutant might also be an effect of cyst-cells dividing slower. The defective egg chamber however still manages to grow. Furthermore, the observation of Orb protein in region 2 of the germarium and in the stage 1 egg chamber could mean that the oocyte is trying to enter meiosis, or has entered meiosis but is unable to maintain the meiotic state because the Orb accumulation is lost in later stage egg chambers and no oocyte is formed. Oocyte differentiation and maintainance in the meiotic cycle are reliant on microtubule based transport of mRNAs and proteins from the nurse cells to the oocyte. Orb, the germline specific RNA-binding protein starts accumulating in the oocyte at region 2a in a microtubule-dependent manner. orb mutant causes the egg chamber to produce 8 nurse cells and no oocyte, similar to the ago1, dcr-1, drosha and pasha mutant phenotype seen in this study. However, since Orb is still expressed, it could be rule out that the phenotype is cause by loss of orb function. The inability to maintain the accumulation of Orb in the oocyte in later stages of oogenesis could relate to defect on maintaining the meiotic cycle (Azzam, 2012).

These results have shown that a greater proportion of older ago1 flies exhibit the 8-nurse cell phenotype than younger mutant flies. This could be due to the level of Ago1 in older flies decreasing to a certain threshold level to show an obvious phenotype. There is also the possibility that the remaining or leaky (due to hypomorphic allele) Ago1 is diluted through GSC division and maintainance such that GSCs from flies at 14 DAE have less Ago1 than GSCs from flies at 7 DAE. Previous studies suggest that GSC loss in ago1 mutants are age-dependent. This could potentially explain the age-dependent 8-nurse cell phenotype that were observed in ago1 mutants. Self-renewed GSC in the absence of Ago1 could be defective, so cystoblasts produced by defective GSC might not be able to divide normally. Although ago1^k08121 and ago1¹⁴ showed a more severe phenotype in older flies, ago1^EMS, as the strongest allele, showed very severe phenotype even in young flies (Azzam, 2012).

Ago1, Dcr-1, Loquacious and PIWI have roles in small RNA biogenesis and all of them have been shown to be important for germline stem cell maintenance. The role of miRNAs regulating GSC division was first reported by Hatfield (2005) who studied null mutants of dcr-1. A similar study looking at ago1 mutants revealed that Ago1 also regulates the fate of the GSC. Both of these studies showed a similar phenotypic defect in the germline. Furthermore, there are some cases where mutations in individual miRNA genes show phenotypes in the germline cells. The miRNA bantam has been previously found to be important for GSC maintenance. Also, miR-184 controls GSC differentiation, dorsoventral patterning of the egg shell and anteroposterior patterning. Although the effect in the GSC is quite reproducible from previous studies, it is not uncommon to see this in that knockouts of miRNA biogenesis factors. This has been shown quite well in the developing wing primordium where clones lacking mir-9a upregulate dLMO and induce wing notching. This phenotype is however not fully reproducible in dcr-1 and pasha mutant clones. The effect of removing all miRNA could cancel the effect of a single miRNA mutation (Azzam, 2012).

This study shows that the dcr-1, pasha and drosha mutants phenocopy the ago1 mutant during oogenesis. However, one Pasha mutant allele, pasha^LL03360, did not phenocopy ago1 and dcr-1. This mutant is a piggyBac insertion into the 5'UTR of pasha and despite showing a convincing loss of pasha protein in adult neurons, it is possible that the allele may only be hypomorphic in the ovary. Pasha has not been studied in the Drosophila germline but it has been shown to play a role in olfactory neuron morphogenesis in the Drosophila adult brain. In that study, Pasha and Dcr-1 were found to be required for arborization of projection neurons but not Ago1. This argues for Ago1-independent roles of Dcr-1 and Pasha. Alternatively, the ago1 mutant used in that study and the current study, ago1^k08121 may not be completely null or the protein from the parental cell could be compensating for the loss of Ago1. Recent studies have suggested that neural processes are exquisitely sensitive to miRNA pathway activity so perhaps a more complete loss of Pasha function is required to produce phenotypic consequences in the ovary compared to neurons. Indeed, the relative phenotypic strength of ago1^k08121 versus ago1^EMS1 and the null mutants of miRNA biogenesis enzymes argues for the hypomorphic nature of ago1^k08121. Mirtrons are another class of small RNAs which bypass Pasha/Drosha processing by utilizing the splicing machinery, but are still processed by Dcr-1 and loaded into Ago1. However, drosha^21K11 and the newly generated pasha^36B2 mutant show the same phenotype, qualitatively and quantitatively, as ago1 and dcr-1 mutants. This argues that the majority of the phenotype we observed is due to loss of canonical miRNAs and that miRtrons have a comparably insignificant role (if any) in the phenotypes analysed. Altogether, this study reaffirms that loss of miRNA function at various stages of biogenesis or effector function has important phenotypic consequences for oogenesis (Azzam, 2012).

RNA helicase Belle/DDX3 regulates transgene expression in Drosophila

Belle (Bel), the Drosophila homolog of the yeast DEAD-box RNA helicase DED1 and human DDX3, has been shown to be required for oogenesis and female fertility. This study reports a novel role of Bel in regulating the expression of transgenes. Abrogation of Bel by mutations or RNAi induces silencing of a variety of P-element-derived transgenes. This silencing effect depends on downregulation of their RNA levels. Genetic studies have revealed that the RNA helicase Spindle-E (Spn-E), a nuage RNA helicase that plays a crucial role in regulating RNA processing and PIWI-interacting RNA (piRNA) biogenesis in germline cells, is required for loss-of-bel-induced transgene silencing. Conversely, Bel abrogation alleviates the nuage-protein mislocalization phenotype in spn-E mutants, suggesting a competitive relationship between these two RNA helicases. Additionally, disruption of the chromatin remodeling factor Mod(mdg4) or the microRNA biogenesis enzyme Dicer-1 (Dcr-1) also alleviates the transgene-silencing phenotypes in bel mutants, suggesting the involvement of chromatin remodeling and microRNA biogenesis in loss-of-bel-induced transgene silencing. Finally genetic inhibition of Bel function was shown to lead to de novo generation of piRNAs from the transgene region inserted in the genome, suggesting a potential piRNA-dependent mechanism that may mediate transgene silencing as Bel function is inhibited (Lo, 2016).

Transgene silencing refers to the activity of various host defense responses that ordinarily act on natural foreign or parasitic sequences such as transposable elements (TEs), viroids, RNA and DNA viruses, and bacterial DNA. Since transgenes or their transcripts can resemble these cellular invaders in a number of ways, they naturally become the targets of host protective reactions. There are at least two distinct host defense systems responsible for silencing transgenes. One performs its effect via de novo DNA methylation at the genome level. The second defense system operates post-transcriptionally to silence transgenes, which involves sequence-specific RNA degradation in the cytoplasm. Therefore, transgene silencing involves complex cell immune systems including epigenetic and RNA silencing mechanisms. Although many factors involved in transgene silencing have been identified, and several mechanisms have been proposed, there remains much to understand regarding this vital aspect of the cell immune system (Lo, 2016).

Drosophila oogenesis, which involves the generation of the female gamete (oocyte), nurse cells, and follicle cells, is an excellent system for the study of TE and transgene silencing. The egg chamber, the developmental unit of oogenesis, contains the germline cells (one oocyte and 15 nurse cells) and a layer of surrounding somatically derived epithelial follicle cells. Both the germline cells and follicle cells can produce small RNAs to silence TE expression. The nuage, a perinuclear structure within Drosophila nurse cells, is an RNA-rich organelle unique to the germline. The nuage is required for the processing and localization of germline mRNAs and for the biogenesis of PIWI-interacting RNAs (piRNAs), a class of small non-coding RNAs that function as the cell immune system for silencing TEs. In D. melanogaster, most primary piRNAs are produced from discrete pericentromeric and telomeric heterochromatic loci (called piRNA clusters) containing damaged repeated TE sequences. In fly germline cells, an additional step of piRNA biogenesis, the 'ping-pong cycle' mechanism, is employed to generate the secondary piRNAs. Multiple factors localized in the nuage of germline cells have been discovered to be essential for secondary piRNA biogenesis, including Aub, AGO3, Spindle-E (Spn-E), and Vasa. In follicle cells, piRNAs are only produced from piRNA clusters (e.g., flamenco ) via PIWI and other related nuclear factors, and there is no secondary piRNA biogenesis involved. Intriguingly, besides piRNA clusters, euchromatic transposon insertion sites have been identified as another origin to produce piRNAs and endo-siRNAs. This mechanism provides another layer of defense to suppress TE activity and can also serve as a way to affect expression of coding genes and microRNA (miRNA) genes adjacent to inserted TEs (Lo, 2016).

Vasa and Spn-E belong to a family of DEAD-box proteins defined by multiple distinct conserved motifs including the D-E-A-D (Asp-Glu-Ala-Asp) motif. Among the identified DEAD-box proteins, one subfamily is highly conserved from yeast to human, which includes orthologs in yeast (DED1), Drosophila (Belle (Bel)), Xenopus (An3), mice (PL10), and humans (DDX3). These DEAD-box subfamily proteins possess the ATP-dependent RNA helicase activity to unwind double-stranded RNA and remodel RNA-protein interactions. Yeast DED1 is a multifunctional protein that functions to regulate multiple stages of RNA processing and translation. DED1 has also been shown to play a specific role in cell-cycle control. DDX3, the human homolog of DED1, is known to be involved in modulating multiple biological processes, including antiviral innate immunity, mitotic chromosome segregation in somatic cells, the suppression of spermatogenesis, G1-S transition of the cell cycle, epithelial-mesenchymal transition (EMT), a bona fide component of the RNAi pathway, TNF-related apoptosis, and WNT signaling (Lo, 2016 and references therein).

Vasa, a paralog of Bel, is required for the formation and function of nuage to suppress TE expression by being involved in the production of piRNAs. Most recently, it has been reported that Vasa is a key component in the piRNA amplifier complex in the nuage and serves as a protein platform to recruit PIWI proteins, the Tudor protein Qin/Kumo and antisense piRNA guides in an ATP-dependent manner for the ping-pong-loop amplification of secondary piRNAs. Bel colocalizes with Vasa in the nuage and at the oocyte posterior during oogenesis, and is required for female fertility. Recent findings have shown that loss of bel delays activation of Notch signaling in follicle cells, which in turn leads to delayed cell differentiation and defects in the switch from the mitotic cycle to the endocycle. However, unlike Vasa, the specific roles of Bel in the nuage of germline cells, and whether it is involved in piRNA biogenesis, remain unknown (Lo, 2016).

From previous studies, it was unexpectedly found that the Gal4-driven expression of a UASp-Bel:GFP transgene was silenced in bel mutant germline cells. This silencing effect was not specific to bel-based transgenes because 13 out of 22 different transgenic lines tested could be silenced in either germline or somatic bel mutant cells, or both. Subsequently the RNA helicase Spn-E, the epigenetic regulator Modifier of mdg4 [Mod(mdg4)] and the miRNA biogenesis enzyme Dcr-1 was identified as crucial factors for this bel-related transgene silencing. Their abrogation could either partially or completely rescue the transgene-silencing phenotype induced by loss of bel. Importantly, small RNA deep sequencing analysis suggests that a piRNA-mediated mechanism is potentially involved in Bel-inactivation-induced transgene silencing. Together, these studies genetically link the function of Bel to Spn-E, Mod(mdg4), and Dcr-1, and suggest that transgene silencing induced by Bel inactivation may involve RNA processing, piRNA, miRNA, and epigenetic mechanisms (Lo, 2016).

This article reports that loss-of-bel function triggers transgene silencing, which occurs through reduction in transgene RNA levels. Furthermore, genetic studies indicate that this transgene silencing effect induced by bel abrogation requires the RNA helicase Spn-E, the insulator modulator Mod(mdg4) and/or the miRNA biosynthesis enzyme Dcr-1. Based on the functional roles of these three molecules, the data suggest that this transgene silencing effect may involve RNA processing, chromatin remodeling and/or miRNA biogenesis. This transgene silencing event occurring under various bel mutant backgrounds implies that Bel may regulate these three molecular mechanisms to sustain transgene expression in the normal physiological condition. Intriguingly, these studies also identified additional complexity in the relationship between Bel and Spn-E because the mislocalization of nuage components in spn-E mutants requires Bel. Therefore, these findings, taken together, provide new insight into Bel function and expand the molecular interaction network radiating from Bel (Lo, 2016).

These studies show that loss of bel gave rise to transgene silencing via decreased transgene RNA levels. This phenomenon could be attributable to either transcriptional suppression or increased RNA degradation. Some support for an RNA degradation/targeting mechanism comes from the finding that Spn-E is required for transgene silencing induced by loss of bel. In germline nurse cells, Spn-E, which is located in the cytoplasmic nuage, is crucial for properly maintaining the subcellular localization of piRNA-related protein factors in the nuage, the ping-pong reaction of piRNA biogenesis, and silencing of TEs. Spn-E is also required for the proper localization of RNA transcripts (e.g., Bicoid and Oskar) during oogenesis, which might be related to its role in organizing a cytoskeletal framework. Therefore, it is plausible that the Spn-E-dependent RNA processing activity and/or Spn-E-generated piRNAs mediate transgene RNA degradation. In addition, it is possible that piRNAs can also elicit transcriptional silencing of transgenes based on their nuclear epigenetic role in TE silencing. Another striking finding from these studies shows that Bel is involved in disrupting the subcellular localization of nuage components when Spn-E is abrogated. In contrast, loss of Bel alone had no impact on the localization of piRNA-related nuage protein components. These findings suggest that there could be a competitive relationship between Bel and Spn-E, where these two molecules negatively regulate each other. This hypothesis is also supported by observations that Spn-E is required for transgene silencing when Bel is abrogated. According to these findings, a model is envisioned that Bel may function as a negative regulator for ping-pong-cycle-mediated piRNA biogenesis via disrupting the nuage localization of piRNA-related proteins as Spn-E function is abrogated, whereas Spn-E may be aberrantly activated by loss of Bel, in turn leading to transgene silencing. Whether piRNAs generated from the Spn-E-mediated ping-pong cycle are involved in transgene silencing is currently unknown. However, a recent study has shown that piRNAs can be generated directly from the transposon-derived transgene insertion area located in the euchromatic genome, which have been proposed to be involved in transgene silencing. This finding links piRNAs to transgene silencing. Preliminary small RNA deep sequencing analysis showed that the viable trans-heterozygous bel74407/neo30 mutant ovaries, which manifest a partial transgene silencing phenotype, displayed no significant defect in overall piRNA biogenesis from piRNA clusters and the ping-pong cycle, indicating that unlike Vasa, Bel is not involved in regulating piRNA generation. This finding is consistent with the result that homozygous bel mutants had no defect in nuage protein localization, which is different from other piRNA-related nuage proteins whose defects can significantly disturb the localization of other nuage protein components. Nevertheless, deep sequencing analysis also identified de novo piRNAs generated in bel74407/neo30 mutant ovaries (but not in wild-type ovaries) that could be mapped to the integrated P-element-derived transgene sequence area (P[LacW]). This result is in line with previous findings and indicates that the de novo generation of piRNAs from the inserted transgene region in the genome occurs under the bel mutant background. Given that Bel is a paralog of Vasa and the current genetic findings also suggest a competitive relationship between Bel and Spn-E, loss of Bel may disrupt its normal regulation of some small RNA-related helicases and co-factors, which in turn aberrantly activates the small RNA pathway(s). Therefore, it is possible that loss of Bel may promote de novo piRNA biogenesis from the transgene insertion sites by freeing these small RNA regulators and provoking the activation of their related small RNA pathway(s), which is one possible mechanism leading to transgene silencing. Since it was not possible create viable mutant progeny bearing mutations at both bel and spn-E gene loci, it is still uncertain whether Spn-E is involved in regulating de novo piRNA biogenesis from transgene insertion sites. Although it was not possible to elucidate the detailed mechanism due to technical hurdles, exploring the regulatory roles of Bel and Spn-E in this new type of piRNA biogenesis will be key, interesting research for understanding molecular mechanisms underlying transgene silencing (Lo, 2016).

Another unexpected finding is that the spn-E mutant rescue of transgene silencing associated with loss of bel also occurs in somatic follicle cells. This indicates that in addition to its well-known function in germline cells, Spn-E may have a somatic function. As Spn-E is not implicated in somatic piRNA biogenesis (e.g., flamenco), it is uncertain how Spn-E mediates somatic transgene silencing and whether piRNAs participate in this event. Therefore, it will be important in the future to unravel whether the mechanism by which Spn-E facilitates transgene silencing in bel mutant somatic cells is the same as that in germline cells (Lo, 2016).

The common feature for transgenes silenced by Bel abrogation is their P-element-based integration into genomic DNA. The observation that some P-element-based transgenes were not silenced in bel mutant cells raises an interesting question about what factors can determine whether a transgene can be silenced or not. From genetic analysis of a series of transgenes, it was observed that six examined transgenes (ci-LacZ, dMyc-LacZ, dom-LacZ, C306-Gal4, ptc-Gal4, Tj-Gal4), which were generated by the insertion of two different P-element-derived vector sequences (P[LacW] and P[GawB]), were silenced under the bel mutant background. In contrast, two transgenes (histone-GFP, histone-RFP) generated by the insertion of P[His2Av]-derived vector sequences were not silenced by Bel inactivation. Although these data are not a conclusive result, they imply that the inserted transgene sequence itself, not the insertion location in the genome, may be a critical determinant for Bel-dependent transgene silencing since this silencing phenotype seems to be transgene-specific and a change in the transgene insertion location in the genome has no influence on whether this transgene can be silenced or not when Bel function is inhibited. It is possible that the transgene sequence determines a local chromosomal conformation and whether the transgene can be silenced under the bel mutant background is determined by whether its chromosomal structure can be recognized by epigenetic regulators involved in transgene silencing. Although further investigations are still needed to verify this hypothesis due to limited cases in this study, it raises the possibility that epigenetic regulation at the chromatin level may be involved in Bel-dependent transgene silencing. Indeed, besides Spn-E, the genetic study identified Mod(mdg4) as another crucial factor required for transgene silencing in both germline and somatic cells when Bel is abrogated. The mod(mdg4) gene encodes multiple nuclear factors through trans-splicing and this protein family is functionally involved in the modification of the properties of insulators, which are genomic elements that regulate gene expression. Mod(mdg4) proteins can function as chromatin modulators engaged in the organization of highly ordered chromatin domains. The involvement of Mod(mdg4) in transgene silencing suggests that nuclear epigenetic events are also crucial for induction of transgene silencing when Bel is inactivated. However, the role of Mod(mdg4) in transgene silencing might also be indirect, such as through its regulation of other genes that could contribute to silencing. Nevertheless, the findings suggest that the co-ordination between nuclear and cytoplasmic events mediated by Mod(mdg4) and Spn-E, respectively, is mandatory for induction of transgene silencing when Bel is functionally inhibited (Lo, 2016).

The miRNA biogenesis enzyme Dcr-1 is the third factor identified from these studies that is crucial for transgene silencing in the absence of Bel. Interestingly, the block in transgene silencing in this case (double mutant for bel and Dcr-1) only occurred in germline cells, but not in somatic cells. This discovery raises the possibility that there are additional miRNA-targeted proteins present in germline cells, but not in somatic cells, and they can interact with factors essential for germline transgene silencing. If this is a case, aberrantly elevated levels of these miRNA-targeted proteins in Dcr-1 mutant germline cells might interfere with transgene silencing. Besides this possible indirect role, another possibility is that the Dcr-1-dependent miRNA pathway may play a direct role in transgene silencing in germline cells as Bel is abrogated. The miRNA pathway has been shown to be implicated in transgene silencing in Drosophila S2 cells. Although the silencing mechanism is unclear, this finding raises a possible direct role of the Dcr-1-dependent miRNA pathway in bel-mutant transgene silencing. A study from Zhou has shown that Bel proteins were cofractionated with the miRNA-dependent RNA-induced silencing complexes (miRISCs) and co-immunoprecipitated with Ago1, the protein component of miRISCs. This finding suggests a compelling possibility that Bel may be directly involved in the miRNA pathway to regulate miRISC-dependent RNA silencing and Bel inactivation may result in the aberrant functionality of miRISC and its related RNA silencing. Since miRNAs can target mRNAs via their short seed sequences, a possibility which cannot be ruled out is that some miRNAs may directly target transgene RNAs to regulate their levels. Future investigation is needed to reveal which possibility is more relevant (Lo, 2016).

In conclusion, these findings provide novel insights into the regulatory role of Bel in the expression of transgenes in Drosophila and its functional linkage to crucial factors implicated in RNA processing, chromatin remodeling and miRNA biogenesis. These findings further advance understanding of the complex cellular functions of Bel. These studies of the role for Bel in transgene expression may have important, future implications for understanding the regulation of expression of newly invaded or transposed TEs and virus-retrotransposon DNA chimeras generated from viral infection as they may share the similar scenario as transgene integration (Lo, 2016).

EVOLUTIONARY HOMOLOGS

Processing of microRNAs upstream of Dicer

The RNase III family of double-stranded RNA-specific endonucleases is characterized by the presence of a highly conserved 9 amino acid stretch in their catalytic center known as the RNase III signature motif. The drosha gene, encoding a new member of this family, was isolated in Drosophila melanogaster. Characterization of this gene revealed the presence of two RNase III signature motifs in its sequence that may indicate that it is capable of forming an active catalytic center as a monomer. The Drosha protein also contains an 825 amino acid N-terminus with an unknown function. A search for the known homologues of the Drosha protein revealed that it has a similarity to two adjacent annotated genes identified during C. elegans genome sequencing. Analysis of the genomic region of these genes by the Fgenesh program and sequencing of the EST cDNA clone derived from it revealed that this region encodes only one gene. This newly identified gene in nematode genome shares a high similarity to Drosophila Drosha throughout its entire protein sequence. A potential Drosha homologue is also found among the deposited human cDNA sequences. A comparison of these Drosha proteins to other members of the RNase III family indicates that they form a new group of proteins within this family (Filippov, 2000).

Hundreds of small RNAs of approximately 22 nucleotides, collectively named microRNAs (miRNAs), have been discovered recently in animals and plants. Although their functions are being unravelled, their mechanism of biogenesis remains poorly understood. miRNAs are transcribed as long primary transcripts (pri-miRNAs) whose maturation occurs through sequential processing events: the nuclear processing of the pri-miRNAs into stem-loop precursors of approximately 70 nucleotides (pre-miRNAs), and the cytoplasmic processing of pre-miRNAs into mature miRNAs. Dicer, a member of the RNase III superfamily of bidentate nucleases, mediates the latter step, whereas the processing enzyme for the former step is unknown. Another RNase III, human Drosha, has been identified as the core nuclease that executes the initiation step of miRNA processing in the nucleus. Immunopurified Drosha cleaved pri-miRNA to release pre-miRNA in vitro. Furthermore, RNA interference of Drosha resulted in the strong accumulation of pri-miRNA and the reduction of pre-miRNA and mature miRNA in vivo. Thus, the two RNase III proteins, Drosha and Dicer, may collaborate in the stepwise processing of miRNAs, and have key roles in miRNA-mediated gene regulation in processes such as development and differentiation (Lee, 2003).

MicroRNAs (miRNAs) represent a family of small noncoding RNAs that are found in plants and animals. miRNAs are expressed in a developmentally and tissue-specific manner and regulate the translational efficiency and stability of partial or fully sequence-complementary mRNAs. miRNAs are excised in a stepwise process from double-stranded RNA precursors that are embedded in long RNA polymerase II primary transcripts (pri-miRNA). Drosha RNase III catalyzes the first excision event, the release in the nucleus of a hairpin RNA (pre-miRNA), which is followed by export of the pre-miRNA to the cytoplasm and further processing by Dicer to mature miRNAs. The human DGCR8, the DiGeorge syndrome critical region gene 8, and its Drosophila melanogaster homolog have been characterized. Biochemical and cell-based readouts are provided to demonstrate the requirement of DGCR8 for the maturation of miRNA primary transcripts. RNAi knockdown experiments of fly and human DGCR8 results in accumulation of pri-miRNAs and reduction of pre-miRNAs and mature miRNAs. These results suggest that DGCR8 and Drosha interact in human cells and reside in a functional pri-miRNA processing complex (Landthaler, 2004).

MicroRNAs (miRNAs) are a growing family of small non-protein-coding regulatory genes that regulate the expression of homologous target-gene transcripts. They have been implicated in the control of cell death and proliferation in flies, haematopoietic lineage differentiation in mammals, neuronal patterning in nematodes and leaf and flower development in plants. miRNAs are processed by the RNA-mediated interference machinery. Drosha is an RNase III enzyme that has been implicated in miRNA processing. Human Drosha is a component of two multi-protein complexes. The larger complex contains multiple classes of RNA-associated proteins including RNA helicases, proteins that bind double-stranded RNA, novel heterogeneous nuclear ribonucleoproteins and the Ewing's sarcoma family of proteins. The smaller complex is composed of Drosha and the double-stranded-RNA-binding protein, DGCR8, the product of a gene deleted in DiGeorge syndrome. In vivo knock-down and in vitro reconstitution studies have revealed that both components of this smaller complex, termed Microprocessor, are necessary and sufficient in mediating the genesis of miRNAs from the primary miRNA transcript (Gregory, 2004).

Mature microRNAs (miRNAs) are generated via a two-step processing pathway to yield approximately 22-nucleotide small RNAs that regulate gene expression at the post-transcriptional level. Initial cleavage is catalysed by Drosha, a nuclease of the RNase III family, which acts on primary miRNA transcripts (pri-miRNAs) in the nucleus. Here it is shown that Drosha exists in a multiprotein complex, the Microprocessor, and begin the process of deconstructing that complex into its constituent components. Along with Drosha, the Microprocessor also contains Pasha (partner of Drosha), a double-stranded RNA binding protein. Suppression of Pasha expression in Drosophila cells or Caenorhabditis elegans interferes with pri-miRNA processing, leading to an accumulation of pri-miRNAs and a reduction in mature miRNAs. Finally, depletion or mutation of pash-1 in C. elegans causes de-repression of a let-7 reporter and the appearance of phenotypic defects overlapping those observed upon examination of worms with lesions in Dicer (dcr-1) or Drosha (drsh-1). Considered together, these results indicate a role for Pasha in miRNA maturation and miRNA-mediated gene regulation (Denli, 2004).

A critical step during human microRNA maturation is the processing of the primary microRNA transcript by the nuclear RNaseIII enzyme Drosha to generate the 60-nucleotide precursor microRNA hairpin. How Drosha recognizes primary RNA substrates and selects its cleavage sites has remained a mystery, especially given that the known targets for Drosha processing show no discernable sequence homology. Human Drosha selectively cleaves RNA hairpins bearing a large (10 nucleotides) terminal loop. From the junction of the loop and the adjacent stem, Drosha then cleaves approximately two helical RNA turns into the stem to produce the precursor microRNA. Beyond the precursor microRNA cleavage sites, approximately one helix turn of stem extension is also essential for efficient processing. While the sites of Drosha cleavage are determined largely by the distance from the terminal loop, variations in stem structure and sequence around the cleavage site can fine-tune the actual cleavage sites chosen (Zeng, 2005).

Plant Dicer homologs

The short integument (sin1) mutation causes a female-specific infertility, and a defect in the control of time to flowering in Arabidopsis. Female sterility of Sin minus plants is due to abnormal ovule integument development and aberrant differentiation of the megagametophyte in a subset of ovules. An additional defect of sin1 mutants is the production of an increased number of vegetative leaf and inflorescence primordia leading to delayed flowering. The delayed flowering phenotype of sin1-1 is not due to a defect in the perception of day length periodicity or in gibberellic acid metabolism. Phenotypes of double mutant combinations of sin1 with terminalflower (tfl1) indicate that SIN1 activity is required for precocious floral induction typical in a tfl1 mutant. Unexpectedly, sin1-1 tfl1-1 plants do not make pollen, thus revealing a novel role for TFL1 in the anther. Early flowers of sin1-1 ap1-1 double mutants are transformed to long inflorescence-like shoots. A genetic model for the role of SIN1 in flowering time control is proposed (A. Ray, 1996).

Maternal gene products deposited in an animal egg determine the polarity of embryonic axes and regulate embryonic cell-cell communication important for morphogenesis. The first maternal-effect embryo-defective mutation discovered in a plant is reported in this study. Recessive mutations in the SHORT INTEGUMENT (SIN1) gene in Arabidopsis were previously shown to influence ovule development and flowering time. A sin1 mutation has a pronounced maternal effect on zygotic embryo development. A homozygous sin1 mutant embryo is normal when nursed by a sin1/+ heterozygous maternal sporophyte. Strikingly, a sin1 or a sin1/+ embryo that is nursed by a sin1 homozygous maternal sporophyte develops morphogenetic defects in the apical-basal and radial axes. The defects resemble those seen in some zygotic-effect embryonic pattern formation mutants. These results imply that in maternal cells the SIN1 gene either codes for or controls the production of a diffusible morphogen necessary for proper zygotic embryogenesis (S. Ray, 1996).

Arabidopsis thaliana floral meristems are determinate structures that produce a defined number of organs, after which cell division ceases. A new recessive mutant, carpel factory (caf), converts the floral meristems to an indeterminate state. They produce extra whorls of stamens, and an indefinite number of carpels. Thus, CAF appears to suppress cell division in floral meristems. The function of CAF is partially redundant with the function of the CLAVATA (CLV) and SUPERMAN (SUP) genes, because caf clv and caf sup double mutants show dramatically enhanced floral meristem over-proliferation. caf mutant plants also show other defects, including absence of axillary inflorescence meristems, and abnormally shaped leaves and floral organs. The CAF gene was cloned and found to encode a putative protein of 1909 amino acids containing an N-terminal DExH/DEAD-box type RNA helicase domain attached to a C-terminal RNaseIII-like domain. A very similar protein of unknown function is encoded by a fungal and an animal genome. Helicase proteins are involved in a number of processes, including specific mRNA localization and mRNA splicing. RNase III proteins are involved in the processing of rRNA and some mRNA molecules. Thus CAF may act through some type of RNA processing event(s). CAF gives rise to two major transcripts of 2.5 and 6.2 kb. In situ hybridization experiments show that CAF RNA is expressed throughout all shoot tissues (Jacobsen, 1999).

In metazoans, microRNAs, or miRNAs, constitute a growing family of small regulatory RNAs that are usually 19–25 nucleotides in length. They are processed from longer precursor RNAs that fold into stem-loop structures by the ribonuclease Dicer and are thought to regulate gene expression by base pairing with RNAs of protein-coding genes. In Arabidopsis thaliana, mutations in CARPEL FACTORY (CAF), a Dicer homolog, and those in a novel gene, HEN1, result in similar, multifaceted developmental defects, suggesting a similar function of the two genes, possibly in miRNA metabolism. To investigate the potential functions of CAF and HEN1 in miRNA metabolism, attempts were made to isolate miRNAs from Arabidopsis and examine their accumulation during plant development in wild-type plants and in hen1-1 and caf-1 mutant plants. Eleven miRNAs were isolated, some of which have potential homologs in tobacco, rice, and maize. The putative precursors of these miRNAs have the capacity to form stable stem-loop structures. The accumulation of these miRNAs appears to be spatially or temporally controlled in plant development, and their abundance is greatly reduced in caf-1 and hen1-1 mutants. HEN1 homologs are found in bacterial, fungal, and metazoan genomes. It is concluded that miRNAs are present in both plant and animal kingdoms. An evolutionarily conserved mechanism involving a protein, known as Dicer in animals and CAF in Arabidopsis, operates in miRNA metabolism. HEN1 is a new player in miRNA accumulation in Arabidopsis, and HEN1 homologs in metazoans may have a similar function. The developmental defects associated with caf-1 and hen1-1 mutations and the patterns of miRNA accumulation suggest that miRNAs play fundamental roles in plant development (Park, 2002).

Formation of microRNA (miRNA) requires an RNaseIII domain-containing protein, termed Dicer-1 in animals and Dicer-like1 (Dcl1) in plants, to catalyze processing of an RNA precursor with a fold-back structure. Loss-of-function dcl1 mutants of Arabidopsis have low levels of miRNA and exhibit a range of developmental phenotypes in vegetative, reproductive, and embryonic tissues. Dcl1 mRNA occurs in multiple forms, including truncated molecules that result from aberrant pre-mRNA processing. Both full-length and truncated forms accumulate to relatively low levels in plants containing a functional Dcl1 gene. However, in dcl1 mutant plants, dcl1 RNA forms accumulated to levels several-fold higher than those in Dcl1 plants. Elevated levels of Dcl1 RNAs are also detected in miRNA-defective hen1 mutant plants and in plants expressing a virus-encoded suppressor of RNA silencing (P1/HC-Pro), which inhibits miRNA-guided degradation of target mRNAs. A miRNA (miR162) target sequence was predicted near the middle of Dcl1 mRNA, and a Dcl1-derived RNA with the properties of a miR162-guided cleavage product was identified and mapped. These results indicate that Dcl1 mRNA is subject to negative feedback regulation through the activity of a miRNA (Xie, 2003).

In plants and invertebrates, viral-derived siRNAs processed by the RNaseIII Dicer guide Argonaute (AGO) proteins as part of antiviral RNA-induced silencing complexes (RISC). As a counterdefense, viruses produce suppressor proteins (VSRs) that inhibit the host silencing machinery, but their mechanisms of action and cellular targets remain largely unknown. This study shows that the Turnip crinckle virus (TCV) capsid, the P38 protein, acts as a homodimer, or multiples thereof, to mimic host-encoded glycine/tryptophane (GW)-containing proteins normally required for RISC assembly/function in diverse organisms. The P38 GW residues bind directly and specifically to Arabidopsis AGO1, which, in addition to its role in endogenous microRNA-mediated silencing, is identified as a major effector of TCV-derived siRNAs. Point mutations in the P38 GW residues are sufficient to abolish TCV virulence, which is restored in Arabidopsis ago1 hypomorphic mutants, uncovering both physical and genetic interactions between the two proteins. It was further shown how AGO1 quenching by P38 profoundly impacts the cellular availability of the four Arabidopsis Dicers, uncovering an AGO1-dependent, homeostatic network that functionally connects these factors together. The likely widespread occurrence and expected consequences of GW protein mimicry on host silencing pathways are discussed in the context of innate and adaptive immunity in plants and metazoans (Azevedo, 2010).

Dicer function in yeast

The Schizosaccharomyces pombe genome encodes only one of each of the three major classes of proteins implicated in RNA silencing: Dicer (Dcr1), RNA-dependent RNA polymerase (RdRP; Rdp1), and Argonaute (Ago1). These three proteins are required for silencing at centromeres and for the initiation of transcriptionally silent heterochromatin at the mating-type locus. The introduction of a double-stranded RNA (dsRNA) hairpin corresponding to a green fluorescent protein (GFP) transgene triggers classical RNA interference (RNAi) in S. pombe. That is, GFP silencing triggered by dsRNA reflects a change in the steady-state concentration of GFP mRNA, but not in the rate of GFP transcription. RNAi in S. pombe requires dcr1, rdp1, and ago1, but does not require chp1, tas3, or swi6, genes required for transcriptional silencing. Thus, the RNAi machinery in S. pombe can direct both transcriptional and posttranscriptional silencing using a single Dicer, RdRP, and Argonaute protein. These findings suggest that these three proteins fulfill a common biochemical function in distinct siRNA-directed silencing pathways (Sigova, 2004).

This study demonstrates that a dsRNA derived from a hairpin transcript can trigger posttranscriptional silencing of a corresponding mRNA in S. pombe. A similar hairpin transcript, corresponding to the ura4 locus has also been shown (Schramke, 2003) to trigger transcriptional silencing. In both studies, silencing triggered by a hairpin transcript require the RNAi machinery -- Dcr1, Rdp1, and Ago1. Transcriptional silencing, unlike posttranscriptional silencing, requires components of the transcriptional silencing apparatus: Chp1, Tas3, or Swi6. Robust silencing by both pathways requires the chromodomain protein Clr4, which appears to play a role in siRNA biogenesis or stability. Why does the GFP hairpin construct presented in this study trigger exclusively posttranscriptional silencing, whereas the previously studied ura4 hairpin triggered transcriptional silencing? One possible explanation is that the GFP hairpin used here includes an efficiently spliced intron between the two arms of the hairpin. It is presumed that splicing of the intron promotes the accumulation of GFP dsRNA in the cytoplasm. In contrast, the ura4 hairpin construct of Schramke (2003) contains an unspliced spacer sequence between the hairpin arms. Thus, the ura4 hairpin may be localized largely to the nucleus. A difference in subcellular localization might explain the different results obtained by the two studies. Alternatively, silencing of ura4 by the ura4-specific hairpin might comprise a mixture of transcriptional and posttranscriptional silencing. In this case, transcriptional silencing might not occur at the adh1 locus, even if the GFP hairpin-derived siRNAs trigger histone modification, perhaps because the gene is strongly expressed or is in a region of the genome otherwise refractory to heterochromatin formation. Nonetheless, the current data, together with those of Schramke (2003), clearly show that at least two distinct silencing responses can be initiated by a common RNAi machinery, without resorting to specialized forms of Dicer, RdRP, or Argonaute proteins. The demonstration that fission yeast contain a functional RNAi pathway now provides a simplified, genetically tractable model in which to study how the nature of the silencing trigger or of the silencing target determines the silencing pathway evoked -- posttranscriptional or transcriptional (Sigova, 2004).

Dicer promotes transcription termination at sites of replication stress to maintain genome stability

Nuclear RNAi is an important regulator of transcription and epigenetic modification, but the underlying mechanisms remain elusive. Using a genome-wide approach in the fission yeast S. pombe, this study found that Dcr1, but not other components of the canonical RNAi pathway, promotes the release of Pol II from the 3' end of highly transcribed genes, and, surprisingly, from antisense transcription of rRNA and tRNA genes, which are normally transcribed by Pol I and Pol III. These Dcr1-terminated loci correspond to sites of replication stress and DNA damage, likely resulting from transcription-replication collisions. At the rDNA loci, release of Pol II facilitates DNA replication and prevents homologous recombination, which would otherwise lead to loss of rDNA repeats especially during meiosis. These results reveal a novel role for Dcr1-mediated transcription termination in genome maintenance and may account for widespread regulation of genome stability by nuclear RNAi in higher eukaryotes (Castel, 2014).

Enzymatic activity of Dicer

RNA silencing phenomena, known as post-transcriptional gene silencing in plants, quelling in fungi, and RNA interference (RNAi) in animals, are mediated by double-stranded RNA (dsRNA) and mechanistically intersect at the ribonuclease Dicer. The 218 kDa human Dicer has been cloned: its ribonuclease activity and dsRNA-binding properties have been characterized. The recombinant enzyme generates approximately 21-23 nucleotide products from dsRNA. Processing of the microRNA let-7 precursor by Dicer produces an apparently mature let-7 RNA. Mg(2+) was required for dsRNase activity, but not for dsRNA binding, thereby uncoupling these reaction steps. ATP is dispensable for dsRNase activity in vitro. The Dicer.dsRNA complex formed at high KCl concentrations is catalytically inactive, suggesting that ionic interactions are involved in dsRNA cleavage. The putative dsRNA-binding domain located at the C-terminus of Dicer binds dsRNA in vitro. Human Dicer expressed in mammalian cells colocalizes with calreticulin, a resident protein of the endoplasmic reticulum. Availability of the recombinant Dicer protein will help improve understanding of RNA silencing and other Dicer-related processes (Provost, 2002).

In animals, the double-stranded RNA-specific endonuclease Dicer produces two classes of functionally distinct, tiny RNAs: microRNAs (miRNAs) and small interfering RNAs (siRNAs). miRNAs regulate mRNA translation, whereas siRNAs direct RNA destruction via the RNA interference (RNAi) pathway. In human cell extracts, the miRNA let-7 naturally enters the RNAi pathway, which suggests that only the degree of complementarity between a miRNA and its RNA target determines its function. Human let-7 is a component of a previously identified, miRNA-containing ribonucleoprotein particle, which is an RNAi enzyme complex. Each let-7-containing complex directs multiple rounds of RNA cleavage, which explains the remarkable efficiency of the RNAi pathway in human cells (Hutvagner, 2002).

The bidentate RNase III Dicer cleaves microRNA precursors to generate the 21-23 nt long mature RNAs. These precursors are 60-80 nt long; they fold into a characteristic stem-loop structure and they are generated by an unknown mechanism. To gain insights into the biogenesis of microRNAs, the precise 5' and 3' ends of the let-7 precursors in human cells have been characterized. They harbor a 5'-phosphate and a 3'-OH and remarkably, they contain a 1-4 nt 3' overhang. These features are characteristic of RNase III cleavage products. Since these precursors are present in both the nucleus and the cytoplasm of human cells, these results suggest that they are generated in the nucleus by the nuclear RNase III. Additionally, these precursors fit the minihelix export motif and are thus likely exported by this pathway (Basyuk, 2003)

Dicer is a multidomain ribonuclease that processes double-stranded RNAs (dsRNAs) to 21 nt small interfering RNAs (siRNAs) during RNA interference, and excises microRNAs from precursor hairpins. Dicer contains two domains related to the bacterial dsRNA-specific endonuclease, RNase III, which is known to function as a homodimer. Based on an X-ray structure of the Aquifex aeolicus RNase III, models of the enzyme interaction with dsRNA, and its cleavage at two composite catalytic centers, have been proposed. Mutations were generated in human Dicer and Escherichia coli RNase III residues implicated in the catalysis, and their effect on RNA processing was studed. The results indicate that both enzymes have only one processing center, containing two RNA cleavage sites and generating products with 2 nt 3' overhangs. Based on these and other data, it is proposed that Dicer functions through intramolecular dimerization of its two RNase III domains, assisted by the flanking RNA binding domains, PAZ and dsRBD (Zhang, 2004).

TRBP, a regulator of cellular PKR and HIV-1 virus expression, interacts with Dicer and functions in RNA silencing

Dicer is a key enzyme involved in RNA interference (RNAi) and microRNA (miRNA) pathways. It is required for biogenesis of miRNAs and small interfering RNAs (siRNAs), and also has a role in the effector steps of RNA silencing. Apart from Argonautes, no proteins are known to associate with Dicer in mammalian cells. This work describes the identification of TRBP (human immunodeficiency virus (HIV-1) transactivating response (TAR) RNA-binding protein) as a protein partner of human Dicer. TRBP is required for optimal RNA silencing mediated by siRNAs and endogenous miRNAs, and it facilitates cleavage of pre-miRNA in vitro. TRBP had previously been assigned several functions, including inhibition of the interferon-induced double-stranded RNA-regulated protein kinase PKR and modulation of HIV-1 gene expression by association with TAR. The TRBP-Dicer interaction shown raises interesting questions about the potential interplay between RNAi and interferon-PKR pathways (Haase, 2005).

Mutation of the C. elegans Dicer homolog

To obtain direct evidence that Dicer is involved in gene silencing, the effectiveness of RNAi in a C. elegansstrain containing a null mutation in the Dicer homolog (dcr-1), was examined. DCR-1 is encoded by an 8165-base pair (bp) gene in C. elegans and contains an NH₂-terminal DExH/DEAD-box type RNA helicase domain, two RNase III-like domains, and a COOH-terminal dsRNA binding motif. Animals with a deletion in dcr-1 that removes a 2470-bp fragment spanning a region from exon 13 to intron 18 were obtained from the C. elegans gene knockout consortium. The deletion removes the NH₂-terminal portion of the first RNase III domain and is also predicted to introduce multiple stop codons into the reading frame. dcr-1(-/-) animals are sterile, suggesting that DCR-1 has an essential role in vivo and also emphasizing that the deletion creates a loss-of-function allele (Knight, 2001).

In C. elegans, RNAi is typically initiated by injecting or feeding dsRNA, and gene silencing is subsequently observed in the F₁ progeny. Because dcr-1(-/-) animals are sterile and do not give rise to progeny, a transgenic line was used in which RNAi could be monitored in individual animals, without waiting for subsequent generations. The line was constructed by microinjecting DNA encoding green fluorescent protein (GFP) (sur5::GFP), as well as a previously described vector containing an RNA hairpin matching the GFP sequence, under the control of a heat shock promoter [hsp16-2_pGFP(IR)]. In the transgenic line isolated, heat shock produces an easily discernable RNAi phenotype in heat-shocked animals, so it is not necessary to analyze progeny. The transgenic line was made with dcr-1(+/-) animals and dcr-1(-/-) progeny were examined for RNAi resistance after heat shock. Whereas wild-type animals exhibit robust RNAi measured by a loss in GFP fluorescence, animals homozygous for the dcr-1 deletion are RNAi defective and continue to exhibit a strong fluorescence. These results are consistent with the idea that dcr-1 is required for RNAi (Knight, 2001).

Gene silencing by RNAi is known to involve the degradation of the targeted mRNA. To obtain molecular evidence that dcr-1 is required for RNAi, as well as to monitor the effects of the dcr-1 deletion on RNAi of other genes, semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) was used to measure mRNA levels after RNAi. When dsRNA corresponding to the mpk-1 gene was injected into L4 worms, wild-type animals exhibited reduced mpk-1 mRNA levels, whereas mpk-1 mRNA remained abundant in dcr-1(-/-) animals . These results, and similar results with dsRNA to gld-1, again suggest that DCR-1 is required for RNAi. However, when animals were injected with dsRNA to unc-54, decreased levels of unc-54 mRNA were observed in both wild-type and dcr-1(-/-) animals, consistent with a normal RNAi response (Knight, 2001).

In the above experiments, dcr-1(-/-) animals were resistant to RNAi of genes expressed in the germ line (mpk-1 and gld-1) but showed a normal RNAi response for unc-54, a somatic gene. Thus, dcr-1 may be similar to the genes mut-7 and rde-2, or ego-1, which are required for RNAi of germ line expressed genes, but not for somatic genes. Consistent with this idea, it was found that dcr-1 mutants showed a normal RNAi response when dsRNA corresponding to another somatic gene, unc-22, was injected. dcr-1(-/-) animals were also sensitive to RNAi when fed bacteria expressing unc -22 dsRNA (Knight, 2001).

It was also noticed that the requirement of RNAi for the dcr-1 gene depended on the method used to deliver the dsRNA. For example, dcr-1 was required for RNAi of the GFP transgene by the heat shock-inducible RNA hairpin. However, when gfpdsRNA was injected into the dcr-1(-/-) animals carrying the GFP transgene, 100% of dcr-1(-/-) animals had reduced fluorescence (n = 14). The data indicate that dcr-1(-/-) animals are defective for RNAi in some but not all cases. Possibly, gene silencing by dsRNA can occur by multiple pathways, some that require DCR-1 and some that do not. Alternatively, the RNAi defects of dcr-1(-/-) animals may be partially rescued by maternal dicer (mRNA or protein) that persists in the F₁ progeny. However, if this is the case, the maternal DCR-1 must not be available, or sufficient, to rescue all RNAi (Knight, 2001).

To begin to understand the role of dcr-1 in germ line development, germ line morphology was compared in wild-type and dcr-1(-/-) adult hermaphrodites using differential interference contrast microscopy and DAPI epifluorescence staining. Normally, in adult wild-type animals, the germ line develops in a defined and largely invariant manner. Moving from the distal region proximally, germ cells proliferate, enter meiosis, and differentiate into oocytes in the loop region and proximal gonad. Oocytes are fertilized as they are pulled through the spermatheca into the uterus. In dcr-1(-/-) animals, no gross defects were observed in chromosome morphology in the distal region of the gonad; however, several defects are seen in the proximal region. Elongated oocytes are found even before the loop of the gonad. Furthermore, as they migrate proximally, oocytes appear misshapen, lack clear delineation, and remain unfertilized. Nuclei in proximal oocytes also appear abnormal, often appearing nonspherical. DAPI staining reveals areas of intense staining in enlarged proximal nuclei suggestive of DNA replication without cell division (endomitosis). Vulval bursting was observed in many, but not all, dcr-1(-/-) animals. The burst vulva phenotype is also observed in animals containing a mutation in let-7 (see Drosophila microRNA encoding gene let-7), which encodes a 21- to 22-nt RNA important for developmental timing. Because the mature let-7 RNA is similar to the size of putative Dicer products and is thought to be processed from a base-paired hairpin, the burst vulva phenotype may indicate that let-7 processing is defective in the dcr-1 mutants. Taken together, the phenotypes of dcr-1(-/-) animals indicate that DCR-1 has multiple and important roles in vivo (Knight, 2001).

The C. elegans Dicer homolog plays a role in temporal development

RNAi is a gene-silencing phenomenon triggered by double-stranded (ds) RNA and involves the generation of 21 to 26 nt RNA segments that guide mRNA destruction. In Caenorhabditis elegans, lin-4 and let-7 encode small temporal RNAs (stRNAs) of 22 nt that regulate stage-specific development. Inactivation of genes related to RNAi pathway genes, a homolog of Drosophila Dicer (dcr-1), and two homologs of rde-1 (alg-1 and alg-2), cause heterochronic phenotypes similar to lin-4 and let-7 mutations. dcr-1, alg-1, and alg-2 are necessary for the maturation and activity of the lin-4 and let-7 stRNAs. These findings suggest that a common processing machinery generates guide RNAs that mediate both RNAi and endogenous gene regulation (Grishok, 2001).

Genetic studies in C. elegans have identified several genes essential for RNA interference. Probable null mutations in rde-1 (for RNAi defective) cause a complete lack of RNAi but no other discernible phenotypes. rde-1 encodes a 1020 amino acid protein that is a member of a large family of proteins found in a wide range of eukaryotes. Members of the RDE-1 family have two conserved domains of unknown biochemical function. The 300 amino acid PIWI domain located in the C-terminal region of these homologs shows the highest degree of sequence conservation. The 110 amino acid PAZ domain is located N-terminal to the PIWI domain and is also found in the Dicer family of proteins. RDE-1 homologs in the fungus, Neurospora, and the plant, Arabidopsis, have also been implicated in PTGS (post-transcriptional gene silencing) mechanisms suggesting that RDE-1 family members not only share conserved structures but also have conserved functions in gene silencing in three kingdoms of eukaryotic organisms (Grishok, 2001 and references therein).

Mutations in rde-1 homologs have also been shown to have developmental consequences. For example, in Drosophila, the ago1 gene is required for embryogenesis (Kataoka, 2001), the piwi gene is required for the maintenance of the germline stem cell population, and aubergine is required for the proper expression of the germline determinant Oskar (Wilson, 1996). Additionally, aubergine (also known as Sting) has been implicated in the PTGS-like suppression of the repetitive Stellate locus in the Drosophila germline (Schmidt, 1999). In Arabidopsis two very similar genes, argonaute (ago1) and pinhead/zwille, are required for stem cell patterning of the plant meristem. argonaute is also necessary for PTGS in Arabidopsis. The C. elegans genome contains 23 homologs of rde-1 including orthologs of both piwi and ago1. Previous studies have shown that the C. elegans piwi and ago1 orthologs have germline and possibly additional developmental functions. The pleiotropic nature of the defects associated with loss-of-function mutations in members of this family could reflect discrete regulatory functions in numerous developmental events or alternatively might reflect a more general misregulation of silencing mechanisms that are necessary to insure proper stem cell maintenance and differentiation (Grishok, 2001 and references therein).

cDNA clones for 14 rde-1 homologs were tested for developmental functions by RNAi. dsRNAs derived from two closely related genes, F48F7.1 and T07D3.7, which have been named alg-1 and alg-2 (for argonaute like genes), induce developmental phenotypes in the progeny of injected animals, including a tendency to burst at the vulva, and a lack of the adult specific alae, longitudinal stripes that run the length of the cuticle on both sides of the adult animal. In addition these dsRNAs induce incompletely penetrant slow growth and germline abnormalities. The other 12 genes assayed did not exhibit discernable developmental phenotypes (Grishok, 2001).

The alg-1 and alg-2 DNA sequences are 80% identical at the nucleotide level, suggesting a recent duplication of these genes, although they map to distinct chromosomes. This level of similarity is within the range where partial cross-interference is expected in RNAi assays. To target only alg-1 or alg-2, dsRNAs were prepared from short 5' unique segments of each gene. The dsRNA prepared from the unique segment of alg-1 produces the same vulval bursting phenotype, although at a reduced frequency relative to that observed with longer dsRNAs. No RNAi phenotype was observed after injections of the unique segment of alg-2 (Grishok, 2001).

A deletion allele of alg-2 was obtained from the C. elegans gene knockout consortium. This allele, alg-2(ok304), is an out-of-frame deletion that removes the nucleotides encoding amino acids 34-374, including the PAZ domain, and terminates after encoding 8 additional amino acids from reading frame two. It is therefore likely to be a null allele of alg-2. The RNAi experiments suggest that alg-2 may be a nonessential gene, and consistent with this finding the alg-2(ok304) homozygotes are viable and show, at most, subtle defects in fertility and development (Grishok, 2001).

It was next asked if alg-1 and alg-2 might have overlapping functions; dsRNAs prepared from both genes were coinjected and alg-1 dsRNA was injected into alg-2(ok304) homozygotes. Consistent with a shared function, coinjection of alg-1 and alg-2 dsRNAs causes enhanced larval lethality and also induces an embryonic lethal phenotype. Injection of alg-1 dsRNA into alg-2(ok304) homozygous animals results in a fully penetrant embryonic lethal phenotype identical to that observed in the double RNAi experiment. No such synergy was observed when alg-1 dsRNA was injected with dsRNAs prepared from other rde-1 family members. These findings indicate that alg-1 and alg-2 have overlapping functions in both embryogenesis and larval development. Efficient induction of the larval developmental phenotypes require the injection of full-length alg-1 dsRNA, a procedure that appears to partially inhibit alg-2. Therefore, animals produced in such experiments are referred to as 'alg-1/alg-2' RNAi animals (Grishok, 2001).

Finally, alg-1 and alg-2 were assayed for possible roles in RNAi. The alg-2(ok304) homozygotes are fully sensitive to RNAi, and likewise the inhibition of alg-1 or alg-2 by RNAi does not suppress RNAi targeting a second gene. These findings suggest that alg-1 and alg-2 are not necessary for RNAi. Nevertheless, it remains possible that these genes might have some redundant function in RNAi with rde-1 or with other members of this gene family (Grishok, 2001).

The C. elegans gene K12H4.8, which has been named dcr-1, is predicted to encode a protein related to the Drosophila Dicer (Bernstein, 2001) and the Arabidopsis Carpel Factory (Jacobsen, 1999) proteins implicated in RNAi and regulation of development, respectively. A previous study has shown that RNA interference of Drosophila Dicer can induce a partial loss of RNAi (Bernstein, 2001). RNAi of C. elegans dcr-1 was used to assess its role in developmental control and RNA interference. dcr-1(RNAi) induces developmental abnormalities during larval growth that are very similar to those induced by alg-1/alg-2(RNAi). These include a protruding and non-functional vulva, and a tendency to burst at the vulva shortly after the molt from the larval to the adult stage. In addition, dcr-1(RNAi) animals frequently exhibited faint or missing adult-specific alae (Grishok, 2001).

Although the phenotypes induced by dcr-1(RNAi) were similar to those induced by alg-1/alg-2(RNAi), dcr-1(RNAi) phenotypes were less penetrant. For example, 91% of the alg-1/alg-2(RNAi) animals lacked the adult-specific alae while only 19% of the dcr-1(RNAi) animals completely lacked the alae. This finding could indicate that dcr-1 has only a relatively minor role in the specification of the alae; alternatively, it might reflect a difficulty in inhibiting dcr-1 function via RNAi. For example, if dcr-1 is required for RNAi in C. elegans as it appears to be in Drosophila, then the use of RNAi to target dcr-1 may, at best, diminish its activity (Grishok, 2001).

The dcr-1(RNAi) phenotype was compared to the phenotype of animals homozygous for mutations in dcr-1. Three noncomplementing mutant strains were obtained that define the dcr-1 locus. Two of these, let-740(s2624) and let-740(s2795), were identified in an extensive genetic screen for mutations balanced by the free duplication sDp3. The third allele, dcr-1(ok247), was made by the C. elegans gene knockout consortium. The let-740(s2624) and let-740(s2795) mutations result in premature stop codons while dcr-1(ok247) is an out-of-frame deletion allele removing residues 708 through 1321 and terminating after expression of 15 amino acid residues from intronic sequences. All of these lesions are likely to severely disrupt DCR-1 protein expression; the s2624 allele would encode a protein of only 59 amino acids lacking all of the recognizable functional motifs, while the latter two alleles would encode truncated proteins lacking the PAZ, RNase III, and dsRBP domains. All three mutant dcr-1 strains exhibit a similar, fully penetrant, sterile phenotype. Homozygous hermaphrodites produce germ cells, including both sperm and oocytes, but for unknown reasons fail to produce embryos. In addition, all three strains exhibit adult cuticle and vulval defects identical to the defects induced by dcr-1(RNAi), including a protruding vulva and occasional vulval bursting as well as faint or missing alae. Because the let-740 mutations are allelic to dcr-1(ok247), the more descriptive name, dcr-1, will be used to refer to this gene (Grishok, 2001).

The severity of the phenotypes observed in the dcr-1 homozygous mutants is dependent on the maternal genotype, suggesting that dcr-1(+) activity is provided maternally. If dcr-1(+) activity is provided maternally, then RNAi of dcr-1 into a dcr-1 heterozygous mother might be expected to enhance the cuticle defects or cause additional phenotypes in the homozygous mutant progeny of the injected animal. Consistent with this possibility, the homozygous mutant class of progeny from dcr-1 heterozygous mothers injected with dcr-1 dsRNA arrests as embryos at a developmental stage similar to that observed in the double RNAi targeting alg-1 and alg-2. These findings suggest that maternal dcr-1(+) activity rescues essential functions of dcr-1 in the homozygous embryos and larvae and that RNAi of dcr-1 depletes this maternal activity. Because RNAi of dcr-1 efficiently inhibits dcr-1 activities required for larval development without inducing sterility or embryonic lethality, dcr-1(RNAi) was used for the subsequent developmental studies (Grishok, 2001).

Finally, it was asked if homozygous dcr-1 mutants are sensitive to RNAi. The conceptually straightforward experiment of assaying RNAi in the complete absence of dcr-1 is, unfortunately, not feasible since dcr-1 is required for viability of the animal. The best experiments that can be done are to assay for sensitivity to RNAi in animals where dcr-1 activity has been decreased. dcr-1(ok247) homozygous animals were tested for sensitivity to dsRNA delivered by injection into their mother or directly into the homozyous L4 larvae. In both assays nearly normal levels of RNAi were observed. This observation could indicate that maternal dcr-1(+) activity can rescue RNAi in dcr-1 homozygous mutant progeny just as it appears to rescue the developmental and alae defects described above. Consistent with this idea, other RNAi pathway mutants including rde-1 and rde-4 homozygotes are strongly rescued by one maternal dose of rde(+) activity. Because dsRNA targeting dcr-1 induces strong larval developmental defects, it was next asked if dcr-1(RNAi) might sufficiently reduce dcr-1 activity to cause an RNAi-deficient phenotype. For this assay, dcr-1 dsRNA was injected into adult hermaphrodites and then assayed for sensitivity to RNAi targeting a second gene. In experiments targeting two different genes, a significant reduction of RNAi was observed among the progeny of dcr-1(RNAi) animals but not among control animals injected with unrelated dsRNAs. These results support the findings from Bernstein (2001) that implicate Drosophila Dicer in RNAi and suggest that DCR-1 may have a similar activity in C. elegans (Grishok, 2001).

The combination of vulval and adult cuticle maturation defects caused by RNAi of alg-1/alg-2 and dcr-1 is reminiscent of phenotypes resulting from mutations in the genes lin-4 and let-7. The lin-4 and let-7 genes promote transitions from earlier to later cell fates and, thus, mutations in these genes cause reiteration of cell divisions typical of earlier larval stages, a hallmark of genes that regulate developmental timing (such genes have been termed 'heterochronic genes'). For example, loss-of-function mutations in let-7 result in a failure of larval seam cells in the hypodermis to progress to the adult-specific program of terminal differentiation indicated by the production of the adult-specific alae -- instead, the cells repeat the late larval type of divisions. These reiterated divisions contribute to an unstable vulval structure and failure to form a cuticle with adult alae (Grishok, 2001).

The developmental defects in alg-1/alg-2 and dcr-1 RNAi animals also result from temporal misspecifications in the seam cell lineages. To aid in the observation of seam cell divisions, a transgenic strain was utilized that drives GFP expression specifically in the seam cell nuclei. Normally, the ten seam cells present at hatching divide to generate 16 cells during the second larval stage. Although these 16 cells divide at the succeeding third and fourth larval transitions, only one daughter cell maintains the seam cell fate, so that the total number of GFP-expressing seam cells in the adult is 16 (Grishok, 2001).

RNAi of either dcr-1 or alg-1/alg-2 results in adults with extra seam cells that arise from reiterated L2 type divisions. Most progeny of dcr-1 and alg-1/alg-2 dsRNA-injected parents had normal seam cell divisions until the L3 stage, when reiterations of L2 type divisions were common. Many animals showed mixed patterns of stage-specific divisions, a phenotype similar to that observed previously in heterochronic mutants (daf-12, for example). The number of seam cells observed in dcr-1(RNAi) adults ranged from 16 to 33, with an average of 21, and only 15% showed the normal number of 16 seam cells; alg-1/alg-2(RNAi) adults exhibited 18-36 seam cells with an average of 25. The dcr-1 and alg-1/alg-2 (RNAi) progeny also repeated L3 or L4 seam cell division programs into adulthood, when normally these cells would stop dividing and become terminally differentiated (Grishok, 2001).

Inappropriate seam cell division patterns were consistently observed in L3 through later stages in dcr-1(RNAi) and alg-1/alg-2(RNAi) animals. However, because of the likely incomplete RNAi of dcr-1 and the redundancy of alg-1 and alg-2, it was not possible to establish the precise point in larval development where these genes are first required. Additional support that these genes may act earlier in larval development comes from the seam cell division pattern displayed by the more strongly affected animals obtained by coinjecting dsRNAs targeting portions of both alg-1 and alg-2 . In these experiments, reiterations of L1-type divisions were observed, in addition to repetition of later stage patterns (Grishok, 2001).

The similarity of phenotypes described above to those of the heterochronic genes lin-4 and let-7 raised the possibility that alg-1, alg-2, and dcr-1 might act upstream of the lin-4 or let-7 stRNAs or might be necessary for their regulatory activities. The targets of lin-4 and let-7 include the lin-14 and lin-41 mRNAs. Genetic studies suggest that lin-4 and let-7 stRNAs directly regulate lin-14 and lin-41 through complementary sequences in their 3'UTRs. Because the retarded phenotypes of lin-4 and let-7 are caused in part by failure to downregulate their target genes, mutations in lin-14 and lin-41 partially suppress the lin-4 and let-7 mutant phenotypes. To determine if alg-1/alg-2 and dcr-1 RNAi animals exhibit a similar genetic relationship with lin-14 and lin-41 mutants, dsRNA injections were performed in lin-14 and lin-41 mutant backgrounds. Significant suppression of the RNAi-induced alg-1/alg-2 and dcr-1 heterochronic phenotypes was found, including alae and vulval defects, by the lin-14(n179) and lin-41(ma104) nonnull mutations. In addition, the penetrant germline phenotype associated with alg-1/alg-2(RNAi) was partially suppressed by the lin-41 and lin-14 mutations, but the synthetic lethal phenotype associated with double alg-1/alg-2(RNAi) was not suppressed. In control RNAi experiments, the lin-14 and lin-41 mutant strains were fully sensitive to RNAi. These findings are consistent with the idea that the retarded heterochronic phenotypes induced by alg-1/alg-2 and dcr-1(RNAi) are caused, at least in part, by misregulation of lin-14 and lin-41 (Grishok, 2001).

lin-4 and let-7 are expressed as longer, approximately 70 nt RNAs that are predicted to fold into structures containing regions of double-stranded RNA. Because Drosophila Dicer cleaves introduced dsRNAs into fragments of approximately 22 nt (Bernstein, 2001), it was hypothesized that the heterochronic phenotypes caused by dcr-1 (RNAi) may be due to a defect in the processing of the larger, potentially dsRNA, forms of lin-4 and let-7 into the 22 nt stRNAs. To test this idea progeny were collected from mothers subjected to dcr-1(RNAi) and Northern blot analyses were performed to monitor the size and abundance of the lin-4 and let-7 RNAs. Because alg-1/alg-2 (RNAi) causes a similar heterochronic phenotype but acts at an unknown step in the pathway, lin-4 and let-7 processing were also monitored in alg-1/alg-2 (RNAi) animals (Grishok, 2001).

Both dcr-1 and alg-1/alg-2(RNAi) animals exhibited a marked accumulation of the lin-4 long form at both L3-L4 and adult stages. The same RNA preparations from the dcr-1 or alg-1/alg-2 (RNAi) animals were probed for the expression of let-7. It was found that, as with lin-4, let-7 processing depends on dcr-1 activity but, in contrast, does not appear to depend on alg-1/alg-2 activity. lin-4 and let-7 stRNA processing were monitored in dcr-1(ok247) homozygotes and in animals specifically depleted for either alg-1 or alg-2. In this experiment RNAs prepared from each population were simultaneously probed for expression of lin-4 and let-7 RNA. As with dcr-1(RNAi), the ok247 homozygotes exhibit a significant accumulation of both lin-4 and let-7 long forms. A gene-specific dsRNA targeting alg-1 induces accumulation of the pre-lin-4 RNA but not pre-let-7, and similarly, alg-2(ok304) animals exhibits a slight accumulation of pre-lin-4 and little or no accumulation of pre-let-7 (Grishok, 2001).

The quantity of the short forms of the lin-4 and let-7 stRNAs consistently appeared to be reduced in RNA populations prepared from alg-1/alg-2(RNAi), dcr-1(RNAi), and dcr-1(ok247) animals, while control RNA populations prepared from animals undergoing RNAi of the cuticle collagen gene rol-6 exhibited normal levels of lin-4 and let-7 stRNAs. This apparent reduction in let-7 stRNA level was observed even in alg-1/alg-2(RNAi) populations where no significant accumulation of pre-let-7 was observed. These findings suggest that alg-1/alg-2 activities may be more important for the stability or function of let-7 stRNA than for its processing from the larger form. Alternatively, alg-1/alg-2 might also be involved in let-7 processing but the let-7 long form may be less stable, so that unprocessed let-7 does not accumulate in the absence of alg-1/alg-2 activity (Grishok, 2001).

Thus, the efficient processing of the lin-4 and let-7 stRNAs from larger precursors depends on the activity of DCR-1, a C. elegans homolog of the Drosophila multifunctional RNase III related protein, Dicer, that has been shown in Drosophila cell extracts to process dsRNA into siRNAs that can mediate RNAi (Bernstein, 2001). Further, alg-1 and alg-2, two homologs of the RNAi pathway gene rde-1, are required for efficient stRNA expression, and along with dcr-1 function to promote lin-4 and let-7 activities in temporal development. Thus, the expression of the tiny RNAs that mediate RNAi and developmental gene regulation appear to share a requirement for DCR-1 activity, while RDE-1 and its homologs provide parallel functions in these pathways. These findings are consistent with a model in which members of the RDE-1 and DCR-1 families act not only in gene silencing but also with naturally expressed dsRNAs to execute cellular and developmental gene regulatory events (Grishok, 2001).

Although there are compelling similarities between RNAi and developmental regulation by lin-4 and let-7 there are also several important differences. In RNAi, the dsRNAs utilized, typically contain long stretches of perfect base pairing. The stRNA precursors, however, are predicted to contain at most 6, for lin-4, and 13, for let-7, uninterrupted Watson-Crick base pairs. Whereas cleavage of the perfectly base-paired RNAs that initiate RNAi yields both sense and antisense, or potentially double-stranded siRNAs, only one strand of the lin-4 and let-7 stRNAs is detected. Thus, after generation of the mature stRNA, the remaining sequences must undergo rapid degradation (Grishok, 2001).

The RNAi and stRNA pathways also appear to induce distinct outcomes: RNA destruction versus translation inhibition. In RNAi the target mRNA is rapidly degraded. Although the RNase responsible for target RNA destruction is not yet known, it is thought that the antisense strand of the siRNA acts as a guide in mRNA destruction, by base-pairing with the target mRNA. The stRNAs also specifically downregulate the expression of their target genes. Although details of the mechanism by which stRNAs cause decreased expression are unknown, the regulation of lin-14 by lin-4 occurs at the translational level. Upon expression of lin-4 RNA, the levels of LIN-14 protein rapidly decline, but lin-14 mRNA levels remain constant and appear to remain associated with polyribosomes. Because let-7-mediated regulation of LIN-41 protein expression may only occur in a subset of cells, it is, as yet, unclear if the mRNA levels or polyribosome loading of this target is affected by the expression of let-7 RNA (Grishok, 2001).

The distinction between mRNA destruction by RNAi and inhibition of translation by the lin-4 regulatory RNA could reside in the target mRNA sequence or in the particular region of the mRNA targeted. Whereas siRNAs can target sequences anywhere in the mature mRNA, stRNAs pair with specific sites in the 3'UTRs of their target genes. And just as the precursors of the stRNAs have imperfect internal complementarity, the stRNAs contain imperfect complementarity to their target sequences. Imperfect pairing could permit access to RNA nucleotides by sequence-specific RNA binding proteins, or conversely, might reduce the affinity with which a nuclease could cleave the mRNA/stRNA hybrid. Alternatively, both siRNAs and stRNAs may induce similar modifications of their target mRNAs while flanking sequences provide for context dependent interactions that cause inhibition of translation in the case of lin-14 but promote destruction of other mRNAs (Grishok, 2001).

There are 24 members of the RDE-1/AGO1/PIWI family in C. elegans. The degree of conservation between certain members of this family is striking. For example, ALG-1 and ALG-2 exhibit 41% identity with AGO1 from Arabidopsis and 67%-69% identity with AGO1 relatives in animals. The common ancestor of worms and humans appears to have had both an AGO1 ortholog and a second, already-divergent family member that has given rise to the PIWI family of genes. The fact that divergent members of this family, including rde-1, qde-2, and ago-1, all function in gene silencing suggests that PTGS mechanisms represent an important ancestral function of genes within this family (Grishok, 2001).

Developmental functions have also been reported for members of the piwi and ago1 families in both animals and plants. One feature that emerges from studies of these developmental phenotypes is that many of these genes appear to regulate germ cell and stem cell functions. Perhaps germ cells and stem cells have developed PTGS mechanisms for suppressing viral and transposon pathogens that might otherwise degrade the genome and, thus, the totipotency of these cells. The developmental phenotypes associated with mutations in members of the rde-1 gene family could thus reflect a general loss of gene silencing important for stem cell maintenance or differentiation. However, the findings reported in this study suggest an alternative possibility. rde-1-related genes, alg-1 and alg-2, function with natural small RNA cofactors in specific developmental gene regulation events. Thus, it is speculated that the Drosophila genes piwi, aubergine, and ago1, the Arabidopsis gene ago1, and perhaps many other members of this family in C. elegans and other organisms may similarly have small endogenous RNA cofactors with which they function to regulate specific target mRNAs (Grishok, 2001).

While there are 24 members of the rde-1/Argonaute gene family in C. elegans, there are fewer in Arabidopsis, humans, and Drosophila. Only the Piwi and Argonaute subtypes are conserved in many species, while RDE-1 as well as most of the other C. elegans family members are more divergent. Perhaps the family of tiny RNAs that may act with these proteins has also undergone expansion in C. elegans. Whether the ancestral function of RDE-1-related genes was in developmental control or sequence-directed immunity, it is clear that a great potential exists for exploiting these proteins, along with small RNAs as guides, to direct the regulation of specific gene targets in the cell (Grishok, 2001).

Previous work has indicated that RDE-1 plays an upstream role in the initiation of interference in response to dsRNA in C. elegans. Findings described in this study suggest that ALG-1 and ALG-2 may play a similar upstream role in the lin-4 and let-7 stRNA pathways. Thus, distinct members of the extended family of RDE-1 homologs in C. elegans may play specific roles in RNAi and stRNA pathways. It is speculated that one or more of the other C. elegans RDE-1 family members may provide a similar function in cosuppression in C. elegans. One attractive possibility is that these diversified factors provide specificity to their respective pathways. This might involve a role in the recognition of the distinct trigger sequences or in insuring that the processed small RNAs are assembled into distinct downstream complexes. Perhaps members of the RDE-1 family remain associated with the RNA sequences throughout processing and provide specificity needed to ensure that the small RNAs produced are targeted to the appropriate downstream complex, for example, to mediate mRNA destruction versus translation inhibition (Grishok, 2001).

A role for RDE-1 family members in both small RNA production and targeting could explain why the inhibition of alg-1/alg-2 induces such a dramatic effect on lin-4 and let-7 function while at best reducing but not eliminating the processed stRNA. Similarly, recent studies of small RNA accumulation during RNAi suggest that rde-1 is not essential for small RNA production after exposure to dsRNA and yet rde-1(+) activity is absolutely required for interference. Conceivably, dsRNA processing might still occur in the absence of RDE-1 or its homologs but the resulting siRNAs or stRNAs may not be assembled into the appropriate downstream complexes and therefore fail to function. Nevertheless, the finding that alg-1/alg-2(RNAi) dramatically affects the accumulation of the lin-4 precursor supports a role for these factors either upstream of, or at the same step as DCR-1 (Grishok, 2001).

This study shows that dcr-1 is an essential gene and is also required for RNAi in C. elegans. dcr-1, which appears to be a single copy gene in C. elegans, could play a role in dsRNA processing important in many gene silencing and developmental pathways. DCR-1 has several motifs that might be expected in a dsRNA processing enzyme, including a helicase, a dsRNA binding domain, and two RNase III type dsRNA exonuclease domains. Thus, it is proposed that DCR-1 functions in multiple pathways important for developmental and PTGS mechanisms, and may be guided in its processing of distinct substrates by members of the RDE-1 family. Consistent with a relatively specific role for dcr-1, it was found that mature ribosomal RNAs, which are also produced by RNase III type processing, accumulate to normal levels in animals with reduced dcr-1 activity (Grishok, 2001).

The combination of a maternally provided dcr-1 activity and zygotic sterility make it difficult to unambiguously answer the question of whether this protein is absolutely essential for RNAi and stRNA pathways. Nevertheless, the reiteration of L2 fates revealed by the seam cell lineage analysis of dcr-1(RNAi) animals, and the suppression of those phenotypes by mutations in lin-14 or lin-41 are unique phenotypic and genetic signatures that strongly support the model where lin-4 and let-7 processing is dependent on dcr-1(+) activity. Perhaps the embryonic and larval lethal phenotypes associated with dcr-1 inhibition and the developmental phenotypes associated with the Arabidopsis homolog, caf 1, reflect a role for members of this gene family in the processing of other as yet unidentified small regulatory RNAs. Thus, tiny RNAs may function in a broader range of gene regulatory and developmental events than the temporal transitions mediated by the founding members of the class, the lin-4 and let-7 stRNAs (Grishok, 2001).

Double-stranded RNAs can suppress expression of homologous genes through an evolutionarily conserved process named RNA interference (RNAi) or post-transcriptional gene silencing (PTGS). One mechanism underlying silencing is degradation of target mRNAs by an RNP complex, which contains ~22 nt of siRNAs as guides to substrate selection. A bidentate nuclease called Dicer has been implicated as the protein responsible for siRNA production. This study characterizes the C. elegans ortholog of Dicer (K12H4.8; dcr-1) in vivo and in vitro. dcr-1 mutants show a defect in RNAi. Furthermore, a combination of phenotypic abnormalities and RNA analysis suggests a role for dcr-1 in a regulatory pathway comprised of small temporal RNA (let-7) and its target (e.g., lin-41) (Ketting, 2001).

A signature feature of RNA interference and related gene silencing phenomena is the production of small, ~22-nt RNAs termed guide RNAs or siRNAs. These have been observed in plants undergoing cosuppression or virus-induced gene silencing and in C. elegans and Drosophila during RNA interference. In Drosophila, biochemical studies have indicated that siRNAs are produced by nucleolytic digestion of the dsRNA silencing trigger. To test whether siRNAs are produced by an evolutionarily conserved mechanism, a combination of biochemical and genetic approaches was undertaken (Ketting, 2001).

Extracts were prepared from C. elegans embryos and these were tested for Dicer activity, as evidenced by the ability to process long dsRNA into siRNAs. Such an activity is clearly detectable. A comparison of reactions performed in parallel shows that Drosophila and C. elegans extracts produce siRNA species of different sizes. In C. elegans, siRNAs produced in vitro are 23bp in length, consistent with the size of siRNAs found in vivo. Drosophila siRNAs produced in vitro are predominantly 21 bp in length and comigrate with siRNAs that are associated with the RISC enzyme in S₂ cells (Hammond, 2000). As was observed with Drosophila Dicer, longer dsRNAs are processed more efficiently. This correlates with the observation that long dsRNAs are more effective inducers of RNAi than are short dsRNAs (Ketting, 2001 and references therein).

The genome of C. elegans encodes a possible ortholog of the Drosophila Dicer protein, K12H4.8, which shares with Dicer a predicted domain structure comprising (from the N terminus to the C terminus) a helicase domain, a PAZ domain (Cerutti, 2000), dual RNAse III domains, and a double-stranded RNA-binding domain. A polyclonal antiserum was raised to the C terminus of K12H4.8. This antiserum specifically immunoprecipitates from C. elegans embryo extracts an activity that digests dsRNA into siRNAs. These results suggest that K12H4.8 is indeed the functional ortholog of Drosophila Dicer, and therefore this gene is referred to as dcr-1 (Ketting, 2001).

DCR-1, like Drosophila Dicer (Bernstein, 2001), requires ATP for efficient cleavage, and ATP hydrolysis further enhances siRNA production. It has been hypothesized that ATP hydrolysis by the helicase domain might drive a processive cleavage of dsRNA substrates by Dicer (Bernstein, 2001). A prediction of this model is that examination of reaction intermediates might reveal a ladder of products. Indeed, 500-bp dsRNA is shortened by DCR-1 in increments of ~23 nt. Reactions performed in extracts depleted of ATP produce only the first decrement of the ladder. Furthermore, when a partial dsRNA is offered as substrate, the reaction terminates at the point where the RNA becomes single-stranded. These results therefore suggest that DCR-1 converts dsRNA into siRNAs through a processive processing reaction, extracting energy for translocation along the dsRNA from ATP hydrolysis. This proposed mechanism is consistent with the observation that in vitro RNAi in Drosophila embryo extracts leads to cleavage of mRNA at characteristic ~22-nt intervals (Ketting, 2001).

To test the involvement of C. elegans Dicer in RNAi in vivo, a deletion mutant of dcr-1 was isolated. Thus far, screens for RNAi-resistant mutants have yielded viable and fertile mutants. Animals homozygous for the dcr-1 deletion are, however, sterile. Oocytes are abnormal, and no fertilized eggs were detected. These defects can be partially rescued by introduction of a transgene containing a wild-type copy of dcr-1. Fertilized eggs are formed in the presence of this transgene, but the eggs do not hatch, presumably because of loss of the transgene or limited expression of DCR-1 from the transgene in the germ line (Ketting, 2001).

To test whether dcr-1 animals are resistant to RNAi, these worms were fed dsRNA homologous to the unc-22 gene. Surprisingly, a wild-type RNAi response was observed. This might indicate that RNAi can proceed independently of Dicer activity. Alternatively, maternally contributed DCR-1 protein might be sufficient to generate an RNAi response. In fact, at least one other gene required for RNAi (mut-15) displays such a maternal effect. It was expected that maternally contributed Dicer protein would be absent from the germ line of adult dcr-1 homozygotes. It was therefore tested whether RNAi could be used to silence a GFP transgene that is expressed specifically in that tissue. Although RNAi is quite effective at silencing germ-line GFP in wild-type worms, dcr-1 homozygotes are RNAi-resistant, and it is concluded that Dicer is essential for RNAi in at least some tissues. The possibility that there is a second pathway that induces RNAi in the absence of Dicer cannot be excluded; however, the most parsimonious explanation is preferred: that the residual RNAi effects result from persistence of maternal Dicer protein (Ketting, 2001).

In addition to being sterile, dcr-1 homozygotes show a number of additional phenotypic abnormalities. Among these is a defect of the seam cells in the L4-to-adult transition. These cells fail to fuse, and in some cases undergo an additional round of cell division. As a result, the alae are absent in ~60% (38/62) of the dcr-1(pk1531) homozygous animals. Interestingly, this phenotype is also characteristic of loss-of-function mutations in the let-7 gene (Ketting, 2001).

The let-7 gene product (Drosophila homolog: let-7) is a small, noncoding RNA that regulates the timing of developmental events in C. elegans (therefore named small temporal RNA or stRNA. Of interest, the let-7 RNA is 21 nt in length, and it has been hypothesized that the let-7 RNA is produced by post-transcriptional processing of a longer precursor that is predicted to form an extended hairpin structure, which may be a substrate for DCR-1. Regulation by let-7 occurs at the translational level and presumably is mediated by complementary base-pairing between let-7 and the 3'-untranslated regions of target genes (Ketting, 2001 and references therein).

One of the in vivo targets of let-7 is lin-41 (Drosophila homolog: dappled), and the increased expression of this protein in let-7 mutants leads to the burst vulva phenotype. Interestingly, dcr-1 homozygous mutants also display a burst vulva phenotype, up to 80% (17/21), which can be rescued by introducing the wild-type dcr-1 gene. Tests were performed to see if this phenotype can be partially suppressed by down-regulating LIN-41 protein through RNAi, and indeed it can -- after RNAi of lin-41 only 25% burst vulva (5/20) are found. This suggests that the burst vulva phenotype in dcr-1 mutant animals is at least partially caused by an up-regulation of LIN-41, and the epistatic effect is an indication that dcr-1 and lin-41 indeed act in the same pathway. Conversely, hypomorphic alleles of lin-41 have an Egl phenotype (an egg-laying defect), whereas null alleles of lin-41 are sterile owing to the absence of oocytes. Accordingly, different levels of ectopic expression of DCR-1 might, via down-regulation of lin-41, induce an Egl phenotype or sterility. This is indeed what is found. Although the phenotypes described above are not specific enough to directly imply dcr-1 as an actor in the let-7/lin-41 pathway, the phenotypic relationship between animals with altered DCR-1 levels and animals with alterations in the let-7/lin-41 pathway, are suggestive (Ketting, 2001).

To test this more directly two approaches were undertaken (1) Using Drosophila embryo extracts and immunoprecipitates as a source of Dicer, tests were performed to see whether Dicer could process Drosophila let-7 precursor RNA into its mature form in vitro. Indeed, the ~75-nt hairpin was processed into an ~21-nt mature RNA with a disproportionately high efficiency as compared to perfect duplexes of similar size. (2) It was asked whether the dcr-1 mutation had an effect on the levels of let-7 RNA in vivo. Levels of mature let-7 RNA are reduced in dcr-1 mutant animals, and that this reduction is accompanied by an accumulation of the longer let-7 RNA precursor. Together these approaches show that dcr-1 is directly involved in the conversion of the double-stranded let-7 precursor RNA into the active, 21-nt species (Ketting, 2001).

RNAi and PTGS can clearly function to protect the genome against viruses and transposons. In addition, there is some evidence that factors involved in RNAi or PTGS also play a role in proper germ-line development. This study has shown that at least one component of the RNAi machinery in animals, Dicer, also plays a role in generating small RNAs involved in developmental timing (Ketting, 2001).

The mechanisms by which RNAi and stRNAs regulate the expression of target genes are quite distinct. In the former case, mRNAs are destroyed, whereas in the latter, expression is inhibited at the translational level. This raises the possibility that 22-nt RNAs produced by Dicer might act in multiple, distinct regulatory pathways that are not otherwise mechanistically related. Alternatively, the effector machinery may be shared by both processes, with an altered outcome of target recognition. The let-7 RNA is not perfectly homologous to its target substrates, and such a mismatch may inhibit the ability of RISC to cleave its substrates, effectively switching the mode of regulation from degradation to translational repression. It should be noted that let-7 is, most likely, not the only substrate for Dicer that is required for normal development. There may be many other endogenously encoded dsRNAs that are processed by Dicer to produce stRNA molecules, for example, lin-4. For this gene it has been shown that the mismatch between lin-4 and its target is critically required for proper regulation (Ketting, 2001 and references therein).

The 22-nt siRNAs that act in RNAi/PTGS have been found in multiple species. Dicer activity has been detected in extracts of plants and fungi (Nicotiana benthamiana, Neurospora crassa N. crassa and Phytophthora infestans). Thus far, stRNA genes (like let-7) have been identified in animals ranging from C. elegans to humans. Considered together, these observations suggest conserved roles for Dicer proteins in both dsRNA-induced silencing and in regulating developmental timing. Ironically, small temporal RNAs have yet to be identified in plants, in which developmental defects have been associated with mutants in the RNAi machinery. It will therefore be of interest to determine whether small temporal RNAs also regulate developmental timing in plants and to investigate whether this mode of gene regulation might also extend to nondevelopmental programs of gene expression (Ketting, 2001).

Dicer protein interactions

Double-stranded (ds) RNA induces potent gene silencing, termed RNA interference (RNAi). At an early step in RNAi, an RNaseIII-related enzyme, Dicer (DCR-1), processes long-trigger dsRNA into small interfering RNAs (siRNAs). DCR-1 is also required for processing endogenous regulatory RNAs called miRNAs, but how DCR-1 recognizes its endogenous and foreign substrates is not yet understood. The C. elegans RNAi pathway gene, rde-4, encodes a dsRNA binding protein that interacts during RNAi with RNA identical to the trigger dsRNA. RDE-4 protein also interacts in vivo with DCR-1, RDE-1, and a conserved DExH-box helicase. These findings suggest a model in which RDE-4 and RDE-1 function together to detect and retain foreign dsRNA and to present this dsRNA to DCR-1 for processing (Tabara, 2002).

RNA interference (RNAi) is the process of long, double-stranded (ds), RNA-dependent posttranscriptional gene silencing (PTGS). In lower eukaryotes, dsRNA introduced into the cytoplasm is cleaved by the RNaseIII-like enzyme, Dicer, to 21-23 nt RNA (short interfering [si] RNA), which may serve as guide for target mRNA degradation. In mammals, long-dsRNA-dependent PTGS is applicable only to a limited number of cell types, whereas siRNA synthesized in vitro is capable of effectively inducing gene silencing in a wide variety of cells. Although biochemical and genetic analyses in lower eukaryotes show that Dicer and some PIWI family member proteins are essential for long-dsRNA-dependent PTGS, little is known about the molecular mechanisms underlying siRNA-based PTGS. Dicer and eIF2C translation initiation factors belonging to the PIWI family (eIF2C1-4) play an essential role in mammalian siRNA-mediated PTGS, most probably through synergistic interactions. Immunoprecipitation experiments suggest that, in human and mouse cells, complex formation occurs between Dicer and eIF2C1 or 2 and that the PIWI domain of eIF2C is essential for the formation of this complex (Doi, 2003).

C. elegans Dicer interacts with the P-granule component GLH-1 and both regulate germline RNPs

P granules, ribonucleoprotein (RNP) complexes specific to the cytoplasmic side of the nuclear pores of C. elegans germ cells, are implicated in post-transcriptional control of maternally-transcribed mRNAs. This study shows a relationship in C. elegans of Dicer, the riboendonuclease processing enzyme of the RNA interference and microRNA pathways, with GLH-1, a germline-specific RNA helicase and a constitutive component of P granules. Based on results from GST-pull-downs and immunoprecipitations, GLH-1 binds DCR-1 and this binding does not require RNA. Both GLH-1 protein and glh-1 mRNA levels are reduced in the dcr-1^ok247) null mutant background; conversely, a reduction of DCR-1 protein is observed in the glh-1^gk100 deletion strain. Thus, in the C. elegans germline, DCR-1 and GLH-1 are interdependent. In addition, evidence indicates that DCR-1 protein levels, like those of GLH-1, are likely regulated by the Jun N-terminal kinase (JNK), KGB-1. In adult germ cells, DCR-1 is found in uniformly-distributed, small puncta both throughout the cytoplasm and the nucleus, on the inner side of nuclear pores, and associated with P granules. In arrested oocytes, GLH-1 and DCR-1 re-localize to cytoplasmic and cortically-distributed RNP granules and are necessary to recruit other components to these complexes. It is predicted that the GLH-1/DCR-1 complex may function in the transport, deposition, or regulation of maternally-transcribed mRNAs and their associated miRNAs (Beshore, 2010).

Dicer function as a component of RISC

RNA interference is implemented through the action of the RNA-induced silencing complex (RISC). Although Argonaute2 has been identified as the catalytic center of RISC, the RISC polypeptide composition and assembly using short interfering RNA (siRNA) duplexes has remained elusive. RISC is shown to be composed of Dicer, the double-stranded RNA binding protein TRBP, and Argonaute2. This complex can cleave target RNA using precursor microRNA (pre-miRNA) hairpin as the source of siRNA. Although RISC can also utilize duplex siRNA, it displays a nearly 10-fold greater activity using the pre-miRNA Dicer substrate. RISC distinguishes the guide strand of the siRNA from the passenger strand and specifically incorporates the guide strand. Importantly, ATP is not required for miRNA processing, RISC assembly, or multiple rounds of target-RNA cleavage. These results define the composition of RISC and demonstrate that miRNA processing and target-RNA cleavage are coupled (Gregory 2005).

This study shows that, although RISC could utilize the 22 nt duplex as the source of the siRNA, it displays far greater activity once a pre-miRNA, a substrate of Dicer, is used as the source of siRNA. These results strongly support the contention that Dicer cleavage activity is tightly coupled into the effector step of RNAi mediated by Ago2. The coupling of the two enzymatic activities makes ample biological sense since, once the duplex RNA is cleaved by Dicer, it could be unwound and handed over to Ago2 for target-RNA cleavage in a concerted reaction. The data showing a physical and functional coupling of pre-miRNA processing and RISC assembly also provide a mechanistic framework that explains the observations that 27 nt double-stranded RNAs or short hairpin RNAs, both of which are Dicer substrates, are considerably more potent triggers of RNAi than the short duplex siRNA. This study shows that, although RISC can utilize the 22 nt duplex as the source of the siRNA, it displays far greater activity once a pre-miRNA, a substrate of Dicer, is used as the source of siRNA. These results strongly support the contention that Dicer cleavage activity is tightly coupled into the effector step of RNAi mediated by Ago2. The coupling of the two enzymatic activities makes ample biological sense since, once the duplex RNA is cleaved by Dicer, it could be unwound and handed over to Ago2 for target-RNA cleavage in a concerted reaction. The data showing a physical and functional coupling of pre-miRNA processing and RISC assembly also provide a mechanistic framework that explains the observations that 27 nt double-stranded RNAs or short hairpin RNAs, both of which are Dicer substrates, are considerably more potent triggers of RNAi than the short duplex siRNA (Gregory, 2005)

MicroRNAs regulate brain morphogenesis in zebrafish

MicroRNAs (miRNAs) are small RNAs that regulate gene expression posttranscriptionally. To block all miRNA formation in zebrafish, maternal-zygotic dicer (MZdicer) mutants were generated that disrupt the Dicer ribonuclease III and double-stranded RNA-binding domains. Mutant embryos do not process precursor miRNAs into mature miRNAs, but injection of preprocessed miRNAs restores gene silencing, indicating that the disrupted domains are dispensable for later steps in silencing. MZdicer mutants undergo axis formation and differentiate multiple cell types but display abnormal morphogenesis during gastrulation, brain formation, somitogenesis, and heart development. Injection of miR-430 miRNAs rescues the brain defects in MZdicer mutants, revealing essential roles for miRNAs during morphogenesis (Giraldez, 2005).

MicroRNAs are evolutionarily conserved small non-protein-coding RNA gene products that regulate gene expression at the posttranscriptional level. In animals, mature miRNAs are ~22 nucleotides (nt) long and are generated from a primary transcript (termed pri-miRNA) through sequential processing by nucleases belonging to the ribonuclease III (RNaseIII) family. Initially, Drosha cleaves the pre-miRNA and excises a stem-loop precursor of ~70 nt (termed pre-miRNA), which is then cleaved by Dicer. One strand of the processed duplex is incorporated into a silencing complex and guides it to target sequences. This results in the cleavage of target mRNAs and/or the inhibition of their productive translation (Giraldez, 2005).

Several hundred vertebrate miRNAs and several thousand miRNA targets have been predicted or identified, but little is known about miRNA function during development. Clues to vertebrate miRNA function have come from several approaches, including expression analyses, computational prediction of miRNA targets, experimental support of predicted targets, and gain-of-function approaches. These studies have led to the suggestions that vertebrate miRNAs might be involved in processes such as stem cell maintenance or cell fate determination; however, no loss-of-function analysis has assigned a role for a particular miRNA or miRNA family in vivo, and it has been unclear how widespread the role of miRNAs is during vertebrate embryogenesis (Giraldez, 2005).

One approach to reveal the global role of vertebrate miRNAs is to abolish the generation of mature miRNAs with the use of dicer mutants. For example, dicer mutant embryonic stem cells fail to differentiate in vivo and in vitro, and dicer mutant mice die before axis formation, suggesting that mature miRNAs (or other Dicer products) are essential for early mammalian development. In zebrafish, maternal dicer activity has hampered the analysis of the single dicer gene. Mutants for the zygotic function of dicer (Zdicer) retain pre-miRNA processing activity up to 10 days postfertilization, presumably because of maternally contributed dicer. Zdicer mutants have no obvious defects other than a developmental delay at 7 to 10 days postfertilization, a stage when embryogenesis and major steps of organogenesis have been achieved. Hence, the global role of miRNAs during vertebrate embryogenesis is unknown. In light of these observations, zebrafish embryos were generated that lack both maternal and zygotic dicer activity (Giraldez, 2005).

Similar to other model systems, wild-type zebrafish embryos generate mature miRNAs from endogenous or exogenously provided pri-miRNAs, resulting in the post-transcriptional repression of reporter genes. miRNAs induce the cleavage of reporter RNAs with perfectly complementary target sites (PT) in the 3' untranslated region (3'UTR), whereas imperfectly complementary sites (IPT) result in the noneffective translation of reporter mRNAs. Previous biochemical and genetic studies have shown that Dicer is required for the generation of mature miRNAs. To determine whether MZdicer embryos lack mature miRNAs, total RNA from 1-day-old zebrafish embryos was hybridized to a microarray of probes for 120 different zebrafish mature miRNAs. Although such arrays are susceptible to cross-hybridization artifacts, a marked reduction was observed of signals in MZdicer mutants compared with wild-type embryos and zygotic dicer mutants. Of the 120 miRNA probes, 59, 35, and 9 gave a detectable signal in wild-type embryos, Zdicer mutants, and MZdicer mutants, respectively. To test for the presence of mature miRNAs more specifically, Northern blot analyses were performed. Of eight miRNAs present in wild-type embryos, none was detected in MZdicer mutants. These and other experiments suggested that mature miRNAs were not generated in MZdicer mutants (Giraldez, 2005).

The absence of mature miRNAs in MZdicer mutants allowed a determination of their global requirement during early zebrafish development. The MZdicer phenotype notably differs from that of Zdicer mutants, which are indistinguishable from wild-type embryos during these stages. Morphological analysis during the first 5 days of development revealed that axis formation and the regionalization of MZdicer mutants were intact. Major subregions and cell types were present, ranging from forebrain, eye, midbrain, hindbrain, ear, pigment cells, and spinal cord to hatching gland, heart, notochord, somites, and blood. In contrast, morphogenetic processes during gastrulation, somitogenesis, and heart and brain development were severely affected. MZdicer mutants also developed more slowly than wild-type embryos, with 3 to 4 hours of delay within the first 24 hours of development (Giraldez, 2005).

During zebrafish gastrulation, four concomitant cell rearrangements take place: (1) epiboly (spreading of the embryo over the yolk, (2) internalization (formation of mesodermal and endodermal germ layers, (3) convergence (movement of cells toward the dorsal side), and (4) extension (lengthening of the embryo). MZdicer mutants fail to coordinate epiboly and internalization. This results in mutant embryos that had undergone prechordal plate migration corresponding to 80% epiboly in wild-type embryos, yet epiboly movements are delayed to a stage equivalent to 50% to 60% epiboly. MZdicer embryos also display a reduced extension of the axis, resulting in a shortening of the embryo and an accumulation of cells in the head region. Later during development, MZdicer mutants have a reduced posterior yolk extension (Giraldez, 2005).

Neurulation is severely affected in MZdicer embryos. The mutant neural plate gives rise to the neural rod, but the subsequent formation of the neurocoel and neural tube is notably impaired. The formation of the brain ventricles is severely reduced. In wild-type embryos, several constrictions subdivide the brain into distinct regions. These constrictions do not form in MZdicer mutants. For example, the midbrain-hindbrain boundary that is very prominent in wild-type embryos does not form in MZdicer mutants. In addition, retinal development is affected. Defects in the spinal cord are manifested by a rudimentary neurocoel and a reduction of the floor plate in the trunk (Giraldez, 2005).

Despite the gross morphological malformations of the nervous system, gene expression analysis suggested that anterior-posterior and dorsal-ventral patterning are not severely disrupted. Analysis of anterior-posterior and dorsal-ventral markers revealed normal specification of the optic stalk, forebrain, midbrain-hindbrain boundary, otic vesicles, hindbrain rhombomeres, and the dorsal and ventral neural tube (Giraldez, 2005).

Analysis of neuronal differentiation and axonal markers, with the use of HuC and HNK antibodies, revealed mispositioned trigeminal sensory neurons adjacent to the eye. In addition, defasciculation of the postoptic commissure was observed in MZdicer embryos. In the hindbrain, multiple neurons project longitudinal axons anteriorly and posteriorly and form a ladder-like structure on each side of the midline. This scaffold is disrupted and defasciculated in MZdicer mutants, but longitudinal axonal projections are established. In addition, touch-induced escape behavior is severely diminished in MZdicer mutants. Taken together, these results indicate that early patterning and fate specification in the embryonic nervous system are largely unaffected by lack of miRNAs. In contrast, normal brain morphogenesis and neural differentiation and function require Dicer activity (Giraldez, 2005).

During somitogenesis, the paraxial mesoderm becomes segmented. MZdicer embryos formed normally spaced somites and express the muscle marker myoD similar to wild-type embryos. Later in development, the somites acquire a chevron shape in wild-type embryos but form irregular boundaries in MZdicer mutants. Endothelial and hematopoietic precursor cells are present as judged from the expression of the markers fli-1 and scl, respectively, but endocardial fli-1 expression is reduced and blood circulation disrupted in MZdicer mutants. Analysis of the markers pax2a, GFP-nanos-3'UTR, fkd1, cmlc2, and fkd2 revealed that pronephros, germ cells, endoderm, cardiomyocytes, and liver cells, respectively, are specified. MZdicer mutants have contractile cardiomyocytes but the two chambers characteristic of the wild-type heart do not form; instead, a tubular heart and pericardial edema developed (Giraldez, 2005).

Taken together, these results indicated that MZdicer mutant embryos are patterned correctly and have multiple specified cell types but underwent abnormal morphogenesis, in particular during neural development and organogenesis (Giraldez, 2005).

To identify miRNAs that might play important roles during early zebrafish development, small RNAs (~18 to 28 nt) were cloned from eight developmental stages between fertilization and 48 hours of development. These experiments identified miR-430a, miR-430b, and miR-430c as three highly expressed miRNAs, as well as several related species, miR-430d to miR-430h, which were expressed at lower levels. The miR-430 family members each had the same sequence at a segment encompassing nucleotides 2 to 8; this segment is known as the 'seed' and has been shown to be the miRNA segment most important for target recognition. The family members also have strong homology in their 3' region, but differ in their central and terminal nucleotides. Mapping of the miR-430 family to the zebrafish genome revealed a locus composed of multiple copies of the miR-430a,c,b triplet, with more than 90 copies of the miRNAs within 120 kb. miRNA genes are sometimes observed in clusters of about two to seven, which are frequently transcribed as a single polycistronic transcript, but the zebrafish miR-430 cluster has many more miRNAs than reported in other clusters. The miR-430 miRNAs are conserved and clustered in other fish genomes, including Fugu rubripes and Tetraodon nigroviridis. The miR-430 miRNAs belong to a superfamily that includes the vertebrate miR-17-miR-20 family, found in much smaller clusters in mammalian genomes. Despite the sequence similarities of the two families, members of the miR-17-miR-20 family derive from the opposite arm of their precursors; this suggests convergent rather than divergent origins of the two families. The miR-430 RNAs might share evolutionary origins with some of the miRNAs expressed specifically in mammalian embryonic stem cells, including miR-302 and miR-372, which have the same seed nucleotides and derive from the same arm of the hairpin (Giraldez, 2005).

The miR-430 miRNAs are initially expressed at about 50% epiboly [5 hours postfertilization (hpf)], continue to be expressed during gastrulation and somitogenesis, and then decline at about 48 hpf. Analysis of GFP sensors with perfect target sites for miR-430a or miR-430b suggested that the miR-430 miRNAs are ubiquitously expressed and active during early development (Giraldez, 2005).

miRNA duplexes are still active in MZdicer mutants. This allowed a determination of whether aspects of the MZdicer mutant phenotype can be suppressed by providing specific miRNAs that are normally expressed during early zebrafish development (miR-1, miR-204, miR-96, miR-203, miR-430a, miR-430b, or miR-430c). It was also reasoned that such rescue would unequivocally demonstrate that a particular phenotype is caused by the loss of a specific mature miRNA and not by the lack of small interfering RNAs (siRNAs) or the abnormal accumulation of pre-miRNAs in MZdicer mutants. It was found that injection of miR-430 duplexes (miR-430a, miR-430b, or miR-430c) rescues the brain morphogenesis defects in MZdicer mutants. This rescue is specific, as indicated by two control experiments. (1) Injection of unrelated miRNA duplexes did not cause any rescue. (2) Injection of a miRNA duplex with two point substitutions in the 5' seed did not rescue the MZdicer phenotype. Rescue of MZdicer mutant embryos by miR-430 (MZdicer^+miR-430) results in normal brain ventricles and brain constrictions. For example, the midbrain-hindbrain boundary forms in MZdicer^+miR-430 as in wild-type embryos. Injection of miR-430 also induces a substantial rescue of the neuronal defects observed in MZdicer mutants. MZdicer^+miR-430 also display partially rescued gastrulation, retinal development, somite formation, and touch response. In contrast, the defects in the development of the ear and heart and the lack of circulation were not rescued. Later during development (90 hpf), MZdicer^+miR-430 embryos are developmentally delayed and display reduced growth similar to MZdicer. These results indicate that loss of miR-430 miRNAs accounts for some but not all of the defects observed in MZdicer embryos (Giraldez, 2005).

This study of zebrafish that lack Dicer RNaseIII activity and mature miRNAs provides three major insights into the roles of miRNAs during embryogenesis. (1) The results suggest that mature miRNAs do not have widespread essential roles in fate specification or signaling during early zebrafish development. Phenotypic comparison between MZdicer mutants and embryos with aberrant signaling pathways (Nodal, Hedgehog, Wnt, Notch, CXCR4, FGF, BMP, retinoic acid, or STAT3) suggests that none of these pathways is markedly affected by the absence of miRNAs. For example, MZdicer mutants do not display the phenotypes seen upon an increase or decrease in Nodal or BMP signaling. This suggests that miRNAs might have modulating or tissue-specific rather than obligatory roles in various signaling pathways. Similarly, this study reveals that MZdicer mutants can differentiate multiple cell types during development. This suggests that mature miRNAs are not required to specify the major embryonic cell lineages in zebrafish. The results do not exclude more specific roles in fate specification, such as modulating the choice between highly related cell fates. For example, lsy-6 in Caenorhabditis elegans controls the distinction between two closely related neurons, and mouse miR-181 seems to regulate the ratio of cell types within the lymphocyte lineage. miRNAs might also function at later stages to stabilize and maintain a particular fate. For instance, miRNAs might repress large numbers of target mRNAs to maintain tissue homeostasis by dampening fluctuations in gene expression. However, the transplantation results argue against an absolute requirement for miRNAs in every cell type. In particular, fertile adults were generated from MZdicer mutant donors by germ cell transplantation. This indicates that primordial germ cells, the ultimate stem cells, proliferate and remain pluripotent to form the adult germ line in the absence of miRNAs. Multigeneration transplantation studies are required to determine if the lack of miRNAs has effects on germ cell maintenance. More exhaustive analysis of different cell types and signaling pathways is needed to test for more subtle or later roles of miRNAs in zebrafish, but the current study excludes a general role in signaling, embryonic fate specification, or germ line stem cell development (Giraldez, 2005).

(2) The results suggest important roles for miRNAs during embryonic morphogenesis and differentiation, ranging from epiboly and somitogenesis to heart, ear, and neural development. For example, loss of Dicer leads to defects in the positioning of neurons, the defasciculation of axons, and impaired touch-induced behaviors. Most notably, mutants form a neural rod but fail to generate normal brain ventricles. In addition, the morphological constrictions that subdivide the anterior-posterior axis do not form in the absence of Dicer, despite the regionalization observed by marker analysis. These results reveal essential roles of miRNAs during zebrafish morphogenesis (Giraldez, 2005).

(3) This study identified a previously unknown miRNA family, the absence of which is likely to account for the brain morphogenesis defects in MZdicer mutants. The miR-430 family has more genes than any miRNA family described to date, is conserved in fish, and is part of a superfamily found in other vertebrates. Injection of miR-430 duplexes suppresses the brain morphogenesis defects in MZdicer mutants. This complementation approach can now be applied to determine which miRNAs (or siRNAs) account for the MZdicer phenotypes that cannot be rescued by miR-430. The miR-430 family might inhibit mRNAs that are provided maternally or expressed during early embryogenesis but are detrimental to later steps in morphogenesis. Cell shape changes, cell rearrangements, and fluid dynamics are thought to generate both extrinsic and intrinsic forces that contribute to neural tube and ventricle formation, but the underlying molecular mechanisms are poorly understood. The study of the miR-430 family and its targets therefore provides a genetic entry point to dissect the molecular basis of brain morphogenesis (Giraldez, 2005).

Mammalian Dicer homologs

Members of the ribonuclease III superfamily of double-stranded(ds)-RNA-specific endoribonucleases participate in diverse cellular RNA maturation and degradation pathways. A recently identified eukaryotic RNase III family member, named Dicer, functions in the RNA interference (RNAi) pathway by producing 21-23 bp dsRNAs that target the selective destruction of homologous RNAs. RNAi is operative in animals, plants, and fungi, where it is proposed to inhibit viral reproduction and retroposon movement, as well as to participate in developmental pathways. RNAi functions in mammalian cells, including mouse oocytes and embryos. This article reports the cDNA sequence characterization and expression analysis of the mouse Dicer ortholog. On the basis of the cDNA sequence, the Dicer polypeptide is 1906 amino acids and has a predicted molecular mass of 215 kDa. Mouse Dicer contains a DExH/DEAH helicase motif; a PAZ domain; a tandem repeat of RNase III catalytic domain sequences, and a dsRNA-binding motif. The Dicer gene maps to a single locus on the distal portion of mouse chromosome 12. The Dicer transcript is expressed from the embryonic through adult stages of development. The Dicer transcript is also present in a wide variety of adult mouse organs. The highly conserved set of functional domains and the occurrence of a single-copy gene strongly indicate that the encoded protein is the RNase III ortholog responsible for dsRNA processing in the RNAi pathway (Nicholson, 2002).

Dicer1 functions as a haploinsufficient tumor suppressor

While the global down-regulation of microRNAs (miRNAs) is a common feature of human tumors, its genetic basis is largely undefined. To explore this question, the consequences of conditional Dicer1 mutation (Dicer1 'floxed' or Dicer1^fl) were analyzed on several mouse models of cancer. This study shows that Dicer1 functions as a haploinsufficient tumor suppressor gene. Deletion of a single copy of Dicer1 in tumors from Dicer1^fl/+ animals led to reduced survival compared with controls. These tumors exhibited impaired miRNA processing but failed to lose the wild-type Dicer1 allele. Moreover, tumors from Dicer1^fl/fl animals always maintained one functional Dicer1 allele. Consistent with selection against full loss of Dicer1 expression, enforced Dicer1 deletion caused inhibition of tumorigenesis. Analysis of human cancer genome copy number data reveals frequent deletion of DICER1. Importantly, however, the gene has not been reported to undergo homozygous deletion, suggesting that DICER1 is haploinsufficient in human cancer. These findings suggest Dicer1 may be an important haploinsufficient tumor suppressor gene and, furthermore, that other factors controlling miRNA biogenesis may also function in this manner (Kumar, 2009).

ADAR1 forms a complex with Dicer to promote microRNA processing and RNA-induced gene silencing

Adenosine deaminases acting on RNA (ADARs) are involved in RNA editing that converts adenosine residues to inosine specifically in double-stranded RNAs. This study investigated the interaction of the RNA editing mechanism with the RNA interference (RNAi) machinery and found that ADAR1 forms a complex with Dicer through direct protein-protein interaction. Most importantly, ADAR1 increases the maximum rate (Vmax) of pre-microRNA (miRNA) cleavage by Dicer and facilitates loading of miRNA onto RNA-induced silencing complexes, identifying a new role of ADAR1 in miRNA processing and RNAi mechanisms. ADAR1 differentiates its functions in RNA editing and RNAi by the formation of either ADAR1/ADAR1 homodimer or Dicer/ADAR1 heterodimer complexes, respectively. As expected, the expression of miRNAs is globally inhibited in ADAR1(-/-) mouse embryos, which, in turn, alters the expression of their target genes and might contribute to their embryonic lethal phenotype (Ota, 2013).

Dicer and heterochromatin

RNAi-mediated heterochromatin assembly in fission yeast requires the RNA-induced transcriptional silencing (RITS) complex and a putative RNA-directed RNA polymerase (Rdp1). Rdp1 is associated with two conserved proteins, Hrr1, an RNA helicase, and Cid12, a member of the polyA polymerase family, in a complex that has RNA-directed RNA polymerase activity (RDRC, RNA-directed RNA polymerase complex). RDRC physically interacts with RITS in a manner that requires the Dicer ribonuclease (Dcr1) and the Clr4 histone methyltransferase. Moreover, both complexes are localized to the nucleus and associate with noncoding centromeric RNAs in a Dcr1-dependent manner. In cells lacking Rdp1, Hrr1, or Cid12, RITS complexes are devoid of siRNAs and fail to localize to centromeric DNA repeats to initiate heterochromatin assembly. These findings reveal a physical and functional link between Rdp1 and RITS and suggest that noncoding RNAs provide a platform for siRNA-dependent localization of RNAi complexes to specific chromosome regions (Motamedi, 2004).

RNA interference is a conserved mechanism by which double-stranded RNA is processed into short interfering RNAs (siRNAs) that can trigger both post-transcriptional and transcriptional gene silencing. In fission yeast, the RNA-induced initiation of transcriptional gene silencing (RITS) complex contains Dicer-generated siRNAs and is required for heterochromatic silencing. RITS components, including Argonaute protein, bind to all known heterochromatic loci. At the mating-type region, RITS is recruited to the centromere-homologous repeat cenH in a Dicer-dependent manner, whereas the spreading of RITS across the entire 20-kb silenced domain, as well as its subsequent maintenance, requires heterochromatin machinery including Swi6 and occurs even in the absence of Dicer. Furthermore, these analyses suggest that RNA interference machinery operates in cis as a stable component of heterochromatic domains with RITS tethered to silenced loci by methylation of histone H3 at Lys9. This tethering promotes the processing of transcripts and generation of additional siRNAs for heterochromatin maintenance (Noma, 2004).

RNA interference (RNAi) is a widespread silencing mechanism that acts at both the posttranscriptional and transcriptional levels. This study describes the purification of an RNAi effector complex termed RITS (RNA-induced initiation of transcriptional gene silencing) that is required for heterochromatin assembly in fission yeast. The RITS complex contains Ago1 (the fission yeast Argonaute homolog), Chp1 (a heterochromatin-associated chromodomain protein), and Tas3 (a novel protein). In addition, the complex contains small RNAs that require the Dicer ribonuclease for their production. These small RNAs are homologous to centromeric repeats and are required for the localization of RITS to heterochromatic domains. The results suggest a mechanism for the role of the RNAi machinery and small RNAs in targeting of heterochromatin complexes and epigenetic gene silencing at specific chromosomal loci (Verdel, 2004).

In fission yeast, factors involved in the RNA interference (RNAi) pathway including Argonaute, Dicer, and RNA-dependent RNA polymerase are required for heterochromatin assembly at centromeric repeats and the silent mating-type region. RNA-induced initiation of transcriptional gene silencing (RITS) complex containing the Argonaute protein and small interfering RNAs (siRNAs) localizes to heterochromatic loci and collaborates with heterochromatin assembly factors via a self-enforcing RNAi loop mechanism to couple siRNA generation with heterochromatin formation. The role were investigated of RNA-dependent RNA polymerase (Rdp1) and its polymerase activity in the assembly of heterochromatin. Rdp1, similar to RITS, localizes to all known heterochromatic loci, and its localization at centromeric repeats depends on components of RITS and Dicer as well as heterochromatin assembly factors including Clr4/Suv39h and Swi6/HP1 proteins. A point mutation within the catalytic domain of Rdp1 abolishes its RNA-dependent RNA polymerase activity and results in the loss of transcriptional silencing and heterochromatin at centromeres, together with defects in mitotic chromosome segregation and telomere clustering. Moreover, the RITS complex in the rdp1 mutant does not contain siRNAs, and is delocalized from centromeres. These results not only implicate Rdp1 as an essential component of a self-enforcing RNAi loop but also ascribe a critical role for its RNA-dependent RNA polymerase activity in siRNA production necessary for heterochromatin formation (Sugiyama, 2005).

Heterochromatin formation depends on the RNAi machinery in S. pombe, Tetrahymena, and Drosophila, and links between DNA methylation and RNAi have been reported in plants. However, the original premise was that the RNAi pathway is mainly involved in PTGS. When the mechanism of RNAi was initially reported in C. elegans, it was noted that promoter and intronic sequences were ineffective in dsRNA-mediated gene silencing, thus reasoning that this process occurred post-transcriptionally. While there are examples of RNA-guided chromatin modifications in various organisms, they have, to date, been considered an oddity of the relevant host organism and not a general mechanism of epigenetic control. Evidence is reported in this study of a link between the RNAi pathway and DNA methylation of an endogenous sequence in animals, raising the possibility that RNA-based transcriptional gene silencing is a general event in higher eukaryotic gene regulation (Kanellopoulou, 2005).

Specifically, the data suggest that ablation of Dicer, a central molecule in the RNAi pathway, leads to derepression of normally silenced genetic elements, such as transposons and centromeric heterochromatin. An RNA component seems to be involved in heterochromatin formation in mammalian cells, but the specific nature of this RNA component and its possible link to the RNAi machinery have not been established. The present experiments directly address this issue. Centromeric repeat sequence-derived transcripts are up-regulated in DCR minus cells, suggesting that these regions escape transcriptional gene silencing in the absence of Dicer. However, Northern blot analyses have shown that Dicer extensively processes the largely double-stranded RNA derived from centromeric repeats into smaller RNAs, ranging from 25 to 150 nt. While the apparent lack of centromeric region silencing might reflect failure of DCR minus cells to efficiently convert primary transcripts into smaller dsRNA species, the loss of heterochromatin-related modifications in the centromeric regions of the mutants suggests another possibility. Conceivably, the 25-30-nt species, which are absent in the Dicer-deficient cells, could be analogous to small, centromeric heterochromatin-encoded RNAs cloned from S. pombe. If so, one might hypothesize that these RNAs are incorporated into a mammalian RITS complex and function as guides for TGS of homologous genomic sequences (Kanellopoulou, 2005).

Critical roles for Dicer in the female germline

Dicer is an essential component of RNA interference (RNAi) pathways, which have broad functions in gene regulation and genome organization. Probing the consequences of tissue-restricted Dicer loss in mice indicates a critical role for Dicer during meiosis in the female germline. Mouse oocytes lacking Dicer arrest in meiosis I with multiple disorganized spindles and severe chromosome congression defects. Oogenesis and early development are times of significant post-transcriptional regulation, with controlled mRNA storage, translation, and degradation. These results suggest that Dicer is essential for turnover of a substantial subset of maternal transcripts that are normally lost during oocyte maturation. Furthermore, evidence was found that transposon-derived sequence elements may contribute to the metabolism of maternal transcripts through a Dicer-dependent pathway. These studies identify Dicer as central to a regulatory network that controls oocyte gene expression programs and that promotes genomic integrity in a cell type notoriously susceptible to aneuploidy (Murchison, 2007).

Dicer in embryonic stem cells

Dicer is the enzyme that cleaves double-stranded RNA (dsRNA) into 21-25-nt-long species responsible for sequence-specific RNA-induced gene silencing at the transcriptional, post-transcriptional, or translational level. The dicer-1 gene was disrupted in mouse embryonic stem (ES) cells by conditional gene targeting, and Dicer-null ES cells were generated. These cells are viable, despite being completely defective in RNA interference (RNAi) and the generation of microRNAs (miRNAs). However, the mutant ES cells display severe defects in differentiation both in vitro and in vivo. Epigenetic silencing of centromeric repeat sequences and the expression of homologous small dsRNAs are markedly reduced. Re-expression of Dicer in the knockout cells rescues these phenotypes. These data suggest that Dicer participates in multiple, fundamental biological processes in a mammalian organism, ranging from stem cell differentiation to the maintenance of centromeric heterochromatin structure and centromeric silencing (Kanellopoulou, 2005).

Canonical microRNAs (miRNAs) require two processing steps: the first by the Microprocessor, a complex of the dsRNA-binding protein DGCR8 and Drosha, and the second by a complex of TRBP and Dicer. dgcr8δ/δ mouse embryonic stem cells (mESCs) have less severe phenotypes than dicer1δ/δ mESCs, suggesting a physiological role for Microprocessor-independent, Dicer-dependent small RNAs. To identify these small RNAs with unusual biogenesis, high-throughput sequencing was performed from wild-type, dgcr8δ/δ, and dicer1δ/δ mESCs. Several of the resulting DGCR8-independent, Dicer-dependent RNAs were noncanonical miRNAs. These derived from mirtrons and a newly identified subclass of miRNA precursors, which appears to be the endogenous counterpart of shRNAs. These analyses also revealed endogenous siRNAs resulting from Dicer cleavage of long hairpins, the vast majority of which originated from one genomic locus with tandem, inverted short interspersed nuclear elements (SINEs). These results extend the known diversity of mammalian small RNA-generating pathways and show that mammalian siRNAs exist in cell types other than oocytes (Babiarz, 2008).

REFERENCES

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