In eukaryotic cells degradation of bulk mRNA in the 5' to 3' direction requires the consecutive action of the decapping complex (consisting of DCP1 and DCP2) and the 5' to 3' exonuclease XRN1. These enzymes are found in discrete cytoplasmic foci known as P-bodies or GW-bodies (because of the accumulation of the GW182 antigen). Proteins acting in other post-transcriptional processes have also been localized to P-bodies. These include SMG5, SMG7, and UPF1, which function in nonsense-mediated mRNA decay (NMD), and the Argonaute proteins, which are essential for RNA interference (RNAi) and the micro-RNA (miRNA) pathway. In addition, XRN1 is required for degradation of mRNAs targeted by NMD and RNAi. To investigate a possible interplay between P-bodies and these post-transcriptional, processes P-body or essential pathway components were depleted from Drosophila cells and the effects of these depletions were analyzed on the expression of reporter constructs, allowing specific monitoring of NMD, RNAi, or miRNA function. The RNA-binding protein GW182 (Gawky) and the DCP1:DCP2 decapping complex are required for miRNA-mediated gene silencing, uncovering a crucial role for P-body components in the miRNA pathway. This analysis also revealed that inhibition of one pathway by depletion of its key effectors does not prevent the functioning of the other pathways, suggesting a lack of interdependence in Drosophila (Rehwinkel, 2005).

In eukaryotic cells, bulk messenger RNA (mRNA) is degraded via two alternative pathways, each of which is initiated by the removal of the poly(A) tail by deadenylases. Following this first step, mRNAs can be degraded from their 3' ends by the exosome, a multimeric complex of 3' to 5' exonucleases. Alternatively, after deadenylation, the cap structure is removed by the DCP1:DCP2 decapping complex, and the mRNA is degraded by the major cytoplasmic 5' to 3' exonuclease XRN1 (Rehwinkel, 2005).

Proteins required for 5' to 3' mRNA degradation (e.g., DCP1, DCP2, and XRN1) colocalize in specialized cytoplasmic bodies or mRNA decay foci, also known as mRNA processing bodies (P-bodies) or GW-bodies, because of the accumulation of the RNA binding protein GW182 in these bodies. Additional components of P-bodies in yeast and/or human cells include the deadenylase Ccr4 (see Drosophila Twin), the cap binding protein eIF4E and its binding partner eIF4E-transporter (eIF4E-T), auxiliary decay factors such as the LSm1-7 complex, Pat1p/Mtr1p, and the putative RNA helicase Dhh1/rck/p54. Among these, human GW182, eIF4E-T, and Dhh1 are required for P-body formation, while the decapping enzymes and XRN1 are dispensable. In addition, mRNA decay intermediates, microRNA (miRNA) targets, and miRNAs have been localized to P-bodies, suggesting that these bodies are sites where translationally silenced mRNAs are stored before undergoing decay (Rehwinkel, 2005 and references therein).

Recently, proteins involved in other post-transcriptional processes have been localized to P-bodies in human cells. These include the proteins SMG5, SMG7, and UPF1 involved in the nonsense-mediated mRNA decay (NMD) pathway and the Argonaute (AGO) proteins that play essential roles in RNA silencing. Moreover, XRN1 is recruited by both the NMD and the RNA interference (RNAi) machineries to degrade targeted mRNAs, suggesting a possible link between NMD, RNAi, and P-bodies. NMD is an mRNA quality control (or surveillance) mechanism that degrades aberrant mRNAs having premature translation termination codons (PTCs), thereby preventing the synthesis of truncated and potentially harmful proteins. Core components of the NMD machinery include the proteins UPF1, UPF2, and UPF3, which form a complex whose function in NMD is conserved. The activity of UPF1 is regulated in multicellular organisms by additional proteins (i.e., SMG1, SMG5, SMG6, and SMG7) that are also required for NMD in all organisms in which orthologs have been characterized (Rehwinkel, 2005 and references therein).

In yeast and human cells, a major decay pathway for NMD substrates involves decapping and 5' to 3' degradation by XRN1. Although degradation of nonsense transcripts in Drosophila is initiated by endonucleolytic cleavage near the PTC, the resulting 3' decay intermediate is also degraded by XRN1. A molecular link between the NMD machinery and the decay enzymes localized in P-bodies is provided by SMG7 in human cells. Indeed, when overexpressed, human SMG7 localizes in P-bodies and recruits both UPF1 and SMG5 to these bodies, suggesting that NMD factors may reside at least transiently in P-bodies. RNA silencing pathways are evolutionarily conserved mechanisms that elicit decay or translational repression of mRNAs selected on the basis of complementarity with small interfering RNAs (siRNAs) or miRNAs, respectively. siRNAs are fully complementary to their targets and elicit mRNA degradation via the RNAi pathway. Animal miRNAs are only partially complementary to their targets and do not generally elicit decay, but repress translation instead (Rehwinkel, 2005 and references therein).

To perform their function, the siRNAs and miRNAs associate with the AGO proteins to form multimeric RNA-induced silencing complexes (RISC). Drosophila AGO1 mediates miRNA function, while AGO2 catalyzes the endonucleoytic cleavage of siRNA targets within the region complementary to the siRNA. Following this initial cleavage, the resulting 5' mRNA fragment is degraded by the exosome, while the 3' fragment is degraded by XRN1. The localization of AGO proteins in P-bodies in human cells provides a possible link between these bodies and silencing pathways (Rehwinkel, 2005 and references therein).

The NMD, the siRNA, and the miRNA pathways are therefore interlinked by the use of common decay enzymes and/or the coexistence of components of these pathways in P-bodies, suggesting a possible interdependence between these post-transcriptional mechanisms. Evidence for a link between NMD and RNAi has been reported in Caenorhabditis elegans where UPF1, SMG5, and SMG6 are required for persistence of RNAi, though not to initiate silencing. In contrast, UPF2, UPF3, and SMG1, which are also essential for NMD, are not required to maintain silencing, suggesting that UPF1, SMG5, and SMG6 may have evolved specialized functions in RNAi (Rehwinkel, 2005 and references therein).

This study investigates the interplay between NMD, RNAi, and the miRNA pathway using the Drosophila Schneider cell line 2 (S2 cells) expressing reporters allowing the monitoring of NMD, RNAi, or miRNA function. To this end, factors involved in NMD (UPF1, UPF2, UPF3, SMG1, SMG5, and SMG6), RNAi (AGO2), or the miRNA pathway (AGO1) were depleted and the effect on the expression of the reporters analyzed. These proteins showed a high degree of functional specificity. To determine the role of P-body components in these pathways the DCP1:DCP2 decapping complex, the decapping coactivators LSm1 and LSm3, the 5' to 3' exonuclease XRN1, GW182, and the Drosophila protein CG32016, which shares limited sequence homology with human eIF4E-T, were depleted. The results uncovered a crucial role for GW182 and the DCP1:DCP2 decapping complex in the miRNA pathway (Rehwinkel, 2005).

Components of the NMD, RNAi, and miRNA pathways exhibit functional specificity in Drosophila To investigate a potential role of components of RNA silencing pathways or of P-body components in NMD, use was made of cell lines expressing wild-type or PTC-containing reporter constructs in which the coding regions of the bacterial chloramphenicol acetyl transferase (CAT) or the Drosophila alcohol dehydrogenase (adh) genes were placed downstream of inducible or constitutive promoters. The PTCs were inserted at codon 72 and 83 of the CAT and adh open reading frames, respectively. P-body components and proteins involved in NMD, RNAi, or the miRNA pathway were depleted by treating the cells with double-stranded RNAs (dsRNAs) specific for the different factors. A dsRNA that targets green fluorescent protein (GFP) served as a control. The steady-state levels of the wild-type and PTC-containing mRNAs were analyzed by Northern blot and normalized to those of the endogenous rp49 mRNA (encoding ribosomal protein L32) (Rehwinkel, 2005).

Relative to the expression levels of the wild-type mRNAs, the levels of the corresponding PTC-containing transcripts are reduced because these transcripts are rapidly degraded via the NMD pathway. Depletion of UPF1 inhibits NMD, so the levels of the PTC-containing mRNAs are restored. Depletion of AGO1 or AGO2, both singly and in combination, does not interfere with the NMD pathway, although these depletions do inhibit siRNA- or miRNA-mediated gene silencing. The levels of the CAT wild-type transcript were not affected by the depletions. Similar results were obtained with the NMD reporter based on the adh gene. Together, these results indicate that inhibition of RNAi or of the miRNA pathway does not interfere with NMD. XRN1 is the only P-body component known to be required for degradation of decay intermediates arising from mRNAs undergoing NMD in Drosophila. Nevertheless, in cells depleted of XRN1 the NMD pathway is not inhibited, and only the 3' decay intermediate generated by endonucleolytic cleavage of the mRNA accumulates (Rehwinkel, 2005).

In contrast to XRN1, none of the P-body components tested, including GW182 and the DCP1:DCP2 decapping complex, affected NMD or the accumulation of the 3' decay intermediate. The lack of a significant effect of the depletion of the DCP1:DCP2 complex was confirmed using the adh reporter. The decapping enzymes are certainly involved in NMD in yeast and human cells because the major decay pathway for NMD substrates is initiated by decapping in these organisms (for review, see Conti, 2005). Thus, it is possible that the requirement for P-body components and/or P-body integrity in NMD varies across species (Rehwinkel, 2005).

Two different approaches were used to investigate the RNAi pathway. In one approach, a cell line constitutively expressing the wild-type Drosophila adh gene was treated with a dsRNA complementary to a central region of ~300 nucleotides (nt) of adh mRNA (adh dsRNA). This dsRNA elicits decay of the adh mRNA via the RNAi pathway. Cells were treated with dsRNAs targeting various factors in the presence or absence of adh dsRNA. The steady-state levels of the adh mRNA were analyzed by Northern blot and normalized to those of the rp49 mRNA. In cells treated with GFP dsRNA, the normalized levels of the adh transcript were reduced to 4% after addition of adh dsRNA, relative to the levels detected in the absence of adh dsRNA. In cells depleted of AGO2, a sixfold increase of adh mRNA levels was observed despite the presence of adh dsRNA. In contrast, when AGO1 was depleted, adh dsRNA could still trigger a reduction of adh mRNA levels, though a slight increase in transcript levels was observed. Similarly, depletion of UPF1 did not prevent silencing of adh expression by adh dsRNA. These results indicate that UPF1 is not required for RNAi in Drosophila. Additional NMD components (i.e., UPF2, UPF3, SMG1, SMG5, and SMG6) have been identified, but no SMG7 ortholog has been identified in Drosophila. No significant change was observed in the efficacy of RNAi under the conditions in which NMD was inhibited (Rehwinkel, 2005).

Similarly to the results reported for the NMD pathway, depletion of XRN1 leads to the accumulation of the 3' decay intermediate generated by endonucleolytic cleavage by RISC, while depletion of the DCP1:DCP2 decapping complex does not prevent RNAi or the degradation the 3' decay intermediate. In contrast, depletion of GW182 leads to a modest increase in the adh mRNA level in the presence of adh dsRNA, suggesting that this protein could influence the efficiency of RNAi (Rehwinkel, 2005).

In a second approach, RNAi was triggered by an siRNA instead of a long dsRNA, to uncouple RISC activity from processing of dsRNAs. To this end, S2 cells were transiently transfected with a plasmid expressing firefly luciferase (F-Luc) and an siRNA targeting the luciferase coding sequence (F-Luc siRNA) or a control siRNA. A plasmid encoding Renilla luciferase (RLuc) was included to normalize for transfection efficiencies. Cotransfection of the F-Luc reporter with the F-Luc siRNA led to a 50-fold inhibition of firefly luciferase activity relative to the activity measured when the control siRNA was cotransfected, indicating that F-Luc siRNA effectively silences firefly luciferase expression (Rehwinkel, 2005).

The results obtained with the luciferase reporter correlate well with those obtained with adh mRNA, in spite of differences between the methods used to detect changes in reporter levels (RNA levels vs. protein levels), and the nature of the RNA trigger (long dsRNA vs. siRNA). Indeed, depletion of AGO2 impaired silencing of firefly luciferase expression by the F-Luc siRNA, leading to an eightfold increase in firefly luciferase activity relative to the activity of the Renilla control. Depletion of AGO1 led to a twofold increase of firefly luciferase activity (Rehwinkel, 2005).

The observation that depletion of AGO2, but not AGO1, significantly inhibits RNAi is in agreement with previous reports showing that only AGO2-containing RISC is able to catalyze mRNA cleavage triggered by siRNAs. The results together with these observations indicate that Drosophila AGO1 and AGO2 are not redundant (Rehwinkel, 2005).

Depletion of GW182 or the DCP1:DCP2 complex led to a 1.5- to twofold increase of the firefly luciferase activity, although RNAi was not abolished. These results together with those obtained with the adh reporter suggest that GW182 and the DCP1:DCP2 complex are not absolutely required for RNAi but may modulate siRNA function (Rehwinkel, 2005).

Finally, depletion of core NMD components does not inhibit the silencing of firefly luciferase expression by F-Luc siRNA. The results are consistent with results from C. elegans showing that NMD per se is not required for the establishment of silencing (Rehwinkel, 2005).

To investigate the miRNA pathway firefly luciferase reporters were generated in which the coding region of firefly luciferase is flanked by the 3' UTRs of the Drosophila genes CG10011 or Vha68-1. These genes were identified as miRNA targets in a genome-wide analysis of mRNAs regulated by AGO1. The 3' UTR of CG10011 mRNA contains two binding sites for miR-12, while the 3’ UTR of Vha68-1 has two binding sites for miR-9b. Expression of the firefly luciferase construct fused to the 3' UTR of CG10011 (F-Luc-CG10011) was strongly reduced by cotransfection of a plasmid expressing the primary (pri) miR-12 transcript, but not pri-miR-9. Conversely, expression of the firefly luciferase reporter fused to the 3' UTR of Vha68-1 (FLuc-Vha68-1) was inhibited by cotransfection of pri-miR-9b, but not of primiR-12 (Rehwinkel, 2005).

Silencing of luciferase expression by the cognate miRNAs was prevented in cells depleted of AGO1. Indeed, despite the presence of the transfected miRNAs, in cells depleted of AGO1 an 11-fold and a 16-fold increase of firefly luciferase expression was observed from the FLuc- CG10011 and F-Luc-Vha68-1 reporters, respectively. Notably, the firefly luciferase activity measured in AGO1-depleted cells in the presence of the transfected miRNAs was at least twofold higher than the activity measured in control cells in the absence of exogenously added miRNAs. Since endogenous miR-9b and miR-12 are expressed in S2 cells, these results suggest that depletion of AGO1 also suppresses silencing mediated by the endogenous miRNAs. Depletion of AGO2 does not suppress the effect of coexpressing the reporters with the cognate miRNAs. These results provide additional evidence supporting the conclusion that the siRNA and miRNA pathways are not interdependent (Rehwinkel, 2005).

miRNA-mediated silencing of firefly luciferase expression was not affected by depletion of UPF1 or by the additional NMD factors (i.e., UPF2, UPF3, SMG1, SMG5, and SMG6). Thus, the individual NMD factors and NMD per se are not required for miRNA function. Unexpectedly, although the efficiency of NMD and RNAi was unaffected or only modestly affected in cells depleted of GW182 or the DCP1:DCP2 complex, miRNA-mediated silencing of firefly luciferase expression was effectively relieved in these cells. In the presence of cognate miRNAs, depletion of GW182 resulted in a sixfold increase of firefly luciferase expression. Therefore, despite the presence of transfected miRNAs, firefly luciferase activity in GW182-depleted cells was similar to that measured in controls cells in the absence of transfected miRNAs. Codepletion of DCP1 and DCP2 led to a fourfold increase of firefly luciferase expression. Finally, depletion of CG32016 resulted in a twofold increase of firefy luciferase activity, but only for the F-Luc-Vha68-1 reporter, suggesting that this effect may not be significant (Rehwinkel, 2005).

To investigate whether depletion of GW182 affects RISC activity directly, as opposed to interfering with miRNA processing, use was made of a tethering assay. This assay involves the expression of a lN-fusion of AGO1 that binds with high affinity to five BoxB sites (5-BoxB) in the 3’ UTR of a firefly luciferase reporter mRNA. When AGO1 is tethered to this reporter transcript, luciferase expression is inhibited relative to the activity measured in cells expressing the lN-peptide alone. The inhibition was partially relieved in cells depleted of GW182 but not of AGO2. It is concluded that GW182 and the decapping DCP1: DCP2 complex play a critical role in the effector step of the miRNA pathway. These results are in agreement with the observation that Argonaute proteins localize to P-bodies and interact with DCP1 and DCP2 independently of RNA or of P-body integrity (Rehwinkel, 2005).

Thus, despite convergence in P-bodies, NMD, RNAi, and the miRNA pathway are not interdependent in Drosophila. This conclusion is based on the observation that the inhibition of one pathway by depleting key effectors may slightly interfere with, but does not significantly inhibit, the functioning of the other pathways. The lack of interdependence between RNAi and the miRNA pathway is further supported by the observation that knockouts of AGO1 or AGO2 in Drosophila have different phenotypes. Nevertheless, cross-talk between the RNAi and the miRNA pathways may still occur at the initiation step, since Dicer-1 plays a role in RISC assembly (Rehwinkel, 2005).

Biochemical and genetic approaches in several organisms have led to the identification of essential components of the miRNA pathway. These include AGO1 and the enzymes required for miRNA processing, such as Drosha and Dicer-1 and their respective cofactors, Pasha and Loqs. However, the mechanisms by which miRNAs inhibit protein expression without affecting mRNA levels are not completely understood. Recent evidence suggests that translation initiation is inhibited and that the targeted mRNAs are stored in P-bodies, where they are maintained in a silenced state either by associating with proteins that prevent translation or possibly by removal of the cap structure. This study identified the P-body components GW182 and the DCP1:DCP2 decapping complex as proteins required for the miRNA pathway. The precise molecular mechanism by which these proteins participate in this pathway remains to be established. These proteins may have an indirect role in the miRNA pathway by affecting P-body integrity. Alternatively, these proteins may play a direct role in this pathway by escorting miRNA targets to P-bodies or facilitating mRNP remodeling steps required for the silencing of these targets. Consistent with a direct role for the DCP1:DCP2 decapping complex, and thus for the cap structure, in miRNA function is the observation that mRNAs translated via a cap-independent mechanism are not subject to miRNA-mediated silencing. In conclusion, the results uncover an important role for the P-body components, GW182 and the DCP1:DCP2 complex, in miRNA-mediated gene silencing (Rehwinkel, 2005).

MicroRNAs (miRNAs) silence the expression of target genes post-transcriptionally. Their function is mediated by the Argonaute proteins (AGOs), which colocalize to P-bodies with mRNA degradation enzymes. Mammalian P-bodies are also marked by the GW182 protein, which interacts with the AGOs and is required for miRNA function. Depletion of GW182 (Gawky) leads to changes in mRNA expression profiles strikingly similar to those observed in cells depleted of the essential Drosophila miRNA effector AGO1, indicating that GW182 functions in the miRNA pathway. When GW182 is bound to a reporter transcript, it silences its expression, bypassing the requirement for AGO1. Silencing by GW182 is effected by changes in protein expression and mRNA stability. Similarly, miRNAs silence gene expression by repressing protein expression and/or by promoting mRNA decay, and both mechanisms require GW182. mRNA degradation, but not translational repression, by GW182 or miRNAs is inhibited in cells depleted of CAF1, NOT1, or the decapping DCP1:DCP2 complex. The N-terminal GW repeats of GW182 interact with the PIWI domain of AGO1. These findings indicate that GW182 links the miRNA pathway to mRNA degradation by interacting with AGO1 and promoting decay of at least a subset of miRNA targets (Behm-Ansmant, 2006).

To accomplish their regulatory function miRNAs associate with the Argonaute proteins to form RNA-induced silencing complexes (RISCs), which elicit decay or translational repression of complementary mRNA targets. In plants, miRNAs are often fully complementary to their targets, and elicit mRNA decay. In contrast, animal miRNAs are only partially complementary to their targets, and silence gene expression by mechanisms that remain elusive. Recent studies have shown that miRNAs silence gene expression by inhibiting translation initiation at an early stage involving the cap structure; mRNAs translated via cap-independent mechanisms escape miRNA-mediated silencing. Other studies have suggested that translation inhibition occurs after initiation, based on the observation that miRNAs and some targets remain associated with polysomes. In addition, animal miRNAs can also induce significant degradation of mRNA targets despite imperfect mRNA-miRNA base-pairing (Behm-Ansmant, 2006 and references therein).

The existence of a link between the miRNA pathway and mRNA decay is supported by the observation that mammalian Argonaute proteins (AGO1-AGO4), miRNAs, and miRNA targets colocalize to cytoplasmic foci known as P-bodies. These mRNA processing bodies are discrete cytoplasmic domains where proteins required for bulk mRNA degradation in the 5'-to-3' direction accumulate (e.g., the decapping DCP1:DCP2 complex and the 5'-to-3' exonuclease XRN1). Additional components of P-bodies in yeast and/or human cells include the CCR4:NOT deadenylase complex, auxiliary decapping factors (e.g., the LSm1-7 complex and Pat1p/Mtr1p), the cap-binding protein eIF4E, and the RNA helicase Dhh1/Me31B involved in translational repression. In metazoa, P-bodies are also marked by the presence of GW182, a protein with glycine-tryptophan repeats (GW repeats) required for P-body integrity (Behm-Ansmant, 2006 and references therein).

The presence of Argonaute proteins, miRNAs, and miRNA targets in P-bodies has led to a model in which translationally silenced mRNAs are sequestered to these bodies, where they may undergo decay. At present, it is unclear whether the localization in P-bodies is the cause or consequence of the translational repression, though several lines of evidence point to a direct role for P-body components in miRNA-mediated gene silencing. First, DCP1, GW182, and its paralog TNRC6B associate with AGO1 and AGO2 in human cells. Second, depletion of GW182 in human cells impairs both miRNA function and mRNA decay triggered by complementary short interfering RNAs (siRNAs). Similarly, miRNA function is impaired in Drosophila Schneider cells (S2 cells) depleted of GW182 or the decapping DCP1:DCP2 complex (Rehwinkel, 2005). Finally, the Caenorhabditis elegans protein AIN-1, which is related to GW182, is required for gene regulation by at least a subset of miRNAs (Behm-Ansmant, 2006 and references therein).

In Drosophila, siRNA-guided endonucleolytic cleavage of mRNAs (RNA interference [RNAi]) is mediated by AGO2, while gene silencing by miRNAs is mediated by AGO1. That siRNAs and miRNAs enter separate pathways in Drosophila is further supported by the observation that depletion of GW182 inhibits miRNA-mediated, but not siRNA-mediated gene silencing (Rehwinkel, 2005). The precise role of GW182 in the miRNA pathway is unknown. GW182 could have an indirect role by affecting P-body integrity. Alternatively, it could be more directly involved, localizing miRNA targets to P-bodies or facilitating the mRNP remodeling steps required for the silencing and/or decay of these targets (Behm-Ansmant, 2006 and references therein).

This study investigates the role of Drosophila GW182 in the miRNA pathway. Depletion of GW182 leads to changes in mRNA expression profiles strikingly similar to those observed in cells depleted of AGO1, indicating that GW182 is a genuine component of the miRNA pathway. In cells in which miRNA-mediated gene silencing is suppressed by depletion of AGO1, GW182 can still silence the expression of bound mRNAs, suggesting that GW182 acts downstream of AGO1. It is further shown that GW182 triggers silencing of bound transcripts by inhibiting protein expression and promoting mRNA decay via a deadenylation and decapping mechanism. Finally, evidence is provided that mRNA degradation by miRNAs requires GW182, the CCR4:NOT deadenylase, and the DCP1:DCP2 decapping complexes. Together with the observation that GW182 interacts with AGO1, these results indicate that binding of GW182 to miRNA targets induces silencing and can trigger mRNA degradation, providing an explanation for the observed changes in mRNA levels, at least for a subset of animal miRNA targets (Behm-Ansmant, 2006).

These results indicate that GW182 is a genuine component of RNA silencing pathways, associating with the Argonaute proteins and with components of the mRNA decay machinery and, providing a molecular link between RNA silencing and mRNA degradation. Depletion of GW182 or AGO1 from Drosophila cells leads to correlated changes in mRNA expression profiles, indicating that these proteins act in the same pathway. Transcripts commonly up-regulated by AGO1 and GW182 are enriched in predicted and validated miRNA targets. These results, together with the observation that GW182 associates with AGO1, identify GW182 as a component of the miRNA pathway (Behm-Ansmant, 2006).

GW182 belongs to a protein family with GW repeats, a central UBA domain, and a C-terminal RRM. Multiple sequence alignment of all proteins possessing these domains revealed that there are three paralogs (TNRC6A/GW182, TNRC6B, and TNRC6C) in vertebrates, a single ortholog in insects, and no orthologs in worms or fungi. At present, it is unclear whether the vertebrate paralogs have redundant functions, but both GW182 and TNRC6B have been shown to associate with human AGO1 and AGO2 (Behm-Ansmant, 2006).

In Drosophila, GW182 interacts with AGO1 in vivo and in vitro. No stable interaction with AGO2 was detected under the same conditions, suggesting that AGO2 may act independently of GW182. This is consistent with the observation that depletion of GW182 does not affect siRNA-guided mRNA cleavage or RNAi, which is mediated exclusively by AGO2 in Drosophila. Nevertheless, since AGO2 also regulates the expression levels of a subset of miRNA targets (Rehwinkel, 2006), the lack of interaction with GW182 raises the question of whether this regulation occurs by a similar or different mechanism from that mediated by AGO1. Further studies are needed to elucidate the mechanism by which Drosophila AGO2 regulates the expression of a subset of miRNA targets (Behm-Ansmant, 2006).

The N-terminal GW repeat region of GW182 encompasses two highly conserved motifs (I and II) and is expanded in vertebrates. This region is shorter in insects and bears similarity to the GW-like regions in the C. elegans protein AIN-1, involved in the miRNA pathway. However, AIN-1 does not contain UBA, Q-rich, or RRM domains. This lack of common domain architecture suggests that AIN-1 represents a functional analog. Nevertheless, the observation that C. elegans AIN-1 also localizes to P-bodies and interacts with AGO1 (i.e., worm ALG-1), and the finding that the N-terminal GW repeats of Drosophila GW182 interact with the PIWI domain of AGO1, suggest a conserved role for these repeats in mediating the interaction with Argonaute proteins. It would be of interest to determine the molecular basis of the specific interaction between the N-terminal GW repeats of GW182 and the PIWI domain of AGOs, and whether this interaction affects the catalytical activity of the domain (Behm-Ansmant, 2006).

Apart from the interaction with AGO1, the N-terminal repeats and the UBA and Q-rich domains contribute to the localization of GW182 in P-bodies, which is in turn required for P-body integrity. This suggests that GW182 may act as a molecular scaffold bringing together AGO1-containing RISCs and mRNA decay enzymes, possibly nucleating the assembly of P-bodies. Understanding the precise role of the various GW182 domains in the interaction with mRNA decay enzymes and AGO1 as well as in P-body integrity awaits further biochemical characterization (Behm-Ansmant, 2006).

Tethering GW182 to a reporter transcript silences its expression, bypassing the requirement for AGO1. Silencing by GW182 occurs by two distinct mechanisms: repression of protein expression, and mRNA degradation. It remains to be elucidated how GW182 represses translation. mRNA degradation by GW182 is inhibited in cells depleted of CAF1, NOT1, or the DCP1:DCP2 complex, indicating that GW182 promotes mRNA deadenylation and decapping. Thus, binding of GW182 appears to be a point of no return, which marks transcripts as targets for degradation (Behm-Ansmant, 2006).

More studies are needed to determine whether decapping triggered by GW182 requires prior deadenylation or whether these two events occur independently. The observation that mRNA levels are fully restored in cells depleted of DCP1:DCP2, suggests that deadenylation followed by 3'-to-5' exonucleolytic degradation is unlikely to represent a major pathway by which these mRNAs are degraded. Future studies should also reveal the identity of the nuclease(s) acting downstream of the decapping enzymes (Behm-Ansmant, 2006).

Previous studies indicate that miRNAs can reduce the levels of the targeted transcripts, and not just the expression of the translated protein. Consistently, transcripts up-regulated in cells depleted of AGO1 or GW182 are enriched in predicted and validated miRNA targets. In this paper further evidence is provided indicating that miRNAs silence gene expression by two mechanisms: one mechanism involving translational silencing, and one involving mRNA degradation. The contribution of these mechanisms to miRNA-mediated gene silencing appears to differ for each miRNA:target pair. Indeed, of the three reporters analyzed, Nerfin is silenced mainly at the translational level, silencing of the CG10011 reporter can be attributed to mRNA degradation, while Vha68-1 is regulated both at the translational and mRNA levels. Regardless of the extent of the contribution of these two mechanisms to silencing, both require AGO1 and GW182, because the levels of the mRNA reporter and luciferase activity are restored in cells depleted of any of these two proteins (Behm-Ansmant, 2006).

In contrast, although the levels of the mRNA reporter are restored in cells depleted of CAF1 or NOT1, translational repression is not fully relieved, indicating that deadenylation is required for mRNA decay, but not for translational silencing by miRNAs. In agreement with this, two reports published while this manuscript was in preparation have shown that miRNAs trigger accelerated deadenylation of their targets (Giraldez, 2006; Wu, 2006). This study extends these observations further by demonstrating: 1) deadenylation is mediated by the CCR4:NOT complex; 2) decapping is also required for miRNA target degradation; and 3) both deadenylation and decapping triggered by miRNAs requires GW182 (Behm-Ansmant, 2006).

Based on the results presented in this study and the observations that GW182 associates with AGO1 and is required for miRNA-mediated gene silencing, the following model is proposed: AGO1-containing RISCs binds to mRNA targets by means of base-pairing interactions with miRNAs; AGO1 may then recruit GW182, which marks the transcripts as targets for decay via a deadenylation and decapping mechanism (Behm-Ansmant, 2006).

A question that remains open is whether miRNA-mediated translational repression is the cause of mRNA degradation or whether these represent two independent mechanism by which miRNAs silence gene expression as proposed by Wu (2006). Indeed, changes in mRNA levels are not observed for all miRNA targets (Rehwinkel, 2006), suggesting that inhibition of translation is not always followed by mRNA decay. Conversely, depletion of CAF1 or NOT1 prevents mRNA decay but does not relieve translational silencing, suggesting that these two processes are independent (Behm-Ansmant, 2006).

An important finding is that miRNAs elicit degradation to different extents. One possible explanation is that the extent of degradation depends on the stability of the miRNA:mRNA duplexes. Also, the extent of degradation might depend on the particular set of proteins associated with a given target. For instance, some targets may assemble with a set of proteins that antagonize degradation. Finally, GW182 might interact only with a subset of AGO1-containing RISCs, as suggested for AIN-1. A major challenge will be to identify the specific features of miRNA targets and/or RISC complexes that lead to regulation of gene expression at the level of translation or at the level of mRNA stability (Behm-Ansmant, 2006).

The 3' termini of eukaryotic mRNAs influence transcript stability, translation efficiency, and subcellular localization. This study reports that a subset of developmental regulatory genes, enriched in critical RNA-processing factors, exhibits synchronous lengthening of their 3' UTRs during embryogenesis. The resulting UTRs are up to 20-fold longer than those found on typical Drosophila mRNAs. The large mRNAs emerge shortly after the onset of zygotic transcription, with several of these genes acquiring additional, phased UTR extensions later in embryogenesis. These extended 3' UTR sequences are selectively expressed in neural tissues and contain putative recognition motifs for the translational repressor, Pumilio, which also exhibits the 3' lengthening phenomenon documented in this study. These findings suggest a previously unknown mode of posttranscriptional regulation that may contribute to the complexity of neurogenesis or neural function (Hilgers, 2011).

This study identified ~30 genes that exhibit developmental regulation of their 3' UTRs. As a class, the expressed transcripts contain some of the longest 3' UTRs in the Drosophila genome and are comparable to the largest 3' UTRs known in mammals. All of the genes undergo this posttranscriptional transition shortly after the onset of zygotic transcription, with the first detection of the long isoforms at 2-4 h AF. Perhaps the loss or gain of specialized RNA-processing factors during the MZT leads to the extension of the 3' UTRs. Alternatively, depletion of one or more components of the general mRNA poly(A) machinery at the MZT or in neural tissues could lead to weakened poly(A) and mRNA cleavage efficiency, therefore promoting the synthesis of longer transcripts. Such a mechanism, diminished levels of the essential poly(A) factor Cstf-64, promotes the formation of longer isoforms of IgM in B lymphocytes (Hilgers, 2011).

Previous studies suggest that Drosophila 3' UTRs are longest during early development. The genes identified in this study do not conform to this general trend but are consistent with recent whole-genome studies in vertebrates that suggest a statistical enrichment for longer 3' UTRs at later stages in development. In mammals, the expression of long 3' UTR isoforms has been correlated with the loss of cell proliferation and the onset of differentiation. The genes described in this study do not fit this model and may instead be responding to a specific developmental cue during neurogenesis. The key correlation for the large 3' extensions identified in this study is neural expression, irrespective of the state of proliferation. However, the occurrence of 3' elongation events at additional genes in other tissues cannot be excluded because the datasets used for this analysis made use of whole-embryo RNA samples at various developmental stages (Hilgers, 2011).

A significant fraction of the genes with extended 3' UTRs encode proteins implicated in RNA binding or processing, including ago1, adar, pumilio, brat, mei-P26, shep, imp, fne, and elav. Some of these genes, like ago1, are broadly expressed in a variety of tissues. Nonetheless, the extended isoforms of ago1 mRNAs are specifically enriched in neural tissues, a known hotbed of posttranscriptional regulation, including regulation by miRNAs and differential splicing. For example, Dscam is thought to produce tens of thousands of spliced isoforms in the Drosophila CNS. Furthermore, in Drosophila, directed transport of mRNAs, like bicoid, requires functional elements within the 3' UTR. Whether RNA binding factors such as Pum participate in a network of cross-regulation by repression, activation, or transport awaits further study (Hilgers, 2011).

It is currently unclear whether the long forms of mRNAs as seen in mammalian cells produce less protein than the short forms in Drosophila. However, enrichment of Pum recognition motifs in the extended 3' UTRs of elav, brat, and pumilio suggests regulation by repression because Pum and Brat are known to form localized translation repression complexes essential for anterior-posterior body patterning in early embryogenesis. Such regulation may have particular relevance in the Drosophila nervous system because Pum is required for dendrite morphogenesis. It is proposed that neural-specific isoforms of the genes identified in this study comprise elements of an interactive RNA-processing network that mediates some of the distinctive posttranscriptional processes seen in the nervous system (Hilgers, 2011).

This is one of the few studies to use miRNP IP to identify miRNA-regulated mRNAs, and may be the first from adult fly tissues. The data fit well with those derived from the PicTar algorithm and should allow a comparison of different miRNA target prediction algorithms (Kadener, 2009).

The second approach for studying specific miRNA expression relies on cell type-specific inhibition of miRNA synthesis pathways in vivo followed by RNA analysis on tiling arrays. Although very sensitive in identifying many circadian miRNAs, the strategy probably still fails to identify low abundance miRNAs or miRNAs present in small numbers of circadian cells. However, they should be detectable with the same approach, but after a cell purification or cell sorting step. This sensitivity issue is the reason the broad tim-gal4 driver was used rather than the more restricted pdf-gal4 driver. Tim-gal4 is expressed strongly in all circadian tissues of the fly head, including circadian neurons, eyes, fat body, and antennae. This broad expression also explains the strong effect of TIM-Dcr IR on the AGO1 IP enrichment. Consistent with data indicating that core clock components work similarly in both central (brain) and peripheral tissues, bantam overexpression slows the clock pace in both locations: in the central brain as demonstrated by behavior, and in the periphery as demonstrated by luciferase assays (Kadener, 2009).

Intersecting the Ago1 IP data with the tiling array data from Tim-DroshaIR/PashaIR flies as well as with the published fly head miRNA data led to a selection of 10 candidate circadian miRNAs. Since this analysis only used miRNAs with PicTar target predictions and therefore screened only half of the known miRNA population, 10 is likely to be an underestimate. In contrast, of the 27 miRNAs identified as expressed in circadian cells by the Tim-DroshaIR/PashaIR approach, 23 have mRNAs with PicTar predictions in the Ago IP data. This suggests that 10 is not a gross underestimate (Kadener, 2009).

Some of these 10 miRNAs are likely responsible for the decrease in locomotor activity rhythm strength due to inhibition of the miRNA pathway. It is notable that there are no prior reports of a miRNA contribution to circadian behavior in Drosophila and only a single report in mammals. This may be related to the fact that an effect was only manifest at 29°C and with the addition of the UAS-CYC-VP16 transgene. The failure to observe a phenotype in Tim-DcrIR flies at 25°C may reflect a relatively weak effect of the dicer-1 IR transgene on miRNA expression, consistent with the fact that miRNA biosynthesis is not rate-limiting for miRNA-mediated translational regulation. Nonetheless, it is likely that the lack of a circadian defect in Tim-DcrIR flies is not a consequence of inadequate inhibitory transgene expression. This is because the same strain (Tim-DcrIR) still displays normal rhythms even after increasing the temperature to 29°C. Moreover, Tim-Dcr seems to strongly down-regulate the miRNA pathway, as illustrated by the accumulation of pre-bantam and the substantial change in the AGO1 IP profile (Kadener, 2009).

It is therefore suspected that the additional requirement for UAS-CYC-VP16 reflects more than just an increase in UAS-dcr 1 IR expression. It is possible that the transcription and translation of key circadian core components are tightly connected and may buffer each other. Such a regulatory feature could explain why a major increase in transcription, like that caused by the CYC-VP16 transgene, results in only a modest increase in mRNA abundance and probably an even more modest increase in translated protein. A comparable explanation posits that inhibition of the miRNA pathway by the UAS-dcr 1 IR transgene leads to an increase in the translation of circadian repressors, which could then decrease circadian transcription. The use of UAS-CYC-VP16 as well as 29°C might be required to push the system sufficiently far from equilibrium so that pacemaker regulatory mechanisms can no longer compensate for the change in miRNA levels. This type of regulation fits recent data demonstrating that a Drosophila miRNA can function as a buffering agent against environmental perturbations during development (Li, 2009). In any case, the observed behavioral defects observed in Tim-DcrIR-CYCVP16 flies are likely a consequence of down-regulation of several circadian-relevant miRNAs (Kadener, 2009).

Behavioral, genetic, and biochemical evidence indicates that bantam contributes to clk mRNA translational regulation as well as more generally to circadian pacemaker regulation: bantam is highly expressed in circadian tissues, and overexpression with either tim-gal4 or pdf-gal4 significantly lengthens circadian period. The milder effect of the pdf driver may be due to its lower strength in pacemaker cells relative to tim-gal4 and/or to an additional contribution from non-PDF cells to period determination (Kadener, 2009).

Although the period phenotype could be misleading -- due, for example, to an effect of bantam overexpression on a circadian output pathway -- strains with a completely normal central pacemaker do not manifest altered periods, by definition. Another possibility, that bantam overexpression renders the circadian neurons sick or unhealthy, would be expected to result in weak rhythms or arrhythmicity rather than in strong rhythms with long periods. The central pacemaker is therefore the most parsimonious explanation, especially because of the good correlation between the behavioral and the molecular data; i.e., the tim-luciferase results. Unfortunately, the bantam deletion is embryonic lethal, precluding a straightforward behavioral assay of the null phenotype (Kadener, 2009).

The effect of bantam on clk mRNA translation was aided by the finding that the clk 3' UTR extends >700 bases downstream from its predicted 3' end. This error is attributed to priming by oligo (dT) within an A-rich region present near this annotated 3' end. Consistent with this interpretation, a strongly conserved cleavage and polyadenylation site is present near the end of the clk-lg isoform; no obvious site is in the vicinity of the annotated clk 3' end. In addition, RNA protection data indicate that all fly head clk transcripts extend well beyond the annotated clk 3' end. Taken together with the 3' RACE data, these results demonstrate that the clk 3' UTR is significantly longer than previously indicated. Importantly, two of the three clk 3' UTR bantam-binding sites are located downstream from the annotated 3' end (Kadener, 2009).

These clk 3' UTR bantam sites appear to be major circadian targets of bantam in flies. First, clk mRNA is strongly associated with RISC. Second, bantam is strongly expressed in the circadian cells, as demonstrated by the accumulation of precursors of this miRNA when Dicer-1, drosha, or pasha was down-regulated in fly circadian tissues. Third, the effect of bantam (lengthening of the circadian period) resembles the period effect observed in flies carrying fewer genomic copies of clk, and it is opposite to the period effect observed in flies with additional clk copies. Fourth, the three evolutionarily conserved bantam sites are necessary for circadian rhythmicity. Nonetheless, the period effect due to bantam overexpression may be due to effects on other mRNAs in addition to clk (Kadener, 2009).

It is concluded that miRNAs have a role in the central pacemaker and, more specifically, that bantam regulates the central clock component clk. Whereas previous studies have identified miRNAs relevant to circadian rhythms, this one identifies a mRNA-miRNA pair involved in the core timekeeping process. Given the in vivo methods used to study miRNA function (including principally in neuronal tissue), it is suspected that they will have a broad impact on the study of miRNAs and their roles in regulating diverse aspects of Drosophila behavior (Kadener, 2009).

MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) guide Argonaute proteins to silence mRNA expression. Argonaute binding alters the properties of an RNA guide, creating functional domains. The domains established by Argonaute - the anchor, seed, central, 3' supplementary, and tail regions - have distinct biochemical properties that explain the differences between how animal miRNAs and siRNAs bind their targets. Extensive complementarity between an siRNA and its target slows the rate at which fly Argonaute2 (Ago2) binds to and dissociates from the target. Highlighting its role in antiviral defense, fly Ago2 dissociates so slowly from extensively complementary target RNAs that essentially every fully paired target is cleaved. Conversely, mouse AGO2, which mainly mediates miRNA-directed repression, dissociates rapidly and with similar rates for fully paired and seed-matched targets. These data narrow the range of biochemically reasonable models (see Model for RISC Function) for how Argonaute-bound siRNAs and miRNAs find, bind, and regulate their targets (Wee, 2012).

Argonaute divides a small RNA guide into anchor, seed, central, 3' supplementary, and tail functional domains. Nucleotides in the anchor (g1) and tail (g18-g21) facilitate Argonaute loading and help secure the siRNA or miRNA guide to Argonaute after the passenger or miRNA^∗ strand has been removed. But these terminal domains are unlikely to base pair with a target RNA, even when pairing is predicted by their sequences. In contrast, central base pairing (g9-g12) between the guide and target is required for efficient target cleavage. Mismatches in this central region prevent RISC from attaining a catalytically competent conformation. For fly Ago2-RISC, achieving this conformation takes more time than seed pairing alone. The data show that nearly every fly Ago2-RISC that reaches this conformation cleaves its RNA target rather than releasing it. For mouse AGO2-RISC, a slow catalytic rate often allows the target to escape before being sliced (Wee, 2012).

In contrast, most miRNA:Argonaute complexes rapidly bind to and dissociate from their RNA targets via their seed. Even when RISC binds a target through both its seed and 3' supplementary regions, it dissociates nearly as rapidly as for seed-only binding. Thus, the properties of RISC are essentially the same for both the typical seed-only and the less common seed plus 3' supplementary pairing targets. That the rates of association and dissociation are so similar for these two binding modes suggests that pairing between a target and the 3' supplementary region of a miRNA does not require winding the target RNA around the guide, side-stepping the topological problem that must be solved for siRNAs to direct RISC to cleave a target (Wee, 2012).

The finding that miRNAs use so little of their sequence to identify their regulatory targets surprised the biological community. The data show that miRNA-programmed RISC binds with a strength and binding site size similar to those of high affinity RNA-binding proteins. It is siRNA-programmed RISC whose behavior should surprise us: it binds highly complementary targets far less tightly than a comparable antisense RNA because Argonaute reduces the contribution of most of its nucleotides to target binding (Wee, 2012).

What do the physical properties of RISC teach about its cellular function? miRNAs and siRNAs are typically present in cells at dramatically different concentrations. For example, in flies in which the white gene is silenced by RNAi, the abundance of all antisense white siRNAs combined is less than that of any of 29 most abundant miRNAs. Previously, the ability of siRNAs to function at low abundance has been ascribed to the catalytic nature of RNAi. To achieve a concentration 10-fold greater than the K_D for siRNA-like binding (3.7 pM for fly Ago2-RISC) would require only ∼5 molecules of RISC in ovarian terminal filament cells (∼200 μm³ and ∼11 molecules in a cultured S2 cell (∼500 μm³). Thus, even for Argonaute proteins with no endonuclease activity, small numbers of molecules of RISC can repress highly complementary targets; endonuclease activity is only needed when a small amount of RISC must repress a larger amount of target. The combination of high affinity and catalytic turnover helps explain why the siRNA-directed RNAi pathway provides an effective defense against viral infection in plants and invertebrate animals (Wee, 2012).

Animal miRNAs nearly always repress their targets by binding rather than endonucleolytic cleavage. This explains why animal cells express miRNAs at such high levels. Recent data suggest that only the most abundant cellular miRNAs mediate target repression. The data provide a biochemical explanation for this observation (Wee, 2012).

Consider two abundant miRNAs in a cultured HeLa cell (∼5,000 μm³: miR-21 (4 nM; and the let-7 miRNA family, nine highly related miRNAs sharing a common seed sequence (∼3 nM). Both miRNAs are present at a concentration greater than the K_D measured for seed matched targets for fly (∼210 pM) or mouse (∼26 pM) Ago2-RISC. Assuming a mean target mRNA abundance of ten molecules per cell and 50 different mRNA targets per miRNA, miR-21 and let-7 each regulate ∼500 (170 pM) total target mRNA molecules per HeLa cell. Under these conditions, nearly every miR-21 or let-7 target mRNA (∼95%-99%) with an accessible seed match will be bound by the complementary miRNA-programmed RISC (Wee, 2012).

Target repression by miRNAs can be reduced by the presence of competitor RNAs containing miRNA binding sites that titrate miRNA-RISC away from the mRNAs it regulates. The fundamental properties of RISC make specific predictions about how the activity of specific miRNAs can be inhibited by the expression of these competitor transcripts. The effect of such competitor RNAs reflects the concentration of both the miRNA and miRNA-binding sites, as well as the affinity of miRNA-RISC for those sites. For abundant miRNAs such as miR-21 or the let-7 family, the expression of competitor RNAs containing miRNA binding sites -- even highly complementary binding sites -- will have little impact on the regulation of their target genes in flies or mammals. Doubling the expression of mRNAs repressed by miR-21, for example, would require ∼7.8 nM seed only competitor and ∼4.0 nM fully paired competitor for fly Ago2-RISC. For mouse AGO2-RISC, it would still require ∼7.7 nM seed only competitor and ∼7.2 nM of the fully paired competitor. Taken together, this translates to ∼22,400 copies of seed only competitor and ∼12,000-21,700 copies of fully paired competitor. If the competitor contained one miRNA-binding site, it would comprise 12%-50% of all the mRNA in the cell (Wee, 2012).

In contrast, doubling the expression of the mRNA targets for an intermediate (mir-93; ∼140 pM) or a low abundance miRNA (mir-24; 7.3 pM) would require just 600-800 additional seed-matching sites. For mir-93 whose abundance confers the ability to bind to ∼60% of all potential targets, the competitor must be as abundant as the sum of all the target mRNAs (∼500 copies). Low abundance miRNAs like mir-24 are unlikely to contribute much biologically meaningful regulation because they are present at a concentration less than their K_D for seed-matching targets in both flies and mammals: <4% of miR-24 targets are expected to be bound by the miRNA at any given time. Using the conservative assumption that every bound miRNA-RISC completely represses an mRNA target, miR-24 is predicted to reduce the expression of the average seed-matched target by <4% (Wee, 2012).

Thus, the proposal that 'competing endogenous RNAs' ('ceRNAs') sequester miRNAs, derepressing the authentic targets of that miRNA, applies only to a small subset of miRNAs whose cellular concentration and target abundance meet a narrow range of values. The miRNAs with the largest impact on gene expression -- the most abundant miRNAs -- are not predicted to be regulatable by endogenous, transcribed seed-matched competitor transcripts. Consistent with this view, viral and experimental inhibition of specific miRNA function by transcribed RNA requires the use of extensively complementary miRNA-binding sites that recruit a cellular pathway that actively degrades the targeted miRNA. Absent this target directed, catalytic destruction of miRNAs, RNAs of ordinary abundance are unlikely to compete with mRNAs for binding abundant, biologically functional miRNAs (Wee, 2012).

In Drosophila, the transcription factor Gcm/Glide plays a key role in cell fate determination and cellular differentiation. In light of its crucial biological impact, major efforts have been put for analyzing its properties as master regulator, from both structural and functional points of view. However, the lack of efficient antibodies specific to the Gcm protein precluded thorough analyses of its regulation and activity in vivo. In order to relieve such restraints, an epitope-tagging approach to 'FLAG'-recognize and analyze the functional protein was performed both in vitro (exogenous Gcm) and in vivo (endogenous protein). This study reveals a tight interconnection between the small RNA and the Gcm pathways. AGO1 and miR-1 are Gcm targets whereas miR-279 negatively controls Gcm expression. This study also identifies a novel cell population, peritracheal cells, expressing and requiring Gcm/Glide. Peritracheal cells are non-neuronal neurosecretory cells that are essential in ecdysis. In addition to emphasizing the importance of following the distribution and the activity of endogenous proteins in vivo, this study provides new insights and a novel frame to understand the Gcm biology (Laneve, 2013).

The pivotal role of Gcm in orchestrating cell fate specification involves a stringent regulation of its pathway, where multi-level control mechanisms converge. The FLAG-tagged Gcm tool constitutes a useful sensor to characterize post-transcriptional regulatory events (Laneve, 2013).

First, since several bands appear on Western Blots revealing the Gcm–FLAG product and the GCMa vertebrate protein is regulated by phosphorylation, which seems to affect its activity and stability, putative Gcm phosphorylation sites were sought in silico, and several were found. Then cell extracts overexpressing Gcm–FLAG (Act-Gal4) were inclubated with calf intestine phosphatase (CIP) prior to SDS-PAGE and observed a diminished band number and density, thus validating the Gcm phosphorylation prediction. Future studies will assess whether phosphorylation contributes to Gcm stability/activity (Laneve, 2013).

Second, miRNAs are endogenously expressed noncoding RNAs that represent key post-transcriptional regulators of gene expression. An online search carried for miRNAs predicted to target the gcm 3'UTR by miRanda, TargetScan and microCosm databases indicated miR-279/286/996 was the main family of putative effector miRNAs. miR-279, the most characterized member in Drosophila, was initially described for suppressing the formation of heterotopic olfactory neurons and as a component of a complex regulatory circuit orchestrated by the pleiotropic transcription factor Prospero (Pros). Finally, it was also shown to regulate the JAK/STAT pathway, driving rest: activity rhythms and modulating the response to morphogen gradients (Laneve, 2013).

To verify a possible role of miR-279, a sensor construct was developed in which the 3'UTR of gcm was inserted downstream to the FLAG-encoding cassette. Such construct (or its site-specific mutant derivative) was co-transfected in S2 cells along with a vector efficiently over-expressing the miRNA by mean of an Act-Gal4 driver (Laneve, 2013).

Western blot analysis demonstrates a specific downregulation exerted by miR-279 on gcm. Such modulation, abolished in a construct carrying a mutant target site, is specifically mediated by the gcm 3'UTR. Interestingly, ectopic expression of miR-286, a second member of the miR-279 family, diverging from mir-279 at the level of its 3′ sequence, failed to silence Gcm–FLAG. Since the 3′ region of microRNAs is known to play a role in the specificity of microRNA-target recognition, this accounts for the selectivity of gcm post-translational control by miR-279 in vitro (Laneve, 2013).

Finally, this study provides in vivo evidence for miRNA-mediated gcm regulation: (1) miR-279 was co-expressed in the embryonic neural territory (sca-Gal4 driver) in the presence of a Luciferase (Luc) reporter carrying its own 3′UTR or the gcm 3′UTR. By qRT-PCR analysis, a specific downregulation mediated by miR-279 on Luc RNA expression was detected only in the latter case, which parallels the in vitro data (2), an analogous gain-of-function strategy was used to analyze the expression of the endogenous gcm (gcm–FLAG^BAC) in the presence or in the absence of overexpressed miR-279 at RNA and protein level by qRT-PCR and Western blot, respectively. Both approaches show a negative effect, thus validating miR-279 for targeting gcm in vivo in neurogenic territories. In sum, this study identified miR-279 as a negative modulator of gcm both in cell cultures and in embryos. The in vivo relevance of miR-279 on the gliogenic Gcm-dependent pathway was probed. Interestingly, when miR-279 was overexpressed (sca-Gal4) in hypomorphic gcm animals that contain reduced number of glia, a further decrease of glial cell number was expected, but the opposite result was found, and this was obtained upon using two allelic combinations. Thus, further regulatory steps compensate for the effects on gcm, thereby highlighting the complex network linked to small RNAs (Laneve, 2013).

Prompted by the above results, it was asked whether the interplay between Gcm and small RNA metabolism is more pervasive than emerged to date, upon establishing the role of Gcm in this pathway miR-1 was identified in an in vivo DAM ID screen and several GBSs were identified in the region upstream to the miR-1 transcription unit. miR-1 expression was evaluated in vivo upon hs-Gal4 driven Gcm activation and an up-regulation was found, where Gcm plays a crucial role for the development of blood cells. The gcm–Gal4 driver was used to overexpress Gcm and, to restrict the effects to the hemocyte precursor anlagen, and embryonic stages 5–9 were analyzed, when gliogenesis still has to start. An increase of miR-1 levels was confirmed and no effect was detected when Gcm expression was triggered by the sca-Gal4 driver. Interestingly, other miRNAs were identified as potential targets in the DAM ID screen; however their expression did not increase upon Gcm forced expression with any of the used drivers. This further validates the data obtained with miR-1 and suggests that the other miRNAs may work at different developmental stages. Finally, constitutive expression of Gcm in S2 cells, where miR-1 is not endogenously expressed, does not induce miR-1expression, likely due to the absence of appropriate co-activators. Overall, these data call for a cell-specific role of Gcm. Future studies will dissect the role of miR-1 in the Gcm pathways (Laneve, 2013).

Finally, the DAM ID screen also identified Argonaute 1 (AGO1) and an in silico inspection revealed the occurrence of several predicted GBSs mapping upstream to the AGO1 transcription unit. AGO1 a member of the Argonaute/PIWI protein family, involved in small RNA-mediated gene regulation. In Drosophila, AGO1 plays a specific role in miRNA biogenesis and function: it directs the unwinding of the intermediate duplex RNA generated during microRNA biosynthetic pathway and it selects one strand as mature microRNA loaded into the RISC (RNA-induced silencing complex) effector complex. AGO1 is broadly expressed in the embryo as well as in the imaginal discs and this, combined with the well known pleiotropic roles exerted by microRNA, accounts for its involvement in multiple developmental pathways. Interestingly, a genetic screen over a sensitized gcm background identified AGO1 as a putative interactor of gcm (Laneve, 2013).

In short, the Drosophila notum carries a fixed number of sensory organs called bristles. gcm^Pyx/+ flies ectopically express gcm in the larval notum, which triggers the differentiation of supernumerary bristles. gcm^Pyx/+ females show, in average, 18,5 bristles instead of the 11/heminotum typical of WT animals. This phenotype constituted the readout to identify putative gcm interacting genes in a dosage sensitive screen. The AGO1 mutation acts as a suppressor of the gcm^Pyx phenotype in double heterozygous conditions (genotype: gcm^Pyx/AGO1⁰⁸¹²¹), showing a positive genetic interaction with gcm (Laneve, 2013).

A microarray profiling also suggested AGO1 as a possible Gcm target in the neurogenic territories; however its upregulation upon Gcm forced expression as well as in gcm loss of function mutations made unclear the role of Gcm. Therefore (Act-Gal4 driver) Gcm or its tagged derivative was expressed in S2 cells, in which AGO1 is endogenously expressed, and this was found to induce AGO1 accumulation, reflecting and following the temporal accumulation of Gcm–FLAG itself. To further corroborate these data, increasing amounts of the Gcm–FLAG-expressing construct were transfected, and the amount of AGO1 was analyzed at the time-points of Gcm–FLAG highest expression (48–72 h after transfection). This revealed a clear correlation between the quantity of Gcm–FLAG and the expression of AGO1 . Furthermore, to exclude any unspecific influence of the FLAG epitope on target recognition, the same assay employing an untagged version of Gcm. Finally, it was verified AGO1 as a Gcm target in vivo: the UAS–gcm transgene was expressed under the control of the heat-shock (hs) inducible driver hs-Gal4 in Drosophila embryos: the Western blot demonstrates a clear up-regulation of AGO1 upon Gcm ectopic expression. In sum, this study provides significant genetic and molecular evidence for a positive interaction between AGO1 and gcm suggesting a mechanism of direct targeting. Interestingly, no modulation of AGO1 was observed upon Gcm forced expression in the nervous system using sca-Gal4. Since Gcm is required in different cell types, more efforts are required to clarify in which functional pathway Gcm controls AGO1. The data provide nevertheless first evidence for a cell-specific factor modulating the expression of AGO1. Indeed, the widespread distribution and function of miRNAs suggest a complex regulatory network controlling AGO1 expression/activity: Gcm can be proposed as a cell-specific component of the small RNA cascade (Laneve, 2013).

It is concluded that the transcriptional activator Gcm constitutes a paradigmatic example of master regulator, acting as a pivotal cell fate determinant and differentiation factor during Drosophila embryogenesis. It is therefore crucial to outline a trustworthy picture of Gcm biology, from expression to function. The present study provides the first characterization of Gcm at the protein level and reports a large set of data on gcm function, regulation and expression, collected both in vitro and in vivo. Specifically, small RNA metabolism was identified as an important element of the Gcm pathway and a novel gcm-dependent cell type essential in development was identified (Laneve, 2013).

Several lines of evidence suggest that some members of the AGO1 superfamily may bind RNA or may be a component of a protein-RNA complex. For example, C. elegans rde-1 is required for RNA interference (Tabara, 1999). Because soluble recombinant AGO1 proteins were not available, an alternative approach was taken to investigate whether AGO1 has an RNA binding activity. Extracts of adult flies were prepared and incubated with poly(A)- or poly(U)-conjugated Sepharose 4B beads. AGO1 protein co-precipitates with the RNA-conjugated beads, but not with unconjugated control ones, indicating that AGO1 binds RNA either directly or indirectly. Although a unit volume of beads was conjugated with a similar amount of, and a similar range of lengths of, either poly(U) or poly(A), AGO1 was recovered more efficiently with poly(U)-beads than with poly(A)-ones. To determine the region required for the association with the RNA-beads, extracts were made of transgenic lines that produced DeltaN or DeltaC. Both forms of AGO1 precipitate with poly(U)-Sepharose, indicating that a deletion of either region alone does not abolish the association of AGO1 with the beads (Kataoka, 2001).

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).

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).

Spinocerebellar ataxia type 2 (SCA-2) is an autosomal dominantly inherited neurodegenerative disease caused by trinucleotide (CAG) expansion in the ATXN2 gene resulting in the lengthening of polyglutamine stretch in the encoded protein ataxin-2. Ataxin-2 is a large conserved protein that carries an Sm-domain and a PAM-2 motif on the C-terminal side of the Sm-domain. The PAM-2 motif mediates interaction with the C-terminal helical domain of the poly(A) binding protein (PABP; see Drosophila pAbp) and is present in several PABP-interacting proteins. Levels of mutant ataxin-2 (with expanded polyglutamine stretch) are higher in brain tissue of patients with SCA-2 compared with that of wild-type ataxin-2 in normal individuals. Further, abnormal expression of Ataxin-2 has been shown to be deleterious in Drosophila (Satterfield, 2002). However, the biological function of ataxin-2 and the mechanism by which lengthening of the polyglutamine stretch in ataxin-2 leads to the disease are not clear (Tharun, 2008 and references therein).

Several studies implicate ataxin-2 in mRNA decay and regulation of translation suggesting that deregulation of these processes could be related to the disease. Ataxin-2 homologs from multiple organisms have been shown to interact with PABP consistent with the presence of PAM-2 motif in Ataxin-2. Depletion of Ataxin-2 homolog in C. elegans affects germline development and this seems to be due to the deregulation of translational repression by GLD-1 and MEX-3 of their mRNA targets. Reducing the expression of ATXN2 in mammalian cells using siRNAs impairs the formation of stress granules which are the sites where untranslated mRNAs are localized during stress. Finally, both in human cells and in Drosophila, Ataxin-2 is associated with the polysomes (Satterfield, 2006). These observations suggest that Ataxin-2 may have a role in translational repression in vivo. Interestingly, enhancement and suppression of Ataxin-2 expression in human cells leads to decrease and increase in the levels of PABP, respectively, without affecting the levels of PABP mRNA. Given that PABP is a key translation factor, it remains to be seen if the effects on translation caused by alterations in Ataxin-2 expression are related to the changes in the levels of PABP. Ataxin-2 overexpression also leads to decrease in the number of P-bodies in human cells. This seems to be related to the ability of Ataxin-2 to interact with Dhh1/RCK/p54, which is one of the decay factors important for P-body assembly in human cells. Importantly, reduction in P-bodies was caused by overexpression of Ataxin-2 irrespective of whether the polyglutamine stretch in Ataxin-2 was normal or long. These observations together suggest that deregulation of mRNA decay and/or translational repression resulting from abnormal expression of Ataxin-2 may be one of the reasons for the disease phenotype and the expanded polyglutamine stretch could contribute to increased expression of the mutant Ataxin-2 in the diseased individuals (Tharun, 2008 and references therein).

Studies in Drosophila suggest that Ataxin-2 is required for microRNA function and synapse-specific long-term olfactory habituation. Local control of mRNA translation has been proposed as a mechanism for regulating synapse-specific plasticity associated with long-term memory. Glomerulus-selective plasticity of Drosophila multiglomerular local interneurons observed during long-term olfactory habituation (LTH) requires the Ataxin-2 protein (Atx2) to function in uniglomerular projection neurons (PNs) postsynaptic to local interneurons (LNs). PN-selective knockdown of Atx2 selectively blocks LTH to odorants to which the PN responds and in addition selectively blocks LTH-associated structural and functional plasticity in odorant-responsive glomeruli. Atx2 has been shown previously to bind DEAD box helicases of the Me31B family, proteins associated with Argonaute (Ago) and microRNA (miRNA) function. Robust transdominant interactions of atx2 with me31B and ago1 indicate that Atx2 functions with miRNA-pathway components for LTH and associated synaptic plasticity. Further direct experiments show that Atx2 is required for miRNA-mediated repression of several translational reporters in vivo. Together, these observations (1) show that Atx2 and miRNA components regulate synapse-specific long-term plasticity in vivo; (2) identify Atx2 as a component of the miRNA pathway; and (3) provide insight into the biological function of Atx2 that is of potential relevance to spinocerebellar ataxia and neurodegenerative disease (McCann, 2011).

In the mammalian brain, single neurons form up to 100,000 different synapses whose weights may be regulated independently during learning. In principle, the synapse-specificity of short-term plasticity may be explained simply by the restriction of signaling events to active synapses. However, synapse-specific long-term plasticity, which depends on products of nuclear gene expression that would be available in a cell-wide manner, clearly depends on distinct synaptic tags that mark only active synapses (McCann, 2011).

Several lines of evidence suggest that activity-regulated local translation of synaptic mRNAs normally stored in a repressed state contributes to the synapse-specificity of long-term plasticity. Consistent with this idea, several translational control molecules, such as fragile X mental retardation 1 protein, Staufen, cytoplasmic polyadenylation element binding protein/Orb2, and the Gld2 polyA polymerase, are required in Drosophila for long-term but not short-term memory. In the context of identified synapses, local protein synthesis is required for cAMP response element-binding protein (CREB)-dependent, synapse-specific long-term plasticity in cultured Aplysia sensorimotor synapses: In this system, postsynaptic translation may trigger a retrograde signal, which in turn stimulates local translation at presynaptic terminals. However, in vivo, the requirement for and regulation of local protein synthesis at synapses remains poorly understood, in part because of the paucity of preparations in which behavioral learning arises from plasticity within a defined, experimentally convenient, neural circuit (McCann, 2011).

Recent work has shown that long-term olfactory habituation (LTH), a phenomenon in which sustained exposure to an odorant results in a decreased behavioral response, arises through plasticity of synapses between local interneurons (LNs) and projection neurons (PNs) in the Drosophila antennal lobe (Das, 2011; Sachse, 2007). Although LTH requires the transcription factor CREB2 to function (globally) in a multiglomerular class of LNs, LTH is odorant selective and associated with glomerulus-selective (and hence local) structural and physiological plasticity. In screening candidate RNA-binding proteins for potential roles in PNs during LTH, Ataxin-2 (Atx2), a molecule of considerable interest for its known involvement in the human neurodegenerative disease spinocerebellar ataxia-2 (SCA2), was identified. Expansion of a polyglutamine tract in human Atx2 from about 22 (normal) to >32 (pathogenic) glutamines causes degeneration of cerebellar Purkinje cells. While Atx2 has been implicated in many different biological functions, it is generally believed to function in RNA regulation. Evidence for this role comes from biochemical and cell biological studies of the protein or its evolutionarily conserved orthologs in Caenorhabditis elegans, Saccharomyces cerevisiae, and Drosophila melanogaster (McCann, 2011).

In C. elegans, Atx2 is required in postembryonic germline cells for appropriate translational control of GLD-1- and MEX-3-target mRNAs. Atx2 binds the RNA regulatory proteins, polyA-binding protein (PABP) and Me31B/RCK/ Dhh1p/CGH-1, through domains also required for its observed assembly with polyribosomes. At a cell biological level, Atx2 function has been shown to regulate the assembly of P-bodies and stress granules, distinct cytoplasmic messenger ribonucleoprotein particles that contain translationally repressed mRNAs, together with the translational repressor Me31B/RCK/Dhh1p (McCann, 2011 and references therein).

Significantly, both the proteins and cytoplasmic structures with which Atx2 associates have been linked to translation repression by microRNAs (miRNAs), a class of small, noncoding RNAs that bind complementary sequences in mRNA 3'UTRs and repress translation via the RNA-induced silencing complex (RISC). Furthermore, miRNAs and miRNA components have been linked either to long-term memory in Drosophila or to sensorimotor synapses (McCann, 2011).

This study shows that (1) Atx2 functions in olfactory projection neurons for LTH as well as associated glomerulus-selective physiological and structural plasticity; (2) Atx2 functions in LTH with the known miRNA-pathway proteins Argonaute 1 (Ago1) and Me31B; and (3) Atx2 is part of a general machinery required for efficient miRNA-mediated translational repression (McCann, 2011).

When tested in a Y-maze apparatus, flies exposed to either 15% CO2 or 5% ethyl butyrate (EB) for 30 min show a reduced aversive response that lasts less than 1 h (short-term habituation, STH). In contrast, flies exposed to 5% CO2 or 20% EB for 4 d show reduced aversion for days (LTH) and reduced odor-evoked responses in respective odor-responsive PNs, together with CREB-dependent growth of odor-responsive glomeruli (V and DM2/DM5, respectively). In this well-defined behavioral and synaptic context, it was asked whether PNs require Atx2 for LTH and associated synapse-specific structural plasticity. Expression of a UAS-Atx2RNAi construct in GH146-expressing neurons responsive to EB but not to CO2 (GH146Gal4/UASAtx2RNAi) completely blocked LTH to EB without altering LTH to CO2. Atx2 knockdown in GH146-expressing PNs blocked LTH to EB but had no effect on either STH to EB or the EB-avoidance response. Similarly, knockdown of Atx2 in the CO2-responsive VPN (VPNGal4;UASAtx2RNAi/+) selectively blocked LTH to CO2 without altering either STH to CO2 or the naive olfactory response to CO2. Thus, Atx2 is selectively required in glomerulus-specific PNs for odorant-selective LTH (McCann, 2011).

Two observations argue that Atx2 functions in adult neurons for LTH. First, both baseline behavioral responses to odorants and STH are normal in animals after Atx2 knockdown in PNs, indicating relatively normal development of the olfactory system. Second and more direct evidence is the selective block in LTH to EB seen following adult-specific knockdown of Atx2 in EB-responsive PNs using the TubGal80ts system (McCann, 2011).

Atx2 knockdown in odor-responsive PNs blocks not only olfactory LTH but also the LTH-associated increase in the volume of behaviorally relevant glomeruli. Thus, following 4 d of EB exposure, GH146Gal4/UAS-Atx2RNAi flies, which do not show LTH, also show no increase in the volume of either the DM5 glomerulus, previously shown to mediate the aversive response to this odorant, or the EB-responsive DM2 glomerulus. In contrast, the same GH146Gal4/UAS-Atx2RNAi flies show normal LTH to CO2 and robust increases in the volume of the VPN glomerulus in response to 4-d CO2 exposure as observed in control flies. Similarly, VPNGal4;UAS-Atx2RNAi/+ flies do not show LTH to CO2 or associated growth of the V glomerulus but display normal EB-induced LTH and EB-associated growth of DM5. Thus, Atx2 is required in specific PNs for the glomerulus-selective structural plasticity that accompanies odorant-selective LTH (McCann, 2011).

Normal LTH to EB is associated with an experience-dependent reduction in the EB-evoked physiological response of DM2 and DM5 PNs. This reduction can be measured in vivo by imaging odor-evoked calcium transients in PNs of flies expressing the genetically encoded calcium sensor, GCaMP3 (McCann, 2011).

To test whether this LTH-associated physiological plasticity requires Atx2 function in PNs, EB-evoked calcium fluxes were imaged and quantified in PN dendrites of 4-d EB-exposed and mock-exposed GH146Gal4, UAS-GCaMP3/UAS-Atx2RNAi flies (which do not show LTH to EB), and these results were compared with normally habituating GH146Gal4,UAS-GCaMP3/+ controls. In DM2 and DM5 of GH146Gal4, UAS-GCaMP3/UAS-Atx2RNAi flies, 4-d EB exposure caused significantly less change in EB (McCann, 2011).

Biochemical interactions of Atx2 orthologs in Drosophila and other organisms point to an interesting potential mechanism through which Atx2 regulates synapse-specific long-term plasticity required for LTH. In particular, Atx2 binding to Me31B and PABP orthologs, which in turn interact with other core miRNA-pathway proteins, GW182 and Argonaute, suggests that Atx2 may regulate miRNA-mediated translational repression directly. Could the function of Atx2 in LTH reflect a role in the miRNA pathway (McCann, 2011)?

To address this question, the possibility of strong dominant genetic interactions between atx2^X1 and mutations affecting core components of the miRNA pathway was examined. First, LTH and STH were examined in ago1^K08121/+; atx2^X1/+ double-heterozygote animals, and these behaviors were compared with those of single-heterozygote controls. The results were striking. Although STH to EB and CO2 was normal in double heterozygote ago1^K08121/+; atx2^X1/+ animals, LTH to both EB and CO2 was completely abolished. In contrast, control +/+; atx2^X1/+ and ago1^K08121/P[atx2+]; atx2^X1/+ animals showed normal LTH to both odorants (McCann, 2011).

The observation that the atx2 genomic rescue construct P[atx2+] restored normal LTH to ago1^K08121/+; atx2^X1/+ flies also shows that altered LTH in the double-heterozygote flies is caused specifically by a defect in atx2. In a similar experiment LTH and STH were examined in me31B^δ2/+; atx2^X1/+ double-heterozygote animals exposed to EB or CO2. Again, these double heterozygotes showed no LTH but normal STH. The defects in LTH were not observed in +/+; atx2^X1/+ or me31B^δ2 /P[atx2+]; atx2^X1/+ animals, further confirming the involvement of atx2. LTH-associated structural plasticity also was blocked in ago1^K08121/+; atx2^X1/+ and me31B^δ2/+; atx2^X1/+ double heterozygotes. Thus, although the V and DM5 glomeruli of +/+; atx2^X1/+ flies showed the expected growth following 4 d of CO2 or EB exposure, respectively, both the EB-evoked increase in DM5 volume and the CO2-induced increase in V was abolished in ago1^K08121/+; atx2^X1/+ and me31B^δ2/+; atx2^X1/+ double heterozygotes. In every instance, the defect in structural plasticity was restored by a wild-type genomic atx2+ transgene: Both ago1^K08121/ P[atx2+]; atx2^X1/+ flies and me31B^δ2/P[atx2+]; atx2^X1/+ flies showed normal odor-induced structural plasticity (McCann, 2011).

These data indicate that Atx2 functions in odorant-selective LTHas well as in glomerulus-selective structural plasticity through a pathway that depends on Ago1 and on Me31B, two proteins previously linked with miRNA-driven translational control. Consistent with this hypothesis, RNAi-based knockdown of Me31B in EB-responsive PNs mimics the effects of Atx2 knockdown, causing a specific defect in LTH to EB (McCann, 2011).

The observed genetic interactions of atx2 mutations with me31B and ago1 mutations point to a likely role for the Atx2 protein in regulating Ago1- and Me31B-dependent, miRNA-mediated translational repression in vivo. To examine this possibility, it was asked if Drosophila Atx2 is required for miRNA-mediated translational repression in wing imaginal discs, a tissue in which the function and activities of endogenous miRNAs can be analyzed conveniently (McCann, 2011).

To reduce levels of endogenous Atx2 in identified subpopulations of wing imaginal disc cells, either a patched Gal4-driven RNAi construct (UAS-Atx2RNAi) was used against atx2 or the Flippase recognition target-Flippase (FRT-FLP) recombinase system to generate genetic-mosaic animals carrying clusters of homozygous atx2^X1/atx2^X1 mutant cells in the wing imaginal discs of hs-flp;+/+; FRT82B, atx2^X1/FRT82B, arm-lacZ. Homozygous mutant atx2^X1/atx2^X1 cells were identified using either an Atx2 antibody or a surrogate, anti-lacZ staining, which here labels all cells except the atx2^X1/atx2^X1 mutant clones generated by mitotic recombination (McCann, 2011).

To examine Atx2 function in the miRNA pathway, such clones were generated in a genetic background that included any one of a number of transgenically encoded, miRNA-dependent translational reporters, and how loss of Atx2 affected GFP expression of these reporters was assessed. Reporters for head involution defective (hid), bantam, mir-12, costal-2, and sickle were used. The hid, sickle, and costal-2 reporters consist of the 3' UTR of hid, sickle, or costal-2, respectively, placed downstream of GFP-coding sequences under the control of a tubulin promoter (McCann, 2011).

The 3' UTR of hid is repressed by endogenous bantam miRNA and that of sickle by miR-2b. The bantam and miR-12 reporters consist of two copies of the bantam target recognition sequence or four copies of the miR-12 target recognition sequence, respectively, also downstream of GFP-coding sequences (McCann, 2011).

Atx2-deficient cells had a noticeable reduction in the intensity of Me31B and Ago1 staining, suggesting that in vivo Drosophila Atx2 is necessary for maintaining Me31B particles potentially involved in miRNA-mediated translational repression. In addition, and consistent with a defect in miRNA function in vivo, cells lacking Atx2 showed distinctly elevated expression of specific miRNA reporters (McCann, 2011).

Clones of atx2^X1/atx2^X1 mutant cells showed clear up-regulation of the hid reporter compared with flanking atx2^X1/+ or +/+ cells. The increased hid reporter levels in atx2^X1/atx2^X1 mutant cells were not observed if similar clones also expressed a wild-type atx2 genomic transgene). This observed genetic rescue confirms that the increase in hid-reporter expression in atx2^X1/atx2^X1 cells is caused by the absence of atx2 and not by any other unknown potential mutation on the FRT82B,atx2^X1 chromosome. Thus, as predicted by its biochemical and genetic interactions with various miRNA-pathway components, Atx2 is required for optimal repression of a miRNA reporter in vivo (McCann, 2011).

Further experiments confirmed that this requirement reflects a broad requirement for Atx2 function for the repression of many different miRNA-target genes. Clones of atx2^X1/atx2^X1 cells also show increased expression of the sickle, bantam BandBŒ), and miR12 reporters. Given that the latter two reporters are regulated by artificial UTRs engineered to be repressed solely through the miRNA pathway, these data strongly argue that Atx2 is broadly required for miRNA function. In contrast to the other four miRNA reporters, costal-2 reporter expression was not increased detectably in atx2^X1/atx2^X1 cells. This result was surprising, because the costal-2 reporter is similar to the other reporters in being repressed by miRNAs through a mechanism that requires Dicer-1 (Dcr-1), the endonuclease involved in miRNA biogenesis. Therefore, Atx2 is required only for repression of a subset of miRNA targets (McCann, 2011).

A model is considered in which Atx2 functions in only one of two miRNA-repression pathways recently distinguished in Drosophila. Although produced by Dcr-1, miRNAs may repress translation by one of two alternative pathways: either through an Ago1-RISC that requires GW182 or through an Ago2 RISC via a poorly understood GW182-independent mechanism. To test the possibility that Atx2 would be required exclusively for the Ago1/GW182-dependent pathway, previously shown to be required for bantam miRNA function, how the various reporters were affected by knockdown of GW182 was examined (McCann, 2011).

To knock down GW182 in identified groups of cells, the FLP-out technique was used to overexpress a transgenic RNAi construct against GW182 in wing imaginal discs expressing hid, miR12, or costal-2 reporters. Cells expressing a GW182RNAi construct (labeled by anti-lacZ staining) showed visibly increased expression of the Atx2-sensitive hid and miR12 reporters but no up-regulation of the Atx2-insenstive costal-2 reporter (McCann, 2011).

To understand why the costal-2 reporter could be insensitive to GW182 (or Atx2) knockdown, the sequence of its 3' UTR was examined and it was found to contain not only target sites for miR12 and miR283, but also two binding sites for miR277. This finding is significant because, unlike the majority of Dcr1-dependent miRNAs, miR277 is loaded preferentially onto Ago2-RISC complexes because of the extensive base-pairing between its miRNA and miRNA* strands. Thus, these observations suggest that Atx2, although necessary for GW182-dependent repression through Ago1-RISC, may not be necessary for Ago2-RISC.dependent repression (McCann, 2011).

This tentative model is supported by the observation that RNAi-induced, Ago-2-dependent eye phenotypes also are not sensitive to knockdown of Atx2. Knockdown of the caspase inhibitor Drosophila inhibitor of apoptosis (DIAP) by GMRGal4- driven expression of UAS-DIAPRNAi results in significantly smaller eyes because of increased apoptosis. The cell-death phenotype is suppressed if Ago2 levels are reduced by simultaneous expression of UAS-Ago2RNAi. However, similar coexpression of a functional UAS-Atx2RNAi (or UAS-GFPRNAi) does not alter phenotypes of GMRGal4;UASDIAPRNAi flies (McCann, 2011).

Taken together with prior evidence that Atx2 associates physically with Me31B and PABP, two proteins required for the Ago1-RISC pathway, these data indicate that Atx2 is part of a core pathway required for miRNA-mediated translational repression. However, Atx2 may be dispensable for repression by the Ago2-RISC pathway (McCann, 2011).

The circuit that underlies LTH has allowed experience-induced, synapse-specific plasticity to be examined in the context of behavioral memory. Previous pioneering work in cultured Aplysia sensorimotor synapses has led to a model in which CREB-dependent nuclear gene expression provides global (cell-wide) control of long-term facilitation. This facilitation can be restricted to specific synapses, in part through the synapse-specific local translation of stored mRNAs, which also is required for long-term plasticity. In the context of olfactory LTH, which is driven by the plasticity of inhibitory LN-PN synapses in the antennal lobe, previous work has shown that CREB function is required globally in a multiglomerular class of LNs for LTH to CO2 and EB. This study now shows that LTH additionally requires Atx2 in postsynaptic PNs for the glomerulus-restricted plasticity necessary for odorant-selective LTH. Knockdown of Atx2 in adult-stage PNs selectively blocks LTH without affecting either basal odorant response or STH. This distinctive phenotype also is shown following Me31B knockdown in PNs or in animals doubly heterozygote for atx2 and ago1 or atx2 and me31B. When taken together with independent observations that Atx2 is required for efficient miRNA function, these very strong genetic interactions point to a role for Atx2 in miRNA-mediated translational control in the regulation of long-term memory (McCann, 2011).

It is hypothesized that, under appropriate circumstances, NMDA receptor activation in PN dendrites may trigger local protein synthesis, perhaps through RISC degradation, giving rise to a retrograde signal from PNs to LNs that in turn stimulates or synergizes with the cell biological changes required for glomerulus- limited, long-term plasticity. The data do not demonstrate that Atx2 and Me31B function in local translation of synaptic mRNAs, but they do show a specific requirement for Atx2 and Me31B for miRNA function and synapse-specific LTH (but not STH) and thus provide a strong argument for local translation of synaptic mRNAs being the underlying mechanism by which these proteins regulate synapse-specific long-term plasticity in vivo (McCann, 2011).

The proposed need for postsynaptic translation and postulate of retrograde signaling are consistent with recent observations and models explaining long-term synaptic facilitation in Aplysia (Wang, 2009; Cai, 2008). In Drosophila, these models may be tested and elaborated through further genetic and in vivo studies and may lead to an understanding of the local and global mechanisms and their interactions that regulate long-term synaptic plasticity (McCann, 2011).

An important finding is that Atx2 is required for translational repression of four different miRNA reporters. Taken together with prior evidence, in particular that Atx2 binds two known components of the miRNA pathway, this finding indicates a wide and general requirement for Atx2 in miRNA-mediated translational repression (McCann, 2011).

However, Atx2 is not required for silencing of the costal-2 reporter, an observation that may be may be explained by costal-2 reporter's repression by a possibly Atx2-independent RISC complex. Previous work has shown that miRNAs partition between two different silencing complexes, Ago1-RISC and Ago2-RISC; in contrast, siRNAs associate almost exclusively with Ago2-RISC. Ago1-RISC and Ago2-RISC silence mRNAs by different mechanisms: Ago1-RISC is characterized by its dependence on GW182. The specific pathway that produces an miRNA or siRNA does not require that small RNA to associate with a particular Ago protein. Thus, although bantam and miR-277 miRNAs are produced by Dcr1, bantam associates exclusively with Ago1-RISC, whereas miR-277 is loaded into the Ago2 pathway. This loading of miR-227 occurs because, in contrast to the bantam microRNA, which has several bulges and mismatches, the duplex precursor to miR-277 strongly resembles an siRNA precursor with a high degree of perfect matching. By demonstrating that loss of Atx2 causes up-regulation of GW182- or Ago1-dependent miRNA reporters, the results identify Atx2 as a frequently used component of the Ago1-GW182 RISC pathway. Loss of Atx2 does not affect repression of the GW182-insensitive costal-2 reporter, possibly repressed via the Ago2-RISC pathway. This observation, combined with the insensitivity of the RNAi pathway to Atx2 knockdown in the Drosophila eye, suggests that Atx2 may not be required for Ago2-RISC function (McCann, 2011).

Atx2's role in the Ago1-miRNA pathway raises the question as to how Atx2 influences miRNA-mediated translational repression. Uncovering Atx2's molecular mechanism of action is complicated by lack of consensus as to how miRNAs regulate gene expression. However, Atx2 is likely to function through its interactions with PABP [mediated by its PABP-interacting motif 2 (PAM2) domain] or Me31B [via its Like Sm (Lsm) and Like-Sm-associated domain (LsmAD) domains] (McCann, 2011).

Three possible models for Atx2 actions are considered. Under appropriate conditions, Atx2 interactions with PABP could help break eukaryotic initiation factor (eIF) 4G eIF4g-PABP interactions required for efficient translational initiation. In addition, directly or through interactions with Me31B, Atx2 may help recruit either of two deadenylase complexes that promote mRNA deadenylation and consequent repression (McCann, 2011).

The identification of Atx2 as a core component of the neuronal and nonneuronal miRNA repression machinery has implications for understanding spinocerebellar ataxias and some forms of amyotrophic lateral sclerosis. Several studies underline the importance of functional components of the miRNA repression machinery in the mammalian brain. It has been demonstrated that miRNA-regulated activities play a role in polyglutamine-induced neurodegeneration. In addition, other work has shown that Atx2 is required for pathogenic forms of Atx1 and Atx3 to induce neurodegeneration in Drosophila, suggesting a potentially common pathway for neuronal loss in different ataxias. Loss of Dcr1 function results in microcephaly and progressive neurodegeneration, consistent with a model in which miRNA function is required for maintaining the adult nervous system (Saba, 2010). Given Atx2's involvement in human neurodegenerative disease, the current findings may help illuminate some of the phenotypes and symptoms of SCA2 patients and also may illuminate possibly common pathways for neuronal loss in different neurodegenerative conditions. If altered miRNA function contributes to neurodegeneration in SCA2 or related diseases, then it is possible that these diseases arise because of altered regulation of a subset of Atx2-target mRNAs in neurons. The identification and study of such target mRNAs may contribute to further understanding and potential therapeutic strategies (McCann, 2011).

microRNAs induce translational repression by binding to partially complementary sites on their target mRNAs. An in vitro system was established that recapitulates translational repression mediated by the two Drosophila Argonaute (Ago) subfamily proteins, Ago1 and Ago2. Ago1-RISC (RNA-induced silencing complex) was shown to represses translation primarily by ATP-dependent shortening of the poly(A) tail of its mRNA targets. Ago1-RISC can also secondarily block a step after cap recognition. In contrast, Ago2-RISC competitively blocks the interaction of eIF4E with eIF4G and inhibits the cap function. The finding that the two Ago proteins in flies regulate translation by different mechanisms may reconcile previous, contradictory explanations for how miRNAs repress protein synthesis (Iwasaki, 2009).

This study shows that in flies both Ago1 and Ago2 can induce translational repression. These findings contradict a previous proposal that only Ago1 can repress translation (Förstemann, 2007). This notion was based on the observation that in S2 cells a central-bulged reporter for endogenous miR-277, which partitions into both Ago1 and Ago2, was derepressed when Ago1, but not Ago2, was absent. However, in flies Ago1- and Ago2-RISC-loading pathways compete in vitro and in vivo; when one Ago is absent, another Ago can function more strongly with more small RNAs loaded. Because the translational repression by Ago1 is stronger, up to ∼8-fold repression for Ago1 and up to ∼2.5-fold repression for Ago2 in an in vitro system) and presumably more irreversible than that of Ago2, it is possible that, in the case of miR-277, the Ago1-mediated repression was enhanced to compensate for the loss of Ago2. In contrast, this study programmed Ago1-RISC or Ago2-RISC in an essentially exclusive manner, which allowed discrimination of their functions (Iwasaki, 2009).

Drosophila Ago1 shortens the poly(A) tail of the target mRNA, and miRNA-directed deadenylation has been reported in other organisms. Because miRNAs can shorten the poly(A) tail even when translation is blocked, deadenylation is not a consequence of translational repression, but a process independent of it. This study found that deadenylation by Ago1 requires ATP, even though ATP is dispensable for the cleavage activity of Ago1-RISC and for the association of Ago1 with GW182, Pop2/Caf1, and Ccr4. Deadenylation catalyzed by the Ccr4-Not complex is per se ATP independent. Thus, the ATP-dependent step after target recognition remains to be identified for Ago1-directed mRNA deadenylation. Such ATP-dependent deadenylation is reminiscent of regulation of the Drosophila mRNA nanos (Iwasaki, 2009).

Kinetic modeling analyses have shown that differences in the rate-limiting steps of translation can have large effects on the outcome of repression. The current data show that poly(A)⁻ mRNAs are less efficiently repressed by Ago1 compared with poly(A)⁺ mRNAs, agreeing with previous reports on miRNA-mediated translational repression in mammals. This can be simply explained by the fact that poly(A)⁺ mRNAs are better translated than poly(A)⁻ mRNAs. At the same time, this is also consistent with the idea that poly(A) and poly(A)-binding protein (PABP) influence the rate-limiting steps in translation, which can affect the ability of repressors to limit translation. Indeed, PABP is involved in multiple key steps of translation initiation, including cap recognition by eIF4E, 48S, and 80S formation and ribosome recycling. Some literature concludes that poly(A) tail is hardly required for miRNA-mediated repression, but the reason for the apparent discrepancy on the poly(A) dependence of repression is obscure. Perhaps repression at a step after cap recognition is significantly stronger and/or translation itself is less dependent on the poly(A) length under these assay protocols compared with other protocols. Such variations can be caused by a number of factors, including time scale of analysis and stoichiometry of RISC, target mRNA, and translational machinery (Iwasaki, 2009).

Most importantly, the current data show that, even when the same protocols and substrates were used (i.e., under conditions with identical rate-limiting steps in translation), there are striking differences between Ago1- and Ago2-mediated translational repression. (1) Repression by Ago1 is accompanied by deadenylation of the target mRNA, whereas repression by Ago2 has no such effect on the target mRNA. (2) Ago1 requires GW182 for translational repression and ATP-dependent deadenylation, whereas Ago2 represses translation independent of GW182. (3) Ago1-RISC blocks a step after cap recognition, whereas Ago2-RISC binds to eIF4E and specifically blocks eIF4E-eIF4G interaction. Of note is that the affinity of Ago2 to eIF4E is dramatically enhanced when Ago2-RISC binds to a target mRNA, which should, in theory, ensure that the Ago2-mediated repression is limited to translation of the cognate target. Because Ago2 exerts no influence on the target mRNA quantitatively and qualitatively, it is tempting to speculate that Ago2 provides rapid, short-lived repression that preserves the target mRNA, allowing its expression to be reactivated later, whereas repression mediated by Ago1 is more irreversible in cells where deadenylation triggers mRNA decay. Although it is not known whether mammalian Ago proteins similarly act by distinct mechanisms, these findings may reconcile previous, contradictory explanations for how miRNAs repress protein synthesis (Iwasaki, 2009).

MicroRNAs silence their complementary target genes via formation of the RNA-induced silencing complex (RISC) that contains an Argonaute (Ago) protein at its core. It was previously proposed that GW182, an Ago-associating protein, directly binds to poly(A)-binding protein (PABP) and interferes with its function, leading to silencing of the target mRNAs. This study shows that Drosophila Ago1-RISC induces silencing via two independent pathways: shortening of the poly(A) tail and pure repression of translation. The data suggest that although PABP generally modulates poly(A) length and translation efficiency, neither PABP function nor GW182-PABP interaction is a prerequisite for these two silencing pathways. Instead, it is proposed that each of the multiple functional domains within GW182 has a potential for silencing, and yet they need to act together in the context of full-length GW182 to exert maximal silencing (Fukaya, 2011).

How much deadenylation/destabilization and translational repression contribute to miRNA-mediated silencing and how much these two interrelated pathways influence each other as a consequence have been subjects of debate. Traditionally, poly(A)-mRNAs including those with a 3' histone stem loop have been used as control reporters that are resistant to deadenylation. However, intrinsically low translation efficiency of poly(A)-mRNAs and a non-canonical translation mode for 3' histone stem-loop mRNAs have complicated the interpretation of observed translational repression. By utilizing the internalized poly(A) sequence this study successfully separated 'pure' translational repression from deadenylation without substantially affecting overall translational efficiency (Fukaya, 2011).

The data show that miRNAs can efficiently repress translation in vitro, independently of deadenylation. Therefore, miRNA-mediated translational repression is not a mere consequence of deadenylation. Remarkably, such pure translational repression efficiently occurs even when PABP function is blocked. Mishima and colleagues (2012) also reached a similar conclusion that miRNAs cause translational repression independently of deadenylation and PABP by using zebrafish embryos as an in vivo model system, suggesting that the PABP-independent translational repression pathway is conserved among species. In contrast, it has been shown that miRNAs can shorten the poly(A) tail of target mRNAs when translation is completely blocked. Therefore, miRNAs induce deadenylation and translational repression entirely independently, even though these two silencing pathways are tightly linked to each other (Fukaya, 2011).

It was previously proposed that GW182-PABP interaction induces deadenylation of miRNA target mRNAs by sequestering the poly(A) tail into the vicinity of deadenylase complexes and/or by reducing the affinity of PABP for the poly(A) tail and exposing it to deadenylase complexes. However, the current data collectively show that GW182-PABP interaction is dispensable for miRNA-mediated deadenylation; at least in the Drosophila in vitro system, 'net' deadenylation by miRNAs was unaffected by blocking of PABP function, even though PABP generally influenced the poly(A) length in the 'background'. This contrasts with a previous report using Krebs-2 in vitro system, where depletion of PABP abolished miRNA-mediated deadenylation but did not apparently affect the overall poly(A) length). Although it is unclear at this point exactly what makes this difference between the two in vitro systems, it is noted that the activity of cytoplasmic poly(A) regulation can vary among cell types. As such, the poly(A) length might be more plastic in fly embryos and embryo-derived S2 cells than in Krebs-2 ascites carcinoma cells. Indeed, it is known that the poly(A) length is actively and dynamically regulated in the cytoplasm during early development as well as in nerve cells. Given that GW182 likely utilizes multiple pathways to silence their target mRNAs, contribution of each pathway to shorten the target poly(A) tail could be different in each system, perhaps depending on how the poly(A) length is maintained (Fukaya, 2011).

The C-terminal part of GW182, containing PAM2, RRM, and C-term, has been termed as the 'silencing domain', based on the fact that deletion of these domains or mutations within them largely impair miRNA-mediated silencing. The finding of the direct interaction between GW182 and PABP has reinforced this idea. However, the quantitative data with normalized protein levels show that not only the C-terminal silencing domain but also the N-terminal (GW repeats and UBA) and central (Q-rich) regions of GW182 can potentially induce silencing. In addition, a novel conserved motif was identified called QSR within the linker between Q-rich and PAM2, and it is expected that there are many other functional motifs in GW182. Indeed, during the review process of this manuscript, three papers were concurrently published showing that GW182 directly binds to NOT1, a deadenylase complex subunit, through various motifs including QSR (therein referred to as CCR4-interacting motif (CIM)-1), two LWG repeats in C-term (CIM-2; missing in fly GW182) and other multiple tryptophan-containing motifs dispersed in N- and C-terminal parts (Braun, 2011; Chekulaeva, 2011; Fabian, 2011). Moreover, Mishima (2012) has identified that another novel conserved motif called P-GL within the linker between PAM2 and RRM in zebrafish GW182, which corresponds to 1063-1067 of fly GW182, plays a key role in PABP-independent translational repression in zebrafish embryo. Most importantly, none of these functional domains alone appears to be sufficient for silencing; they act together to provide a platform for maximal deadenylation and translational repression only in the context of the full-length GW182. Of course, contribution of each functional domain to the overall silencing should vary depending on the difference in rate-limiting step(s). In this regard, the previously proposed model that the interaction between the silencing domain of GW182 and PABP contributes to miRNA-mediated silencing cannot be excluded, but obviously this is not the only mechanism of miRNA action. Further investigation from a comprehensive and unbiased view is required to clarify how multiple functional domains of GW182 together recruit downstream silencing components and exactly how they repress translation (Fukaya, 2011).

In animal gonads, PIWI proteins and their bound 23-30 nt piRNAs guard genome integrity by the sequence specific silencing of transposons. Two branches of piRNA biogenesis, namely primary processing and ping-pong amplification, have been proposed. Despite an overall conceptual understanding of piRNA biogenesis, identity and/or function of the involved players are largely unknown. This study demonstrates an essential role for the female sterility gene shutdown in piRNA biology. Shutdown, an evolutionarily conserved cochaperone collaborates with Hsp90 during piRNA biogenesis, potentially at the loading step of RNAs into PIWI proteins. Shutdown is shown to be essential for both primary and secondary piRNA populations in Drosophila. An extension of this study to previously described piRNA pathway members revealed three distinct groups of biogenesis factors. Together with data on how PIWI proteins are wired into primary and secondary processing, a unified model for piRNA biogenesis is proposed (Olivieri, 2012).

PIWI interacting RNAs (piRNAs) are a class of animal small RNAs. They are bound by PIWI family proteins and guide the sequence specific silencing of selfish genetic elements such as transposable elements (TEs). Defects in the piRNA pathway lead to TE derepression, genomic instability and ultimately sterility (Olivieri, 2012).

In Drosophila, most piRNAs are generated from two sources; on the one hand, these are piRNA cluster transcripts that originate from discrete genomic loci and serve as reservoirs of TE sequences; on the other hand, these are RNAs derived from active TEs that engage - together with cluster transcripts - in a piRNA amplification loop called the ping-pong cycle (Olivieri, 2012).

Two modes of piRNA biogenesis exist: (1) during primary piRNA biogenesis, a single stranded RNA is processed into pre-piRNAs, which are loaded onto PIWI proteins and are subsequently 3' trimmed and methylated, yielding mature piRNA induced silencing complexes (piRISCs). (2) piRISCs with active slicer activity can trigger secondary piRNA biogenesis, where a new piRNA is formed out of the sliced target RNA. In the presence of corresponding sense and antisense precursor RNAs, secondary piRNA biogenesis acts as the ping-pong amplification loop. The two piRNAs engaged in ping-pong have opposite orientation and exhibit a characteristic ten nucleotide 5' overlap (ping-pong signature) (Olivieri, 2012).

Primary and secondary piRNA biogenesis co-occur in germline cells, complicating the genetic and mechanistic dissection of these processes. However, somatic cells of the gonad also harbor a piRNA pathway and this is based exclusively on primary piRNA biogenesis. The Drosophila ovary is therefore ideally suited to identify and characterize factors required for either primary or secondary piRNA biogenesis or both (Olivieri, 2012).

Somatic support cells of the Drosophila ovary express Piwi as the only PIWI family protein. Primary piRNA biogenesis is thought to take place in peri-nuclear Yb-bodies, where the RNA helicases Armitage (Armi) and Yb as well as the TUDOR domain protein Vreteno (Vret) accumulate. In addition to these three factors, the putative mitochondria-localized nuclease Zucchini (Zuc) and the RNA helicase Sister of Yb (SoYb) are essential for piRNA biogenesis in the soma. Formation of mature Piwi-RISC triggers its nuclear import, while failure in piRNA biogenesis results in destabilization of presumably unloaded Piwi. Mature Piwi-RISC triggers TE silencing by an unknown mechanism that requires Piwi's nuclear localization but not its slicer activity (Olivieri, 2012).

With the exception of Yb, the above mentioned biogenesis factors are also essential in germline cells for the formation of Piwi-RISC. Germline cells, however, express two additional PIWI proteins, Aubergine (Aub) and Argonaute 3 (AGO3), which localize to the cytoplasm and are enriched in peri-nuclear nuage. Aub and AGO3 are the main players in the ping-pong cycle. Several factors with essential or modulatory roles in the ping-pong cycle have been identified. These are the RNA helicases Spindle-E and Vasa and the TUDOR domain proteins Krimper, Tejas (Tej), Qin and Tudor (Olivieri, 2012).

The analysis of piRNA populations from wild-type and piRNA pathway mutant ovaries indicated that Piwi is mainly a recipient of primary piRNAs, while Aub and AGO3 are predominantly or exclusively recipients of secondary piRNA biogenesis. Given this, three major questions arise: (1) Are primary and secondary piRNA biogenesis processes genetically and mechanistically separate or do common factors act in both processes? (2) In which processing step do identified piRNA biogenesis factors act? (3) How are the three PIWI family proteins wired into piRNA biogenesis? In other words, are certain PIWI proteins only receiving primary or only secondary piRNAs (Olivieri, 2012)?

This study shows that the female sterility gene shutdown encodes a piRNA biogenesis factor. Shu is required for all piRNA populations in ovaries and it acts downstream of known piRNA biogenesis factors. A comparison of Shu to several other pathway factors led to the definition of three major groups of piRNA biogenesis factors. In combination with data on how PIWI proteins are wired into piRNA biogenesis, a model is proposed that accounts for the distinct association of piRNA subpopulations with specific PIWI proteins in Drosophila (Olivieri, 2012).

The outcome of this work is threefold: (1) The cochaperone Shutdown is essential for the biogenesis of all Drosophila piRNA populations. (2) Three major groups of piRNA biogenesis factors can be distinguished. (3) Piwi and Aub but not AGO3 are loaded with primary piRNAs, explaining how the cell maintains highly specific piRNA populations in the three PIWI proteins (Olivieri, 2012).

A remarkable feature of the shu mutant phenotype is that piRNA populations for every TE collapse. This already points to a common piRNA biogenesis step downstream of the primary and secondary branches. Epistatic analysis in somatic follicle cells is consistent with Shu acting at a late step in piRNA biogenesis: Shu is not required for the localization of any known biogenesis factor to Yb-bodies. On the other hand, Shu's localization to Yb-bodies depends on all other biogenesis factors and even on Piwi, arguing that unloaded Piwi recruits Shu to the Yb-body. Similarly, Shu colocalizes with nonloadable AGO3 in OSCs as well as in ovaries defective of ping-pong in discrete foci that also contain and are dependent on Krimp. Thus, in wild-type and in biogenesis mutants, Shu appears to colocalize with unloaded PIWI proteins (Olivieri, 2012).

Shu's C-terminal TPR domain falls into the class of Hsp90 binders and Hsp90 is important for small RNA loading into Argonaute proteins (Iki, 2010; Iwasaki, 2010; Miyoshi, 2010). In addition, the plant cochaperone Cyp40 interacts with Hsp90 via its TPR domain and is a critical cofactor for small RNA loading into AGO1. The genetic and localization data support an analogous role for Shu and Hsp90 during small RNA loading into PIWI proteins. Clearly, in vitro assays will be crucial to dissect the precise order of events and the molecular role of Shu, especially its PPIase domain (Olivieri, 2012).

A major challenge in the field is to assemble piRNA biogenesis factors into pathways that explain the stereotypic populations of piRNAs in vivo. Advantage was taken of efficient germline specific knockdowns to study the impact of several factors on piRNA populations. Based on levels and localization of PIWI proteins as well as on piRNA populations obtained from several pathway factor knockdowns, three major groups of piRNA biogenesis factors are proposed (Olivieri, 2012).

Group I factors are required for primary piRNA biogenesis but dispensable for secondary biogenesis. In fact, piRNAs that initiated ping-pong in group I knockdowns were amplified and ping-pong signatures of such TEs were strongly increased, presumably as primary piRNAs that do not feed into ping-pong were absent (Olivieri, 2012).

Group II factors are specific for ping-pong, as primary piRNA biogenesis feeding into Piwi was unaffected. An alternative explanation that cannot be excluded is that some or all group II genes are required specifically for Aub biology (primary and secondary) per se. This would similarly leave Piwi bound piRNAs intact and would lead to a collapse in ping-pong. Given the data on Aub loading in OSCs, a model is favored however where the primary biogenesis machineries that feed Aub and Piwi are very similar (Olivieri, 2012).

Finally, group III factors are required for the biogenesis of Piwi/Aub/AGO3 bound piRNAs. The prototypic member of this group is Shu. Loss of Shu affects essentially all piRNA populations to the same extent. It is noted that analysis of piRNA populations from vret mutants indicated a role for this group III factor in primary biogenesis but not ping-pong (Handler, 2011; Zamparini, 2011). The distorted tissue composition of vret mutant ovaries coupled with perdurance of maternal Vret protein or RNA may underlie this discrepancy. The existence of group III factors predicts that primary and secondary piRNA biogenesis feed into a final piRISC maturation step that requires a set of common factors for all PIWI proteins. Given that piRNA biogenesis -- irrespective of the source of the precursor RNA -- requires an RNA loading step as well as a 3' trimming step, the existence of group III factors suggests itself (Olivieri, 2012).

The three proposed groups serve as a rough classification of biogenesis factors. Clearly, at a molecular level, the precise role of each factor within the biogenesis process will vary considerably. Of note, the classification of group I and group II genes extends to the mouse piRNA pathway. The Armi and Zuc orthologs MOV10L1 and PLD6 are required for primary piRNA biogenesis, whereas mouse VAS and TDRD9 (mouse Spn-E) were reported to be dispensable for primary biogenesis but are required for secondary biogenesis pathway (Olivieri, 2012).

The data indicate that Aub is not only loaded via ping-pong, but also via primary piRNA biogenesis. It is also postulate that Aub and Piwi proteins are wired into primary piRNA biogenesis processes in a very similar manner, meaning that they require the same or highly overlapping core factors (e.g., Armi or Zuc). In agreement with this, ectopically expressed Aub is loaded in OSCs that harbor a fully functional primary pathway but lack critical ping-pong factors such as Vas. The genetic requirements for Aub loading in OSCs are identical to those for Piwi. It is extrapolated from this that the core primary biogenesis machinery that loads Piwi in the soma also loads Piwi and Aub in the germline. Analyses of piRNA populations from armi versus piwi or aub-GLKDs support a model where Armi and Zuc are required for the biogenesis of both Piwi and Aub bound primary piRNAs. The possibility is not excluded that - despite a similar biogenesis machinery - populations of primary piRNAs in Aub and Piwi are different. For example, differences in subcellular localizations of PIWI proteins as well as piRNA precursor RNAs might result in such differences (Olivieri, 2012).

In contrast to Aub, AGO3 was unstable in OSCs. Coexpression of Aub or simultaneous knockdown of krimp had no impact on AGO3 stability. It is therefore concluded that primary piRNA biogenesis is incompatible with AGO3. In fact, also in the germline genetic data indicated that AGO3 depends on secondary piRNA biogenesis for being loaded. Blocking AGO3's access to the primary biogenesis machinery would allow the cell to load AGO3 with a unique class of piRNAs if it couples AGO3 loading to a precursor RNA originating from Aub-slicer mediated cleavage of an active TE. This would explain the remarkable bias of AGO3 bound piRNAs being sense and carrying an Adenosine at position ten (Olivieri, 2012).

Interestingly, on a primary sequence level Aub -- despite its significantly different biology -- is more closely related to Piwi than to AGO3. A critical question emanating from this is to which extent Piwi is participating in ping-pong, and if it does not, why. A weak, yet statistically significant, ping-pong signature has been observed between Piwi and AGO3 bound piRNAs. This could mean that there is indeed low level of Piwi-AGO3 ping-pong. An alternative explanation is that the Piwi-AGO3 signal is a misleading computational signal: If Piwi and Aub are loaded via the same primary biogenesis machinery, initiator piRNAs for ping-pong that end up in Aub also end up in Piwi. As primary piRNA biogenesis appears to be nonrandom and preferentially processed piRNAs likely trigger ping-pong more robustly, an 'artificial' AGO3/Piwi ping-pong signature might result (Olivieri, 2012).

What could be the molecular basis of why Piwi does not or only moderately participate in ping-pong? Either, specific features of Aub (e.g., symmetric Arginine methylation) are funneling this protein into ping-pong and similar features are absent on Piwi. Or, the mere sequestration of Piwi into the nucleus prevents Piwi from participating in ping-pong. Notably, N-terminally truncated Piwi that is still loaded but that cannot translocate into the nucleus is enriched in nuage the proposed site of secondary piRNA biogenesis. A simple difference in the subcellular localization of Aub and Piwi might thus contribute to the dramatic differences of piRNA populations residing in Aub or Piwi (Olivieri, 2012).

Argonaute (Ago) proteins are typically recruited to target messenger RNAs via an associated small RNA such as a microRNA (miRNA). This study describe a new mechanism of Ago recruitment through the Drosophila Smaug RNA-binding protein. Smaug interacts with the Ago1 protein, and Ago1 interacts with and is required for the translational repression of the Smaug target, nanos mRNA. The Ago1/nanos mRNA interaction does not require a miRNA, but it does require Smaug. Taken together, these data suggest a model whereby Smaug directly recruits Ago1 to nanos mRNA in a miRNA-independent manner, thereby repressing translation (Pinder, 2013).

The data indicate that Smaug directly recruits Ago1 to nos mRNA in a miRNA-independent fashion. Interestingly, previous work suggests similarities between the mechanisms that Smaug and Ago use to regulate mRNAs. For example, translationally repressed nos mRNA is polysome associated similar to some transcripts that are repressed by Ago proteins. However, it is unclear if Smaug protein participates in repression of nos mRNA that is polysome associated or if this is mediated the RNA-binding protein Glorund, which is another regulator of nos mRNA. Another similarity between Smaug and Ago-mediated mechanisms comes from in vitro translation extracts showing that both Smaug and Ago1 repress translation through steps that function downstream of translation initiation and require ATP. While these similarities are consistent with Smaug-mediated translational repression functioning, at least to some extent, through Ago1, it is noteworthy that several other mechanisms have been shown to have roles in translational repression by Ago protein (Pinder, 2013).

The data also indicate that while Ago1 functions in nos translational repression it does not induce nos mRNA degradation. Thus, in the context of nos mRNA, Ago1 does not induce transcript decay. In contrast, Smaug has a role in both translational repression and degradation of nos message, indicating that some of Smaug's functions are Ago1-independent (Pinder, 2013).

The interaction of Smaug with both Ago1 and Ago2 could reflect a common binding site on Ago proteins that mediates their interaction with Smaug. However, no ectopic Nos protein was seen in Ago2 mutant embryos. This could suggest that while the Smaug/Ago1 interaction is functional, the interaction with Ago2 is not, perhaps because of the different mechanisms that are used by Ago1 and Ago2 to repress translation (Pinder, 2013).

The data presented in this study, along with previous work, indicate that Smaug uses several mechanisms to regulate its target mRNAs. In addition to translational repression mediated by Smaug/Ago1, Smaug interacts with the Cup protein, which in turn interacts with the cap-binding protein eIF4E. eIF4E bound to the 5' cap of an mRNA indirectly recruits the 40S ribosomal subunit to an mRNA through eIF4E's interaction with eIF4G. As Cup blocks the eIF4E/eIF4G interaction, formation of the Smaug/Cup/eIF4E complex on a target mRNA represses translation initiation. Smaug also recruits the Ccr4/Not deadenylase to target mRNAs resulting in removal of the transcripts poly(A) tail, thus repressing translation and/or inducing transcript degradation. Interestingly, Smaug has also been shown to interact with the Piwi-type Ago proteins, Aubergine and Ago3. That study proposed that a complex consisting of Smaug, Aubergine with associated piwiRNAs and the Ccr4/Not deadenylase is recruited to nos mRNA through binding of both Smaug to the nos SREs and piwiRNAs to complementary to sequences in the nos 3' UTR (Pinder, 2013).

These models of Smaug function raise the question as to why one RNA-binding protein uses several mechanisms to repress its target transcripts? One possibility is that by using several mechanisms, Smaug ensures that its targets are efficiently silenced. This might be especially important for a transcript, such as nos, where even low levels of inappropriate expression are lethal to the embryo. Alternatively, Smaug might use different mechanisms to regulate different target mRNAs. Indeed, while Smaug induces the degradation of Hsp83 mRNA, it does not repress Hsp83 translation. In contrast, while Smaug represses nos translation, it has only a modest role in destabilizing nos mRNA. This differential regulation could reflect the fact that the location of the SREs in the target mRNA (e.g., 3' UTR for nos versus open reading frame for Hsp83) and/or more cis-elements within these target mRNAs along with the trans-acting factors that recognize these influence the mechanisms of Smaug function (Pinder, 2013).

While the data indicate that Smaug recruits Ago1 to nos mRNA via a miRNA-independent mechanism, it is still possible that a miRNA bound to Ago1, but not base-paired with nos mRNA, could be important for nos regulation. In this model, the miRNA would be required for Ago1 to repress nos translation at a step downstream of recruitment. For example, miRNA binding to Ago1 might be required to induce allosteric changes in Ago1 that facilitate its interaction with other factors that are required for Ago1-mediated repression. In this model, any miRNA would be sufficient for repression as it is not base-paired with the target transcript. Indeed, it has been suggested that miRNA binding might be involved in allosteric changes that are important for Ago-mediated repression, while other studies have shown that miRNA binding confers substantial structural stability to an Ago protein. It is noted that the Ago1 miRNA-binding mutant used in the present study also fails to interact with GW182, an Ago-binding protein that is essential for translational repression; therefore it could not be used to test for miRNA-independent translational repression of nos mRNA in vivo (Pinder, 2013).

A variety of RNA-binding proteins have been shown to function at numerous steps in miRNA/Ago-mediated post-transcriptional regulation. For example, both a mammalian and Caenorhabditis elegans Pum form a complex that includes an Ago protein, and in vitro experiments showed that the mammalian version of this complex represses translation of a reporter mRNA that carried only Pum-binding sites. The lack of miRNA-binding sites within this reporter suggests that in this in vitro system Pum is able to recruit Ago to an mRNA in a miRNA-independent manner. It is, however, unclear whether this Pum/Ago complex is able to recognize target mRNAs in vivo in the absence of miRNA binding. Indeed, it is suggested that in vivo targeting of the Pum/Ago complex might involve the presence of both Pum and miRNA-binding sites within a transcript. Thus, the data indicating that, in vivo, Smaug can recruit Ago1 to an mRNA in a miRNA-independent fashion, combined with other in vitro data suggesting that Pum can also directly recruit an Ago protein, suggests that other RNA-binding proteins might also function in a similar manner (Pinder, 2013).

In the Drosophila ovary, bone morphogenetic protein (BMP) ligands maintain germline stem cells (GSCs) in an undifferentiated state. The activation of the BMP pathway within GSCs results in the transcriptional repression of the differentiation factor bag of marbles (bam). The Nanos-Pumilio translational repressor complex and the miRNA pathway also help to promote GSC self-renewal. How the activities of different transcriptional and translational regulators are coordinated to keep the GSC in an undifferentiated state remains uncertain. Data presented in this study show that Mei-P26 cell-autonomously regulates GSC maintenance in addition to its previously described role of promoting germline cyst development. Within undifferentiated germ cells, Mei-P26 associates with miRNA pathway components and represses the translation of a shared target mRNA, suggesting that Mei-P26 can enhance miRNA-mediated silencing in specific contexts. In addition, disruption of mei-P26 compromises BMP signaling, resulting in the inappropriate expression of bam in germ cells immediately adjacent to the cap cell niche. Loss of mei-P26 results in premature translation of the BMP antagonist Brat in germline stem cells. These data suggest that Mei-P26 has distinct functions in the ovary and participates in regulating the fates of both GSCs and their differentiating daughters (Li, 2012).

Evidence is provided that Mei-P26 promotes GSC self-renewal in addition to its previously described role in negatively regulating the miRNA pathway during germline cyst development. Disruption of mei-P26 results in a bam-dependent GSC loss phenotype and further characterization reveals that Mei-P26 fosters BMP signal transduction within GSCs by repressing Brat protein expression. In addition, Mei-P26 also appears to participate in the miRNA-mediated silencing of orb mRNA in GSCs. These results indicate that Mei-P26 carries out multiple functions within the Drosophila ovary and might be at the center of a molecular hierarchy that controls the fates of GSCs and their differentiating daughters (Li, 2012).

Three observations suggest that mei-P26 functions within GSCs. First, the average number of GSCs per terminal filament decreases from an average of two to well below one in mei-P26 mutant ovaries. Second, mei-P26 mutant germline clones are rapidly lost from the GSC niche. Third, syncytial cysts and Bam-expressing cells are often observed immediately adjacent to the cap cells in mei-P26 mutant ovaries (Li, 2012).

Research over the last ten years has shown that BMP ligands emanating from cap cells at the anterior of the germarium initiate a signal transduction cascade in GSCs that results in the transcriptional repression of bam. Stem cell daughters one cell diameter away from the cap cell niche express bam, suggesting that a steep gradient of Dpp availability or responsiveness exists between GSCs and cystoblasts. Recent work has shed light on how various mechanisms antagonize BMP signaling in cystoblasts. For example, the ubiquitin ligase Smurf (Lack -- FlyBase) promotes germline differentiation and partners with the serine/threonine kinase Fused to reduce levels of the Dpp receptor Tkv in cystoblasts. The TRIM-NHL domain protein Brat also functions in cystoblasts, serving to translationally repress Mad expression. Notably, inappropriate expression of Brat within GSCs results in a stem cell loss phenotype. Brat itself is translationally repressed in GSCs by the Pumilio-Nanos complex. Mutant phenotypes and co-IP experiments presented in this study support a model in which Mei-P26 partners with Nanos to repress Brat expression in GSCs. This negative regulation of Brat expression protects the BMP signal transduction pathway in GSCs from inappropriate deactivation (Li, 2012).

Mei-P26 appears to enhance miRNA-dependent translational silencing within GSCs based on several lines of experimental evidence. First, co-IP experiments using ovarian extracts from c587-gal4>UAS-dpp and bam mutants suggest that Mei-P26 physically associates with Ago1 and GW182 in undifferentiated germ cells. Second, disruption of mei-P26 results in a GSC loss phenotype, similar to the effects of disrupting components of the miRNA pathway tested to date. Third, Mei-P26 and Ago1 can physically associate with the same target mRNA. Finally, disruption of either Ago1 or mei-P26 results in increased expression of this target in GSCs. The evidence that Mei-P26 promotes miRNA action in certain contexts is consistent with the established activities of its close homologs NHL-2 and TRIM32 (Li, 2012 and references therein).

It is proposed that Mei-P26 regulates GSC self-renewal and early germ cell differentiation through distinct mechanisms. In GSCs, Mei-P26 promotes self-renewal by repressing the expression of Brat and potentially other negative regulators of BMP signal transduction. Within stem cells, Mei-P26 also functions together with miRISC to attenuate the translation of specific mRNAs. miRISC does not appear to target brat mRNA based on clonal data. However, the possiblity cannot be ruled out that the enhancement of miRNA-mediated silencing of some mRNAs by Mei-P26 contributes to stem cell self-renewal. Interestingly, recent findings suggest that Pumilio can function together with the miRNA pathway in certain contexts In BJ primary fibroblasts, Pumilio 1, miR-221 and miR-222 regulate the expression of p27 in a 3' UTR-dependent manner. In response to growth factors, Pumilio 1 becomes phosphorylated, which in turn increases its RNA binding activity. Pumilio 1 binding to p27 mRNA results in a conformational change in the 3' UTR that allows miR-221 and miR-222 to bind more efficiently, resulting in greater repression of p27. Perhaps, together, Drosophila Pumilio, Nanos, Ago1 and Mei-P26 also silence specific messages in specific contexts. Identifying more direct in vivo targets for these proteins within GSCs will be crucial for testing this idea (Li, 2012).

In cystoblasts, Mei-P26 promotes germline cyst development by antagonizing the miRNA pathway. This study shows that Mei-P26 can also promote miRNA translational repression in another cell, the GSC. Evidence is provided that Mei-P26 physically associates with miRISC and co-regulates translation of at least one mRNA, orb, through specific elements within its 3′UTR. In cystoblasts and early developing cysts, the induction of Bam expression might cause Mei-P26 to switch from an miRISC-associated silencer to an miRNA antagonist. How Bam activates this switch is currently under investigation. The finding that Mei-P26 functions in both GSCs and differentiating cysts hints at a mechanism whereby different translational repression programs coordinate changes in cell fate (Li, 2012).

Further work will be needed to determine the specific biochemical function of Mei-P26 when it associates with either the Nanos complex or miRISC. Like other TRIM-NHL domain proteins, Mei-P26 contains a RING domain that may have E3 ubiquitin ligase activity. Based on results presented in this study, it is proposed that Mei-P26 and perhaps other TRIM-NHL domain proteins act as effectors for multiple translational repressor complexes. In this model, Mei-P26 is targeted to specific mRNAs through sequence-directed RNA-binding proteins. Specific protein substrates of Mei-P26 in the germline have not yet been determined but identifying these targets will provide key insights into how Mei-P26 and other related TRIM-NHL domain proteins regulate translational repression. Furthermore, the Mei-P26 complex is likely to target additional mRNAs for silencing in both GSCs and developing cysts. Identifying more of these mRNAs will further elucidate the complex translational regulatory hierarchies that control the balance between stem cell self-renewal and differentiation (Li, 2012).

Because microRNAs (miRNAs) influence the expression of many genes in cells, discovering how the miRNA pathway is regulated is an important area of investigation. This study found that the Drosophila miRNA-induced silencing complex (miRISC) exists in multiple forms. A constitutive form, called G-miRISC, is comprised of Ago1, miRNA, and GW182. Two distinct miRISC complexes that lack GW182 are regulated by mitogenic signaling. Exposure of cells to serum, lipids, or the tumor promoter PMA suppressed formation of these complexes. P-miRISC is comprised of Ago1, miRNA, and Loqs-PB, and it associates with mRNAs assembled into polysomes. The other regulated Ago1 complex associates with membranous organelles and is likely an intermediate in miRISC recycling. The formation of these complexes is correlated with a 5- to 10-fold stronger repression of target gene expression inside cells. Taken together, these results indicate that mitogenic signaling regulates the miRNA effector machinery to attenuate its repressive activities (Wu, 2013).

This study found that different miRISC complexes are present in S2 cells, depending upon extracellular signals received by the cells. A constitutive G-miRISC complex composed of Ago1, miRNA, and GW182 is present under all signaling conditions tested. Other groups have shown that G-miRISC in S2 cells suppresses target mRNAs via inhibition of translation initiation and enhanced mRNA decay. This study found that lipid signaling does not affect G-miRISC but blocks other miRISC complexes from forming. This signaling is likely mediated by PKC because a phorbol ester mimics the effect of lipids on miRISC formation. Signaling blocks the formation of P-miRISC, which contains Ago1, miRNA, and Loqs-PB, but not GW182. P-miRISC represses translation of target mRNAs, which is manifested in polysome association of the complex. Thus, this work reveals a mechanistic shift in miRISC-executed translation repression under the influence of extracellular lipid signals. In the presence of lipid signaling, initiation is inhibited, and this occurs by G-miRISC. In the absence of lipid signaling, it is proposed that cells generate two levels of translational repression: one mediated by G-miRISC that inhibits initiation, and one mediated by P-miRISC that inhibits elongation. It is proposed that each miRISC complex independently represses the same target, and because they act in series (initiation - elongation), the net result on protein synthesis is the product (not sum) of each inhibitory step. This would provide the strongly synergized repression of reporter protein synthesis that was observed after serum withdrawal (Wu, 2013).

P-miRISC resembles the miRNA loading complex (miRLC) complex in terms of subunit composition (Ago1, Loqs-PB), but the two differ in one important way. Whereas miRLC contains premiRNA, P-miRISC contains mature miRNA. Thus, P-miRISC has an inherent potential to engage target mRNAs via base pairing interactions. It is suggested that P-miRISC is formed by the processing and loading of mature miRNA into Ago1 within the miRLC. Rather than releasing Loqs-PB/Dcr-1 and recruiting GW182, the loaded Ago1 retains Loqs-PB and never recruits GW182. P-miRISC can then engage target mRNAs, but its subunit composition dictates a different mode of repression upon the target (Wu, 2013).

Although GW182 and Loqs-PB binding to Ago1 are mutually exclusive, P-miRISC is not simply a default state when GW182 recruitment fails to occur. Knockdown of GW182 was insufficient to induce formation of P-miRISC. Moreover, formation of P-miRISC did not appear to occur at the expense of G-miRISC levels, as measured in sedimentation and immunoprecipitation experiments. This suggests a mechanism in which stable loading of miRNA is limited by the availability of cofactors for Ago1. Under serum-fed conditions, only GW182 is available, whereas both GW182 and Loqs-PB are available under serum-free conditions. This possibly offers a rapid way to modulate miRISC levels without the need for synthesis of more cofactors (Wu, 2013).

The switch in miRISC formation is regulated by PKC, but how this switch occurs is not clear. A recent study demonstrated that the mammalian homolog of Drosophila Ago1 can be phosphorylated by Akt3, which contributes to increased miRISC-mediated translation repression (Horman, 2013). However, no evidence was found for differential phosphorylation of Ago1 in S2 cells. A study of the mammalian ortholog of Loqs-PB, called TRBP, found it to be phosphorylated by ERK kinase in response to PKC. Phosphorylation stabilized miRLC and increased processing of growth-promoting miRNAs. The same mechanism was not shown for Loqs-PB, and examination of the Loqs-PB sequence failed to find strict conservation of those sites (Wu, 2013).

A second Ago1 complex also appears when lipid signaling is absent. Membrane-associated Ago1 likely contains miRNA, but not Loqs-PB or GW182. Association of mammalian Ago proteins with late endosomes has been previously observed. Drosophila Ago1 has also been observed to associate with endosomes in vivo. Endosomes have been proposed to serve as sites for miRISC turnover whereby miRISC continuously associates and releases from endosomes, constituting a mechanism that promotes miRISC recycling onto new targets. Thus, membrane-associated Ago1 may represent an intermediate in miRISC turnover. If so, where does the membrane- associated Ago1 originate? Several lines of evidence suggest that it originates from P-miRISC. First, its appearance precisely correlates with P-miRISC. Second, it is sensitive to puromycin treatment, which also disrupts association of P-miRISC with polysomes. However, membrane-associated Ago1 does not sediment in ribosome-containing fractions. Third, insulin specifically inhibits membrane-associated Ago1, arguing that membrane-associated Ago1 is not an obligate precursor of P-miRISC. The simplest interpretation of these data is that membrane- associated Ago1 is formed from a P-miRISC precursor. If so, then Loqs-PB dissociation must be involved in the conversion because Loqs-PB is not found in the membrane-associated complex. A similar manner of cofactor stripping was observed for GW182, which dissociated from Ago-miRNA complexes when they associated with endosomes. Perhaps, cofactor dissociation is a fundamental part of the recycling mechanism (Wu, 2013).

This model might provide some insights into a long-standing controversy in the miRNA field. Some studies have found evidence for translation initiation as the regulated step, whereas others have found evidence for translation elongation. This work provides a potential explanation for these differences. That is, experimental model systems experiencing diverse extracellular signals might respond accordingly to form distinct types of miRISC complexes, which regulate different steps of translation. Thus, all studies have depicted an accurate picture of miRISC activity because signals that dictate miRISC subunit composition affect its mode of action (Wu, 2013).

MicroRNA (miRNA)-induced silencing complexes (miRISCs) repress translation and promote degradation of miRNA targets. Target degradation occurs through the 5'-to-3' messenger RNA (mRNA) decay pathway, wherein, after shortening of the mRNA poly(A) tail, the removal of the 5' cap structure by decapping triggers irreversible decay of the mRNA body. This study, carried out in Drosophila S2 cells, demonstrates that miRISC enhances the association of the decapping activators DCP1, Me31B and HPat with deadenylated miRNA targets that accumulate when decapping is blocked. DCP1 and Me31B recruitment by miRISC occurs before the completion of deadenylation. Remarkably, miRISC recruits DCP1, Me31B and HPat to engineered miRNA targets transcribed by RNA polymerase III, which lack a cap structure, a protein-coding region and a poly(A) tail. Furthermore, miRISC can trigger decapping and the subsequent degradation of mRNA targets independently of ongoing deadenylation. Thus, miRISC increases the local concentration of the decapping machinery on miRNA targets to facilitate decapping and irreversibly shut down their translation (Nishihara, 2013).

This study demonstrates that miRISCs enhance the association of DCP1, Me31B and HPat with miRNA targets in a miRNA-dependent manner. This association occurs even when the miRNA target lacks a 5' cap structure, an ORF and a poly(A) tail. Furthermore, mRNA reporters that are immune to deadenylation are degraded through decapping in the presence of the miRNA, indicating that miRISCs can promote decapping independently of deadenylation (Nishihara, 2013).

It is known that miRNAs promote the degradation of partially complementary targets through the 5'-to-3' decay pathway. In this pathway, decapping is coupled to deadenylation and does not occur on polyadenylated and fully functional mRNAs. This study investigated whether the decapping of miRNA targets occurs by default, as a consequence of this coupling, or whether miRISCs can also recruit decapping factors independently of deadenylation. miRISCs was shown to enhance the association of DCP1, Me31B and HPat with unadenylated 7SL-derived miRNA targets that have been transcribed by Pol III, indicating that the cap, a poly(A) tail and ongoing deadenylation are not required for the recruitment of decapping factors to miRNA targets. DCP1 association with the Alu-miRNA target reporterers, termed EvAluator reporters, was strictly miRNA dependent and stimulated by GW182. miRNAs and GW182 also stimulated the association of HPat and Me13B with the EvAluator reporters, indicating that these decapping factors interact with miRISC components that are bound to EvAluator RNA. However, DCP1 and Me31B did not interact with isolated AGO1 or GW182 in co-immunoprecipitation assays, suggesting that the interaction of decapping factors with miRISC is indirect or that DCP1 and Me31B recognize AGO1 and GW182 as a complex. Indeed, it is possible that the decapping factors are recruited by the PAN2-PAN3 or CCR4-NOT deadenylase complexes, which interact with GW182 proteins directly. Alternatively, DCP1 and Me31B might recognize AGO1 or GW182 only in a certain conformation that is adopted on target binding. Although HPat did interact with AGO1 and GW182 in co-immunoprecipitation assays, these interactions were apparently not sufficient to enhance the association of HPat and a polyadenylated miRNA target. Nevertheless, it is possible that these interactions contribute to the recruitment of HPat to deadenylated or oligoadenylated targets (Nishihara, 2013).

A previous study in human cells reported that EDC4 co-localized with a specific miRNA target in a miRNA-dependent manner, whereas DCP1 and RCK (the human ortholog of Dm Me31B) associated with the target, regardless of the presence of the miRNA. In agreement with that study, this study observed that decapping factors associate with miRNA targets in the absence of the miRNA; however, it was found that their binding is enhanced by the cognate miRNA. This enhancement was observed for targets that are not degraded or when degradation of the target was partially inhibited and may have escaped detection in co-localization studies (Nishihara, 2013).

A functional implication for the association of decapping factors with miRNA-targets is that miRNA targets can be decapped and degraded even in the absence of a poly(A) tail or ongoing deadenylation. In combination with previously published data, the current results suggest that miRISC has multiple and redundant activities to ensure robust gene regulation: it induces translational repression, deadenylation and decapping, the latter in both a deadenylation-dependent and -independent manner (Nishihara, 2013).

Under which circumstances can deadenylation-independent decapping contribute to silencing? Decapping might play a role in silencing specific miRNA targets when deadenylation is blocked or when decapping is blocked and targets that have undergone deadenylation accumulate. Indeed, deadenylation and decapping can be uncoupled on specific mRNAs, in different cell types and under various cellular conditions, leading to the accumulation of deadenylated repressed mRNAs. These mRNAs can re-enter the translational pool on polyadenylation or might be degraded in a deadenylation-independent manner once decapping resumes. For example, in immature mouse oocytes, DCP2 and DCP1 are not detectable, but their expression increases during oocyte maturation. Consequently, in immature oocytes, many maternal mRNAs (most likely including miRNA targets) accumulate in a deadenylated silenced form. These mRNAs may be polyadenylated and translated at later stages of oogenesis or embryogenesis. However, a fraction of these deadenylated targets may be degraded through decapping when DCP2 and DCP1 are expressed. Additionally, DCP1 and DCP2 are phosphorylated under cellular stress conditions, and DCP1 is hyperphosphorylated during mitosis. Under these conditions, a subset of mRNAs is stabilized, suggesting that DCP1 and DCP2 phosphorylation inhibits decapping. Thus, it is possible that under various stress conditions, miRNA targets accumulate in a deadenylated form because decapping is inhibited and that deadenylation-independent decapping is required for the clearance of these targets on return to normal cellular conditions (Nishihara, 2013).

Notably, in addition to their role in target degradation, decapping activators act as general repressors of translation even in the absence of decapping. Therefore, these factors could play a more direct role in the translational repression of miRNA targets in the absence of mRNA degradation (Nishihara, 2013).

In contrast to translational repression and deadenylation, decapping irreversibly shuts down translation initiation and commits mRNA to full degradation. Thus, decapping prevents the reversal of miRNA-mediated silencing. However, some miRNA targets have been shown to be released from miRNA-mediated repression in response to extracellular signals, suggesting that decapping is somehow blocked for these targets to allow for a fast reversal of their repression. How decapping is prevented in a target-specific manner remains unclear, but it can reasonable be expected that proteins associated with these targets block decapping in cis by preventing DCP2 access to the cap structure. These proteins may bind the cap structure directly or may act indirectly, for example, by stabilizing binding of the cap-binding protein eIF4E to the mRNA. Proteins that act as inhibitors of DCP2-mediated decapping have been described and include Variable Charged X chromosome VCX-A protein, YB-1, Y14 and Dm CUP. Thus, it is possible that additional proteins that prevent the decapping of specific mRNAs are present in eukaryotic cells. Such mRNA-specific decapping regulators would be likely to play an important role in controlling the reversibility of silencing. Alternatively, mRNAs can be recapped in the cytoplasm; however, how recapping is regulated remains unknown (Nishihara, 2013).

In addition to the aforementioned sequence-specific decapping regulators, the cap-binding protein eIF4E acts as a general inhibitor of decapping by limiting DCP2 access to the cap structure. Therefore, for decapping to occur, eIF4E needs to dissociate from the 5' end of the mRNA. This study shows that eIF4E remains bound to at least a fraction of silenced miRNA targets in cells in which decapping is blocked. Furthermore, the DCP2 catalytic mutant did not detectably associate with the mRNA target, even though its overexpression inhibited decapping. These observations suggest that DCP2 does not stably associate with miRNA targets. Similarly, DCP2 did not co-localize with miRNA targets in human cells, although in these cells, EDC4 co-localized with the target in a miRNA-dependent manner. Thus, the process of decapping may involve multiple consecutive steps, including the association of decapping activators with the target mRNA in the absence of DCP2, eIF4E dissociation, DCP2 recruitment and cap hydrolysis. The current results suggest that miRISC facilitates an early decapping step by increasing the local concentration of decapping factors on mRNA targets, promoting decapping independently of deadenylation. Further studies are necessary to determine whether, in addition to recruiting decapping factors, miRISC plays a more direct role in accelerating the chemical catalysis step of decapping (Nishihara, 2013).

Early development depends heavily on accurate control of maternally inherited mRNAs, and yet it remains unknown how maternal microRNAs are regulated during maternal-to-zygotic transition (MZT). This study found that maternal microRNAs are highly adenylated at their 3' ends in mature oocytes and early embryos. Maternal microRNA adenylation is widely conserved in fly, sea urchin, and mouse. This study identified Wispy, a noncanonical poly(A) polymerase, as the enzyme responsible for microRNA adenylation in flies. Knockout of wispy abrogates adenylation and results in microRNA accumulation in eggs, whereas overexpression of Wispy increases adenylation and reduces microRNA levels in S2 cells. Wispy interacts with Ago1 through protein-protein interaction, which may allow the effective and selective adenylation of microRNAs. Thus, adenylation may contribute to the clearance of maternally deposited microRNAs during MZT. This work provides mechanistic insights into the regulation of maternal microRNAs and illustrates the importance of RNA tailing in development. (Lee, 2014).

This study provides mechanistic insights into the regulation of maternally deposited miRNAs. The majority of maternal miRNAs are subject to adenylation during early development in Drosophila. The sequencing and northern blotting data indicate that the levels of adenylated miRNAs begin to drop at ~1.5 hr AEL with an average half-life of ~2 hr. This is substantially shorter than those estimated in mammalian cell lines (>100 hr considering dilution caused by cell division). The results collectively suggest that Wisp induces adenylation and facilitates miRNA downregulation. Thus, miRNA adenylation may provide a molecular basis for the clearance of maternally inherited miRNAs during MZT. MiRNA tailing is often associated with miRNA destabilization. An interesting example is poxvirus whose adenylyl transferase VP55 downregulates host miRNAs. It is conceivable that poxviruses have adopted the cellular adenylation/decay machineries to their own benefits. It is noted that adenylation may have an opposing effect in different contexts, as mammalian poly(A) polymerase GLD2 is known to stabilize certain miRNAs. It is yet unclear why the seemingly related modifications result in such different consequences. To dissect the mechanism behind the link between tailing and decay, it will be important to identify the enzyme that executes decay. Exoribonuclease candidates including exosome components and a known fly miRNA trimming factor, Nibbler, were tested by individual knockdown in S2 cells, it was not possible to the enzyme(s), implying that multiple factors may act redundantly, as in Arabidopsis (Lee, 2014).

It is intriguing that three distant species (fly, sea urchin, and mouse) show a similar pattern of miRNA expression and adenylation. In all species examined, relative contents of miRNA are low in mature oocytes. In frogs and zebrafish, the miRNA proportion was particularly small in oocytes and early embryos. Thus, maternal miRNAs may be cleared out at an early stage(s) of oocyte maturation in frogs and fish so that maternal miRNAs may not be transmitted to the next generation. In flies, maternal miRNA clearance is relatively delayed and overlaps with zygotic transcription. Therefore, while the clearance of maternal miRNA appears to be a universal phenomenon, the precise timing of the event varies among species. Consistently, previous studies have suggested that miRNAs may be inactive in oocytes and early development. Maternal and zygotic Dgcr8 null mouse embryos develop normally to the blastocyst stage, indicating that miRNA function is suppressed in oocytes and early embryos. In parallel, reporter assays for endogenous miRNA activity in mouse oocytes revealed a dramatic decrease in miRNA activity along oocyte maturation. The current data suggest that active clearance of miRNA via adenylation may be necessary for normal gene regulation in early embryo development (Lee, 2014).

Wisp physically associates with Ago1, which may explain why miRNAs are effectively adenylated. However, adenylation frequency varies among miRNA species, and does not show a strong correlation with abundance. Common features were sought among the highly adenylated miRNAs, but fno sequence motif was found that is significantly enriched in adenylated miRNAs. When miR-312 (that is highly adenylated in embryos) and miR-286 (that is not adenylated in embryos) were ectopically in S2 cells, both miRNAs were similarly adenylated by Wisp, suggesting that adenylation is not determined by intrinsic sequences of miRNA. Temporal differential expression was examined in embryos. Due to an inevitable normalization issue, the heatmap does not strictly reflect the changes in absolute abundance. Nevertheless, this analysis was useful to classify miRNAs according to their expression patterns during development. MiRNAs are clustered into three groups. The 'early' miRNAs show higher expression at early stage and decline after major zygotic genome activation (~1.5 hr AEL), indicating that these miRNAs are reduced during MZT. The 'late' miRNAs are mainly induced after zygotic activation in the later stage. The 'biphasic' miRNAs also show the highest levels in late embryos, but they are detected in early stage to some degrees. By comparing adenylation ratios of three groups, it was found that the 'early' group tends to be more frequently adenylated than the other groups. This result was validated by northern blotting of two representative miRNAs. miR-312 (an 'early' miRNA) is highly adenylated and gradually disappears, while miR-286 (a 'late' miRNA) is not modified and is induced zygotically. The data collectively suggest an intriguing possibility that 'early' maternal miRNAs are downregulated during MZT, although the mechanism underlying the preference of adenylation is unclear at this point (Lee, 2014).

It is conceivable that miRNA targets may affect adenylation rate in vivo. MiRNAs bound to complementary targets may be more susceptible to Wisp-mediated adenylation because the 3' end of guide miRNA is thought to be released from the PAZ domain of AGO protein when the miRNA binds to a highly complementary target. Not mutually exclusively, some miRNAs may be more accessible to Wisp than others in vivo. Certain miRNAs may be localized separately from Wisp, and the physical segregation may protect the miRNAs from adenylation and decay. It will be interesting in the future to dissect the mechanism underlying the selectivity of adenylation (Lee, 2014).

Knockout mutation of wisp results in defects in late meiosis and early development. MiRNA adenylation activity of Wisp may at least partly contribute to the phenotype, although currently it is not possible to separate the contribution of miRNA adenylation from that of mRNA polyadenylation. Cytoplasmic polyadenylation of mRNA induces translation, while miRNA adenylation facilitates miRNA decay. Because both events are expected to positively regulate protein synthesis, it will be interesting to ask if the two seemingly independent activities corroborate to enhance translation (Lee, 2014).

Thus far, the extensive miRNA adenylation in oocytes (over 30%) is unprecedented among uninfected cell types. MiRNAs typically show less than 2%-4% of adenylation. But it is conceivable that adenylation may be used for gene regulation in diverse cellular contexts. Some specific cell types and/or subcellular locations may contain highly adenylated miRNA species. It will be interesting in the future to investigate miRNA tailing in various cell types, such as in neurons, as cytoplasmic polyadenylation is known to be active and important for local translation in neural synapses (Lee, 2014).

The solution structures of the Argonaute2 PAZ domain bound to RNA and DNA oligonucleotides is described. The structures reveal a unique mode of single-stranded nucleic acid binding in which the two 3'-terminal nucleotides are buried in a hydrophobic cleft. It is proposed that the PAZ domain contributes to the specific recognition of siRNAs by providing a binding pocket for their characteristic two-nucleotide 3' overhangs (Lingel, 2004).

Argonaute proteins associate with small RNAs that guide mRNA degradation, translational repression, or a combination of both. The human Argonaute family has eight members, four of which (Ago1 through Ago4) are closely related and coexpressed in many cell types. To understand the biological function of the different Ago proteins, attempts were made to determine if Ago1 through Ago4 are associated with miRNAs as well as RISC activity in human cell lines. The results suggest that miRNAs are incorporated indiscriminately of their sequence into Ago1 through Ago4 containing microRNPs (miRNPs). Purification of the FLAG/HA-epitope-tagged Ago containing complexes from different human cell lines revealed that endonuclease activity is exclusively associated with Ago2. Exogenously introduced siRNAs also associate with Ago2 for guiding target RNA cleavage. The specific role of Ago2 in guiding target RNA cleavage was confirmed independently by siRNA-based depletion of individual Ago members in combination with a sensitive positive-readout reporter assay (Meister, 2004).

Argonaute proteins and small interfering RNAs (siRNAs) are the known signature components of the RNA interference effector complex RNA-induced silencing complex (RISC). However, the identity of 'Slicer', the enzyme that cleaves the messenger RNA (mRNA) as directed by the siRNA, has not been resolved. The crystal structure of the Argonaute protein from Pyrococcus furiosus is reported at 2.25Å resolution. The structure reveals a crescent-shaped base made up of the amino-terminal, middle, and PIWI domains. The Piwi Argonaute Zwille (PAZ) domain is held above the base by a 'stalk'-like region. The PIWI domain (named for the protein piwi) is similar to ribonuclease H, with a conserved active site aspartate-aspartate-glutamate motif, strongly implicating Argonaute as 'Slicer'. The architecture of the molecule and the placement of the PAZ and PIWI domains define a groove for substrate binding and suggest a mechanism for siRNA-guided mRNA cleavage (Song, 2004).

RNA silencing regulates gene expression through mRNA degradation, translation repression and chromatin remodelling. The fundamental engines of RNA silencing are RISC and RITS complexes, whose common components are 21-25 nt RNA and an Argonaute protein containing a PIWI domain of unknown function. The crystal structure of an archaeal Piwi protein (AfPiwi) is organised into two domains, one resembling the sugar-binding portion of the lac repressor and another with similarity to RNase H. Invariant residues and a coordinated metal ion lie in a pocket that surrounds the conserved C-terminus of the protein, defining a key functional region in the PIWI domain. Furthermore, two Asp residues, conserved in the majority of Argonaute sequences, align spatially with the catalytic Asp residues of RNase H-like catalytic sites, suggesting that in eukaryotic Argonaute proteins the RNase H-like domain may possess nuclease activity. The conserved region around the C-terminus of the PIWI domain, which is required for small interfering RNA (siRNA) binding to AfPiwi, may function as the receptor site for the obligatory 5' phosphate of siRNAs, thereby specifying the cleavage position of the target mRNA (Parker, 2004).

The slicer activity of the RNA-induced silencing complex resides within its Argonaute (Ago) component, in which the PIWI domain provides the catalytic residues governing guide-strand mediated site-specific cleavage of target RNA. This study reports on structures of ternary complexes of Thermus thermophilus Ago catalytic mutants with 5'-phosphorylated 21-nucleotide guide DNA and complementary target RNAs of 12, 15 and 19 nucleotides in length, which define the molecular basis for Mg(2+)-facilitated site-specific cleavage of the target. Pivot-like domain movements within the Ago scaffold were observed on proceeding from nucleation to propagation steps of guide-target duplex formation, with duplex zippering beyond one turn of the helix requiring the release of the 3'-end of the guide from the PAZ pocket. Cleavage assays on targets of various lengths supported this model, and sugar-phosphate-backbone-modified target strands showed the importance of structural and catalytic divalent metal ions observed in the crystal structures (Wang, 2009).

The current structures of ternary complexes with catalytic mutants of T. thermophilus Ago have defined the positioning of the guide DNA-target RNA A-form duplex relative to the catalytic Asp residues of the RNase H fold of the PIWI domain, thereby establishing the molecular basis for site-specific cleavage at the phosphate bridging the 10'-11' step of the target strand. Further structural studies of ternary complexes with wild-type Ago have identified two Mg2+ cations within the catalytic pocket, located on either side of the cleavable phosphate, thereby positioned to mediate the cleavage chemistry. Both ends of the guide strand are anchored in the ternary complex composed of one turn of the DNA-RNA duplex spanning the seed segment and cleavage site, but consistent with a two-state model, the 3'-end is released from the PAZ pocket after propagation of the guide-target duplex by three additional base pairs. Notably, the guide DNA and target RNA form a regular A-form helix spanning a maximum of 15 base pairs (positions 2-16), with the Ago scaffold undergoing pivot-like domain movements as the target RNA zippers up by pairing with its guide template (Wang, 2009).

The kinetic effects of target site phosphorothioate substitution and 2' modification during Ago-mediated DNA cleavage are rationalized by the crystal structure, and consistent with the mechanism of RNase H cleavage studied in other systems. The absence of amino-acid side chains able to interrogate whether the target presented at the active site is RNA or DNA might suggest that DNA could be a more probable target of this bacterial Ago protein, as seen for other members of the retroviral integrase superfamily in which Ago proteins belong (Wang, 2009).

Fifteen Neurospora crassa mutants defective in 'quelling' or transgene-induced gene silencing have been isolated. These quelling-defective mutants (qde) belonging to three complementation groups have provided insights into the mechanism of posttranscriptional gene silencing in N. crassa. The recessive nature of the qde mutations indicates that the encoded gene products act in trans. When qde genes are mutated in a transgenic-induced silenced strain containing many copies of the transgene, the expression of the endogenous gene is maintained despite the presence of transgene sense RNA, the molecule proposed to trigger quelling. Moreover, the qde mutants failed to show quelling when tested with another gene, suggesting that they may be universally defective in transgene-induced gene silencing. As such, qde genes may be involved in sensing aberrant sense RNA and/or targeting/degrading the native mRNA. The qde mutations may be used to isolate the genes encoding the first components of the quelling mechanism. Moreover, these quelling mutants may be important in applied and basic research for the creation of strains able to overexpress a transgene (Cogoni, 1997).

An allelic series of the novel argonaute mutant (ago1-1 to ago1-6) of the herbaceous plant Arabidopsis thaliana has been isolated. The ago1 mutation pleotropically affects general plant architecture. The apical shoot meristem generates rosette leaves and a single stem, but axillary meristems rarely develop. Rosette leaves lack a leaf blade but still show adaxial/abaxial differentiation. Instead of cauline leaves, filamentous structures without adaxial/abaxial differentiation develop along the stem and an abnormal inflorescence bearing infertile flowers with filamentous organs is produced. Two independent T-DNA insertions into the AGO1 locus led to the isolation of two corresponding genomic sequences as well as a complete cDNA. The AGO1 locus was mapped close to the marker mi291a on chromosome 1. Antisense expression of the cDNA results in a partial mutant phenotype. Sense expression causes some transgenic lines to develop goblet-like leaves and petals. The cDNA encodes a putative 115 kDa protein with sequence similarity to translation products of a novel gene family present in nematodes as well as humans. No specific function has been assigned to these genes. Similar proteins are not encoded by the genomes of yeast or bacteria, suggesting that AGO1 belongs to a novel class of genes with a function specific to multicellular organisms (Bohmert, 1998).

Postembryonic development in higher plants is marked by repetitive organ formation via a self-perpetuating stem cell system, the shoot meristem. Organs are initiated at the shoot meristem periphery, while a central zone harbors the stem cells. The ZWILLE (ZLL) gene of Arabidopsis is specifically required to establish the central-peripheral organization of the embryo apex and this step is critical for shoot meristem self-perpetuation. zll mutants correctly initiate expression of the shoot meristem-specific gene SHOOT MERISTEMLESS in early embryos, but fail to regulate its spatial expression pattern at later embryo stages and initiate differentiated structures in place of stem cells. The ZLL gene was isolated by map-based cloning. It encodes a novel protein, and related sequences are highly conserved in multicellular plants and animals but are absent from bacteria and yeast. It is proposed that ZLL relays positional information required to maintain stem cells of the developing shoot meristem in an undifferentiated state during the transition from embryonic development to repetitive postembryonic organ formation (Moussian, 1998).

Several lines of evidence indicate that the adaxial leaf domain possesses a unique competence to form shoot apical meristems. Factors required for this competence are expected to cause a defect in shoot apical meristem formation when inactivated and to be expressed or active preferentially in the adaxial leaf domain. PINHEAD, a member of a family of proteins that includes the translation factor eIF2C, is required for reliable formation of primary and axillary shoot apical meristems. In addition to high-level expression in the vasculature, low-level PINHEAD expression defines a novel domain of positional identity in the plant. This domain consists of adaxial leaf primordia and the meristem. These findings suggest that the PINHEAD gene product may be a component of a hypothetical meristem forming competence factor. Defects are also described in floral organ number and shape, as well as aberrant embryo and ovule development associated with pinhead mutants, thus elaborating on the role of PINHEAD in Arabidopsis development. In addition, embryos doubly mutant for PINHEAD and ARGONAUTE1, a related, ubiquitously expressed family member, fail to progress to bilateral symmetry and do not accumulate the SHOOT MERISTEMLESS protein. Therefore PINHEAD and ARGONAUTE1 together act to allow wild-type growth and gene expression patterns during embryogenesis (Lynn, 1999).

Introduction of transgene DNA may lead to specific degradation of RNAs that are homologous to the transgene transcribed sequence through phenomena named post-transcriptional gene silencing (PTGS) in plants, quelling in fungi, and RNA interference (RNAi) in animals. PTGS, quelling, and RNAi require a set of related proteins (SGS2, QDE-1, and EGO-1, respectively). Arabidopsis mutants impaired in PTGS have been isolated that are affected at the Argonaute1 (AGO1) locus. AGO1 is similar to QDE-2, required for quelling, and RDE-1, required for RNAi. Sequencing of ago1 mutants has revealed one amino acid essential for PTGS that is also present in QDE-2 and RDE-1 in a highly conserved motif. Taken together, these results confirm the hypothesis that these processes derive from a common ancestral mechanism that controls expression of invading nucleic acid molecules at the post-transcriptional level. As opposed to rde-1 and qde-2 mutants, which are viable, ago1 mutants display several developmental abnormalities, including sterility. These results raise the possibility that PTGS, or at least some of its elements, could participate in the regulation of gene expression during development in plants (Fagard, 2000).

Small RNA molecules have been found to be specifically associated with posttranscriptional gene silencing (PTGS) in both plants and animals. Small sense and antisense RNAs are also involved in PTGS in Neurospora crassa. The accumulation of these RNA molecules depends on the presence of functional qde-1 and qde-3 genes, coding for an RNA dependent RNA polymerase and a helicase respectively, previously shown to be essential for gene silencing, but does not depend on a functional qde-2, coding for an Argonaute family protein, indicating that this gene is involved in a downstream step of the gene silencing pathway. Supporting this idea, a purified QDE2 protein complex was found to contain small RNA molecules, suggesting that QDE2 could be part of a small RNA-directed ribonuclease complex involved in sequence-specific mRNA degradation (Catalonotto, 2002).

Transgene-induced post-transcriptional gene silencing (PTGS) results from specific degradation of RNAs that are homologous with the transgene transcribed sequence. This phenomenon, also known as cosuppression in plants and quelling in fungi, resembles RNA interference (RNAi) in animals. Indeed, cosuppression/quelling/RNAi all require related PAZ/PIWI proteins (AGO1/QDE-2/RDE-1), indicating that these mechanisms are related. Unlike Neurospora crassa qde-2 and Caenorhabditis elegans rde-1 mutants, which are morphologically normal, the 24 known Arabidopsis ago1 mutants display severe developmental abnormalities and are sterile. Hypomorphic Arabidopsis ago1 mutants, described in this study, have been isolated, including fertile ones. These hypomorphic ago1 mutants are defective for PTGS, like null sgs2, sgs3, and ago1 mutants, suggesting that PTGS is more sensitive than development to perturbations in AGO1. Conversely, a mutation in ZWILLE/PINHEAD, another member of the Arabidopsis AGO1 gene family, affects development but not PTGS. Similarly, mutations in ALG-1 and ALG-2, two members of the C. elegans RDE-1 gene family, affect development but not RNAi, indicating that the control of PTGS/RNAi and development by PAZ/PIWI proteins can be uncoupled. Finally, hypomorphic ago1 mutants are hypersensitive to virus infection, confirming the hypothesis that in plants PTGS is a mechanism of defense against viruses (Morel, 2002).

In a number of organisms, transgenes containing transcribed inverted repeats (IRs) that produce hairpin RNA can trigger RNA-mediated silencing, which is associated with 21-24 nucleotide small interfering RNAs (siRNAs). In plants, IR-driven RNA silencing also causes extensive cytosine methylation of homologous DNA in both the transgene 'trigger' and any other homologous DNA sequences -- i.e., 'targets'. Endogenous genomic sequences, including transposable elements and repeated elements, are also subject to RNA-mediated silencing. The RNA silencing gene ARGONAUTE4 (AGO4) is required for maintenance of DNA methylation at several endogenous loci and for the establishment of methylation at the FWA gene. Mutation of AGO4 substantially reduces the maintenance of DNA methylation triggered by IR transgenes, but AGO4 loss-of-function does not block the initiation of DNA methylation by IRs. AGO4 primarily affects non-CG methylation of the target sequences, while the IR trigger sequences lose methylation in all sequence contexts. Finally, AGO4 and the DRM methyltransferase genes are shown to be required for maintenance of siRNAs at a subset of endogenous sequences, but AGO4 is not required for the accumulation of IR-induced siRNAs or a number of endogenous siRNAs, suggesting that AGO4 may function downstream of siRNA production (Zilberman, 2004).

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).

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).

RNA interference (RNAi) acts on long double-stranded RNAs (dsRNAs) in a variety of eukaryotes to generate small interfering RNAs that target homologous messenger RNA, resulting in their destruction. This process is widely used to 'knock-down' the expression of genes of interest to explore phenotypes. In plants, fission yeast, ciliates, flies and mammalian cells, short interfering RNAs (siRNAs) also induce DNA or chromatin modifications at the homologous genomic locus, which can result in transcriptional silencing or sequence elimination. siRNAs may direct DNA or chromatin modification by siRNA-DNA interactions at the homologous locus. Alternatively, they may act by interactions between siRNA and nascent transcript. In fission yeast (Schizosaccharomyces pombe), chromatin modifications are directed by RNAi only if the homologous DNA sequences are transcribed. Furthermore, transcription by exogenous T7 polymerase is not sufficient. Ago1, a component of the RNAi effector RISC/RITS complex, associates with target transcripts and RNA polymerase II. Truncation of the regulatory carboxy-terminal domain (CTD) of RNA pol II disrupts transcriptional silencing, indicating that, like other RNA processing events, RNAi-directed chromatin modification is coupled to transcription (Schramke, 2005).

Small RNAs regulate chromatin modification and transcriptional gene silencing across the eukaryotic kingdom. Although these processes have been well studied, fundamental mechanistic aspects remain obscure. Specifically, it is unclear exactly how small RNA-loaded Argonaute (see Drosophila Ago1) protein complexes target chromatin to mediate silencing (see Drosophila post-transcriptional gene silencing). Using fission yeast, this study demonstrates that transcription of the target locus is essential for RNA-directed formation of heterochromatin. However, high transcriptional activity is inhibitory; thus, a transcriptional window exists that is optimal for silencing. It was found that pre-mRNA splicing is compatible with RNA-directed heterochromatin formation. However, the kinetics of pre-mRNA processing is critical. Introns close to the 5' end of a transcript that are rapidly spliced result in a bistable response whereby the target either remains euchromatic or becomes fully silenced. Together, these results discount siRNA-DNA base pairing in RNA-mediated heterochromatin formation, and the mechanistic insights further reveal guiding paradigms for the design of small RNA-directed chromatin silencing studies in multicellular organisms (Shimada, 2016).

Double-stranded (ds) RNA can induce sequence-specific inhibition of gene function in several organisms. However, both the mechanism and the physiological role of the interference process remain mysterious. In order to study the interference process, C. elegans mutants resistant to dsRNA-mediated interference (RNAi) have been selected. Two loci, rde-1 and rde-4 (Drosophila homolog: r2d2), are defined by mutants strongly resistant to RNAi but with no obvious defects in growth or development. rde-1 is a member of the piwi/sting/argonaute/zwille/eIF2C gene family conserved from plants to vertebrates. Interestingly, several, but not all, RNAi-deficient strains exhibit mobilization of the endogenous transposons. One natural function of RNAi is transposon silencing (Tabara, 1999).

In Caenorhabditis elegans, the introduction of double-stranded RNA triggers sequence-specific genetic interference (RNAi) that is transmitted to offspring. The inheritance properties associated with this phenomenon were examined. Transmission of the interference effect occurs through a dominant extragenic agent. The wild-type activities of the RNAi pathway genes rde-1 and rde-4 (coding for a Dicer homolog) are required for the formation of this interfering agent but were not needed for interference thereafter. In contrast, the rde-2 and mut-7 (coding for a ribonuclease) genes were required downstream for interference. These findings provide evidence for germ line transmission of an extragenic sequence-specific silencing factor and implicate rde-1 and rde-4 in the formation of the inherited agent (Grishok, 2000).

RNA interference (RNAi) is a cellular defense mechanism that uses double-stranded RNA (dsRNA) as a sequence-specific trigger to guide the degradation of homologous single-stranded RNAs. RNAi is a multistep process involving several proteins and at least one type of RNA intermediate, a population of small 21-25 nt RNAs (called siRNAs) that are initially derived from cleavage of the dsRNA trigger. Genetic screens in Caenorhabditis elegans have identified numerous mutations that cause partial or complete loss of RNAi. This work analyzes cleavage of injected dsRNA to produce the initial siRNA population in animals mutant for rde-1 and rde-4, two genes that are essential for RNAi but that are not required for organismal viability or fertility. The results suggest distinct roles for RDE-1 and RDE-4 in the interference process. Although null mutants lacking rde-1 show no phenotypic response to dsRNA, the amount of siRNAs generated from an injected dsRNA trigger is comparable to that of wild-type. By contrast, mutations in rde-4 substantially reduce the population of siRNAs derived from an injected dsRNA trigger. Injection of chemically synthesized 24- or 25-nt siRNAs can circumvent RNAi resistance in rde-4 mutants, whereas no bypass was observed in rde-1 mutants. These results support a model in which RDE-4 is involved before or during production of siRNAs, whereas RDE-1 acts after the siRNAs have been formed (Parrish, 2001).

Posttranscriptional gene silencing in Caenorhabditis elegans results from exposure to double-stranded RNA (dsRNA), a phenomenon designated as RNA interference (RNAi), or from co-suppression, in which transgenic DNA leads to silencing of both the transgene and the endogenous gene. Single-stranded RNA oligomers of antisense polarity can also be potent inducers of gene silencing. As is the case for co-suppression, antisense RNAs act independently of the RNAi genes rde-1 and rde-4 but require the mutator/RNAi gene mut-7 and a putative DEAD box RNA helicase, mut-14. These data favor the hypothesis that gene silencing is accomplished by RNA primer extension using the mRNA as template, leading to dsRNA that is subsequently degraded (Tijsterman, 2002).

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).

Tissue-specific stem cells combine proliferative and asymmetric divisions to balance self-renewal with differentiation. Tight regulation of the orientation and plane of cell division is crucial in this process. This study reports an investigation of the reproducible pattern of anterior-posterior-oriented stem cell-like divisions in the C. elegans seam epithelium. In a genetic screen, an alg-1 Argonaute mutant was identified with additional and abnormally oriented seam cell divisions. ALG-1 is the main subunit of the microRNA-induced silencing complex (miRISC) and was previously shown to regulate the timing of postembryonic development. Time-lapse fluorescence microscopy of developing larvae revealed that reduced alg-1 function successively interferes with Wnt signaling, cell adhesion, cell shape and the orientation and timing of seam cell division. Wnt inactivation, through mig-14 Wntless mutation, was found to disrupt tissue polarity but not anterior-posterior division. However, combined Wnt inhibition and cell shape alteration resulted in disordered orientation of seam cell division, similar to the alg-1 mutant. These findings reveal additional alg-1-regulated processes, uncover a previously unknown function of Wnt ligands in seam tissue polarity, and show that Wnt signaling and geometric cues redundantly control the seam cell division axis (Wildwater, 2011).

PUF (Pumilio/FBF) RNA-binding proteins and Argonaute (Ago) miRNA-binding proteins regulate mRNAs post-transcriptionally, each acting through similar, yet distinct, mechanisms. This study reports that PUF and Ago proteins can also function together in a complex with a core translation elongation factor, eEF1A, to repress translation elongation. Both nematode (C. elegans) and mammalian PUF-Ago-eEF1A complexes were identified, using coimmunoprecipitation and recombinant protein assays. Nematode CSR-1 (Ago) promoted repression of FBF (PUF) target mRNAs in in vivo assays, and the FBF-1-CSR-1 heterodimer inhibited EFT-3 (eEF1A) GTPase activity in vitro. Mammalian PUM2-Ago-eEF1A inhibited translation of nonadenylated and polyadenylated reporter mRNAs in vitro. This repression occurred after translation initiation and led to ribosome accumulation within the open reading frame, roughly at the site where the nascent polypeptide emerged from the ribosomal exit tunnel. Together, these data suggest that a conserved PUF-Ago-eEF1A complex attenuates translation elongation (Friend, 2012).

Argonaute (Ago) proteins mediate posttranscriptional gene repression by binding guide miRNAs to regulate targeted RNAs. To confidently assess Ago-bound small RNAs, a mouse embryonic stem cell system was adapted to express a single epitope-tagged Ago protein family member in an inducible manner. This paper reports the small RNA profile of Ago-deficient cells and shows that Ago-dependent stability is a common feature of mammalian miRNAs. Using this criteria and immunopurification, an Ago-dependent class of noncanonical miRNAs was identified derived from protein-coding gene promoters, which were named transcriptional start site miRNAs (TSS-miRNAs). A subset of promoter-proximal RNA polymerase II (RNAPII) complexes produces hairpin RNAs that are processed in a DiGeorge syndrome critical region gene 8 (Dgcr8)/Drosha-independent but Dicer-dependent manner. TSS-miRNA activity is detectable from endogenous levels and following overexpression of mRNA constructs. Finally, evidence is presented of differential expression and conservation in humans, suggesting important roles in gene regulation (Zamudio, 2014).

Organisms can develop adaptive sequence-specific immunity by reexpressing pathogen-specific small RNAs that guide gene silencing. For example, the C. elegans PIWI-Argonaute/piwi-interacting RNA (piRNA) pathway recruits RNA-dependent RNA polymerase (RdRP) to foreign sequences to amplify a transgenerational small-RNA-induced epigenetic silencing signal (termed RNAe). This study provides evidence that, in addition to an adaptive memory of silenced sequences, C. elegans can also develop an opposing adaptive memory of expressed/self-mRNAs. This mechanism, which can prevent or reverse RNAe, is referred to as RNA-induced epigenetic gene activation (RNAa). CSR-1 (an Argonaute homolog), which engages RdRP-amplified small RNAs complementary to germline-expressed mRNAs, is required for RNAa. A transgene with RNAa activity also exhibits accumulation of cognate CSR-1 small RNAs. These findings suggest that C. elegans adaptively acquires and maintains a transgenerational CSR-1 memory that recognizes and protects self-mRNAs, allowing piRNAs to recognize foreign sequences innately, without the need for prior exposure (Seth, 2013).

Argonaute (AGO) proteins are key nuclease effectors of RNAi. Although purified AGOs can mediate a single round of target RNA cleavage in vitro, accessory factors are required for small interfering RNA (siRNA) loading and to achieve multiple-target turnover. To identify AGO cofactors, the C. elegans AGO WAGO-1, which engages amplified small RNAs during RNAi, was immunoprecipitated. These studies identified a robust association between WAGO-1 and a conserved Vasa ATPase-related protein RDE-12. rde-12 mutants are deficient in RNAi, including viral suppression, and fail to produce amplified secondary siRNAs and certain endogenous siRNAs (endo-siRNAs). RDE-12 colocalizes with WAGO-1 in germline P granules and in cytoplasmic and perinuclear foci in somatic cells. These findings and genetic studies suggest that RDE-12 is first recruited to target mRNA by upstream AGOs [RDE-1 (Drosophila homolog: Ago1) and ERGO-1], where it promotes small RNA amplification and/or WAGO-1 loading. Downstream of these events, RDE-12 forms an RNase-resistant (target mRNA-independent) complex with WAGO-1 and may thus have additional functions in target mRNA surveillance and silencing (Shirayama, 2014).

The argonaute protein family provides central components for RNA interference (RNAi) and related phenomena in a wide variety of organisms. A cDNA clone termed BmAGO2 has been isolated from a Bombyx cell that is homologous to Drosophila ARGONAUTE2, the gene encoding a repressive factor for the recombination repair of extrachromosomal double-strand breaks (DSBs). RNAi-mediated silencing of the BmAGO2 sequence markedly increases homologous recombination (HR) repair of DSBs in episomal DNA, but has no effect on that in chromosomes. Moreover, RNAi for BmAGO2 enhances the integration of linearized DNA into a silkworm chromosome via HR. These results suggest that BmAgo2 protein plays an indispensable role in the repression of extrachromosomal DSB repair (Tsukioka, 2006).

The observation that knockdown of the silkworm Argonaute2 homolog gene, BmAGO2, augments the rate of extrachromosomal HR appears to be explicable by two alternative assumptions: (1) BmAgo2 may act as an inhibitor of HR. This inference comes from the notion that Argonaute protein possibly inhibits the binding of HR-related proteins to the strands via a PAZ domain, which can directly bind to substrate DNA; (2) BmAgo2 is needed to discriminate extrachromosomal DNA from chromosomal DNA and represses extrachromosomal HR repair indirectly. The latter possibility is favored, because Argonaute family proteins have been reported to participate in heterochromatin formation. Argonaute1 is one of the components of the RNA-induced initiation of the transcriptional gene silencing (RITS) complex, which is necessary for heterochromatin assembly. Recent studies indicate that RNAi is involved in heterochromatin formation at the centromere and therefore in chromosome segregation. If BmAgo2 plays a role in the extrachromosomal DNA-specific assembly of heterochromatin, in which HR repair of DSBs are repressed, the decrease in BmAgo2 expression would not affect intrachromosomal HR repair, and this was in fact the case as described above (Tsukioka, 2006).

It is reasonable to predict that cells have defense systems for exogenous DNA, e.g., viral DNA. If such exogenous DNA has a homologous sequence to that of a host genome region, HR between these may frequently cause a partial loss of genomic information. Therefore, the cells would acquire mechanisms to repress the HR repair of extrachromosomal DSBs by using an Argonaute protein. Indeed, baculoviruses, DNA viruses widely isolated from lepidopteran insects, often carry DNA transposable elements, such as piggybac and Tc1-like elements, which apparently originate from the cellular genomes and are inserted into infecting baculovirus genomes. These viruses are to be excluded from the HR-related integration pathway leading to the modification of host genomes (Tsukioka, 2006).

In Xenopus laevis oocytes and early embryos, double-stranded exogenous siRNAs cannot function as microRNA (miRNA) mimics in either deadenylation or guided mRNA cleavage (RNAi). Instead, siRNAs saturate and inactivate maternal Argonaute (Ago) proteins, which are present in low amounts but are needed for Dicer processing of pre-miRNAs at the midblastula transition (MBT). Consequently, siRNAs impair accumulation of newly made miRNAs, such as the abundant embryonic pre-miR-427, but inhibition dissipates upon synthesis of zygotic Ago proteins after MBT. These effects of siRNAs, which are independent of sequence, result in morphological defects at later stages of development. The expression of any of several exogenous human Ago proteins, including catalytically inactive Ago2 (Ago2mut), can overcome the siRNA-mediated inhibition of miR-427 biogenesis and function. However, expression of wild-type, catalytically active hAgo2 is required to elicit RNAi in both early embryos and oocytes using either siRNA or endogenous miRNAs as guides. The lack of endogenous Ago2 endonuclease activity explains why these cells normally are unable to support RNAi. Expression of catalytically active exogenous Ago2, which appears not to perturb normal Xenopus embryonic development, can now be exploited for RNAi in this vertebrate model organism (Lund, 2011).

Gene silencing through RNA interference (RNAi) is carried out by RISC, the RNA-induced silencing complex. RISC contains two signature components, small interfering RNAs (siRNAs) and Argonaute family proteins. The multiple Argonaute proteins present in mammals are both biologically and biochemically distinct, with a single mammalian family member, Argonaute2, being responsible for messenger RNA cleavage activity. This protein is essential for mouse development, and cells lacking Argonaute2 are unable to mount an experimental response to siRNAs. Mutations within a cryptic ribonuclease H domain within Argonaute2, as identified by comparison with the structure of an archeal Argonaute protein, inactivate RISC. Thus, the evidence supports a model in which Argonaute contributes 'Slicer' activity to RISC, providing the catalytic engine for RNAi (Liu, 2004).

The presence of double-stranded RNA (dsRNA) in most eukaryotic cells provokes a sequence-specific silencing response known as RNA interference (RNAi). The dsRNA trigger of this process can be derived from exogenous sources or transcribed from endogenous noncoding RNA genes that produce microRNAs (miRNAs). RNAi begins with the conversion of dsRNA silencing triggers into small RNAs of ~21 to 26 nucleotides (nts) in length. This is accomplished by the processing of triggers by specialized ribonuclease III (RNase III)-family nucleases, Dicer and Drosha. Resulting small RNAs join an effector complex, known as RISC (RNA-induced silencing complex). Silencing by RISC can occur through several mechanisms. In flies, plants, and fungi, dsRNAs can trigger chromatin remodeling and transcriptional gene silencing. RISC can also interfere with protein synthesis, and this is the predominant mechanism used by miRNAs in mammals. However, the best studied mode of RISC action is mRNA cleavage. When programmed with a small RNA that is fully complementary to the substrate RNA, RISC cleaves that RNA at a discrete position, an activity that has been attributed to an unknown RISC component, 'Slicer'. Whether or not RISC cleaves a substrate can be determined by the degree of complementarity between the siRNA and mRNA, since mismatched duplexes are often not processed. However, even for mammalian miRNAs, which normally repress at the level of protein synthesis, cleavage activity can be detected with a substrate that perfectly matches the miRNA sequence. This result prompted the hypothesis that all RISCs are equal, with the outcome of the RISC-substrate interaction being determined largely by the character of the interaction between the small RNA and its substrate (Liu, 2004 and references therein).

RISC contains two signature components. The first is the small RNA, which cofractionates with RISC activity in Drosophila S2 cell extracts, and whose presence correlates with dsRNA-programmed mRNA cleavage in Drosophila embryo lysates. The second is an Argonaute (Ago) protein, which was identified as a component of purified RISC in Drosophila. Additional studies have suggested that Argonautes are also key components of RISC in mammals, fungi, worms, protozoans, and plants (Liu, 2004 and references therein).

Argonautes are often present as multi-protein families and are identified by two characteristic domains, PAZ and PIWI. These proteins mainly segregate into two subfamilies, comprising those that are more similar to either Arabidopsis Argonaute1 or Drosophila Piwi. The Argonaute family was first linked to RNAi through genetic studies in Caenorhabditis elegans, which identified Rde-1 as a gene essential for silencing . Subsequent placement of a Drosophila Argonaute protein in RISC has prompted an exploration of the roles of this protein family. Toward this end, both biochemical and genetic studies of the Ago1 subfamily proteins in mammals have been undertaken (Liu, 2004).

Mammals contain four Argonaute1 subfamily members -- Ago1 to Ago4. Different Argonaute family members in Drosophila preferentially associate with different small RNAs, with Ago1 preferring miRNAs and Ago2 siRNAs. Recent studies of Drosophila melanogaster (Dm) Ago1 and DmAgo2 mutants have strengthened these conclusions. To assess whether mammalian Ago proteins specialized in their interactions with small RNAs, Ago-associated miRNA populations were examined by microarray analysis. Ago1-, Ago2- and Ago3-associated RNAs were hybridized to microarrays that report the expression status of 152 human microRNAs. Patterns of associated RNAs were identical within experimental error in each case. Additionally, each of the tagged Ago proteins associate similarly with a cotransfected siRNA (Liu, 2004).

Previous studies have used tagged siRNAs to affinity purify Argonaute-containing RISC. These preparations, containing mixtures of at least two mammalian Argonautes, were capable of cleaving synthetic mRNAs that were complementary to the tagged siRNA. The ability of purified complexes containing individual Argonaute proteins to catalyze similar cleavages was examined. Unexpectedly, irrespective of the siRNA sequence, only Ago2-containing RISC was able to catalyze cleavage. All three Ago proteins were similarly expressed and bound similar amounts of transfected siRNA (Liu, 2004).

These results demonstrated that mammalian Argonaute complexes are biochemically distinct, with only a single family member being competent for mRNA cleavage. To examine the possibility that Ago proteins might also be biologically specialized, the mouse Ago2 gene was disrupted by targeted insertional mutagenesis. Intercrosses of Ago2 heterozygotes produced only wild-type and heterozygous offspring, strongly suggesting that disruption of Ago2 produced an embryonic-lethal phenotype (Liu, 2004).

Ago2-deficient mice display several developmental abnormalities, beginning approximately halfway through gestation. Both gene-trap and in situ hybridization data of day 9.5 embryos show broad expression of Ago2 in the embryo, with some hot spots of expression in the forebrain, heart, limb buds, and branchial arches. The most prominent phenotype is a defect in neural tube closure, often accompanied by apparent mispatterning of anterior structures, including the forebrain. Roughly half of the embryos display complete failure of neural tube closure in the head region, while all embryos display a wavy neural tube in more caudal regions. Mutant embryos also suffer from apparent cardiac failure. The hearts are enlarged and often accompanied by pronounced swelling of the pericardial cavity. By day 10.5, mutant embryos are severely developmentally delayed compared with wild-type and heterozygous littermates. This large difference in size, like the apparent cardiac failure, may be accounted for by a general nutritional deficiency caused by yolk sac and placental defects; histological analysis reveals abnormalities in these tissues (Liu, 2004).

Not all Argonaute proteins are required for successful mammalian development. Thus, it is unclear why Ago2 should be required for development, while other Ago proteins are dispensable. Ago subfamily members are expressed in overlapping patterns in humans. In situ hybridization demonstrates overlapping expression patterns for Ago2 and Ago3 in mouse embryos. Considered together with the essentially identical patterns of miRNA binding, these results suggest the possibility that the ability of Ago2 to assemble into catalytically active complexes might be critical for mouse development. Although most miRNAs regulate gene expression at the level of protein synthesis, recently miR196 has been demonstrated to cleave the mRNA encoding HoxB8, a developmental regulator. Evolutionary conservation of an essential cleavage-competent RISC in organisms in which miRNAs predominantly act by translational regulation raises the possibility that target cleavage by mammalian miRNAs might be more important and widespread than previously appreciated (Liu, 2004).

Numerous studies have indicated that experimentally triggered RNAi in mammalian cells proceeds through siRNA-directed mRNA cleavage because in many cases, but not all, reiterated binding sites are necessary for repression at the level of protein synthesis. If Ago2 were uniquely capable of assembling into cleavage-competent complexes in mice, then embryos or cells lacking Ago2 might be resistant to experimental RNAi. To address this question, mouse embryo fibroblasts (MEFs) from E10.5 embryos were prepared from Ago2 heterozygous intercrosses. Reverse transcription polymerase chain reaction (RT-PCR) analysis and genotyping revealed that it was possible to obtain wild-type, mutant, and heterozygous MEF populations. Importantly, MEFs also express other Ago proteins, including Ago1 and Ago3. Ago2-null MEFs were unable to repress gene expression in response to an siRNA. This defect could be rescued by the addition of a third plasmid that encoded human Ago2 but not by a plasmid encoding human Ago1 . In contrast, responses were intact for a reporter of repression at the level of protein synthesis, mediated by an siRNA binding to multiple mismatched sites (Liu, 2004).

Because Ago2 is exceptional in its ability to form cleavage-competent complexes, the determinants of this capacity were mapped. Deletion analysis indicated that an intact Ago2 was required for RISC activity. The sequence of highly conserved but cleavage-incompetent Ago proteins was used as a guide to the construction of Ago2 mutants. A series of point mutations were tested, included H634P, H634A, Q633R, Q633A, H682Y, L140W, F704Y, and T744Y. Whereas all of these mutations retain siRNA-binding activity and most retain cleavage activity, changes at Q633 and H634 have a profound effect on target cleavage. Both the Q633R and H634P mutations, in which residues were changed to corresponding residues in Ago1 and Ago3, abolished catalysis. Changing H634 to A634 also inactivated Ago2, whereas a similar change, Q633A, was permissive for cleavage. Thus, even relatively conservative changes can negate the ability of Ago2 to form cleavage-competent RISC (Liu, 2004).

Several possibilities could explain a lack of cleavage activity for Ago2 mutants. Such mutations could interfere with the proper folding of Ago2. However, this seems unlikely because those same residues presumably permit proper folding in closely related Argonaute proteins, and mutant Ago2 proteins retained the ability to interact with siRNAs. Alternatively, cleavage-incompetent Ago2 mutants could lose the ability to interact with the putative Slicer. Finally, Ago2 itself might be Slicer, with the conservative substitutions altering the active center of the enzyme in a way that prevents cleavage (Liu, 2004).

The last possibility predicted that an active enzyme might be reconstituted with relatively pure Ago2 protein. Ago2 was immunoaffinity purified from 293T cells and attempts were made to reconstitute RISC in vitro. Incubation with the double-stranded siRNA produced no appreciable activity, whereas Ago2 could be successfully programmed with single-stranded siRNAs to cleave a complementary substrate. Formation of the active enzyme was unaffected by first washing the immunoprecipitates with up to 2.5 M NaCl or 1 M urea. A 21-nt single-stranded DNA was unable to direct cleavage. Programming could be accomplished with different siRNAs that direct activity against different substrates. RISC is formed though a concerted assembly process in which the RISC-loading complex (RLC) acts in an adenosine triphosphate (ATP)-dependent manner to place one strand of the small RNA into RISC. In vitro reconstitution occurs in the absence of ATP; this suggests that Ago2 could be programmed with siRNAs without a need for the normal assembly process. However, in vitro reconstitution of RISC still requires the essential characteristics of an siRNA. For example, single-stranded siRNAs that lack a 5' phosphate group cannot reconstitute an active enzyme (Liu, 2004).

Although consistent with the possibility that the catalytic activity of RISC is carried within Ago2, these results do not rule out the possibility that a putative Slicer copurifies with Ago2. To demonstrate more conclusively that Ago2 is Slicer, the crystal structure of an Argonaute protein from an archebacterium, Pyrococcus furiosus, was examined. This structure revealed that the PIWI domain folds into a structure analogous to the catalytic domain of RNase H and avian sarcoma virus (ASV) integrase. The notion that such a domain would lie at the center of RISC cleavage is consistent with previous observations. RNase H and integrases cleave their substrates, leaving 5' phosphate and 3' hydroxyl groups through a metal-catalyzed cleavage reaction. Notably, previous studies have strongly indicated that the scissile phosphate in the targeted mRNA is cleaved via a metal ion in RISC to give the same phosphate polarity. In vitro data are consistent with the reconstituted RISC also requiring a divalent metal (Liu, 2004}.

The active center of RNase H and its relatives consists of a catalytic triad of three carboxylate groups contributed by aspartic or glutamic acid. These amino acid residues coordinate the essential metal and activate water molecules for nucleolytic attack. Reference to the known structure of RNase H reveals two aspartate residues in the archeal Ago protein present at the precise spatial locations predicted for formation of an RNase H-like active site. These align with identical residues in the human Ago2 protein. Therefore, to test whether the PIWI domain of Ago2 provides catalytic activity to RISC, the two conserved aspartates, D597 and D669, were changed to alanine, with the prediction that either mutation would inactivate RISC cleavage. Consistent with the hypothesis, the mutant Ago2 proteins were incapable of assembling into a cleavage-competent RISC in vitro or in vivo, despite retaining the ability to bind siRNAs (Liu, 2004).

Considered together, these data provide strong support for the notion that Argonaute proteins are the catalytic components of RISC: (1) The ability to form an active enzyme is restricted to a single mammalian family member, Ago2. This conclusion is supported both by biochemical analysis and by genetic studies in mutant MEFs. (2) Single amino acid substitutions within Ago2 that convert residues to those present in closely related proteins negate RISC cleavage. (3) The structure of the P. furiosis Argonaute protein reveals provocative structural similarities between the PIWI domain and the RNase H domains, providing a hypothesis for the method by which Argonaute cleaves its substrates. This hypothesis was tested by introducing mutations in the predicted Ago2 active site. It is extremely unlikely that such mutations could affect interactions with other proteins, because they are buried within a cleft of Ago (Liu, 2004).

These studies indicate that the Argonaute proteins that are unable to form cleavage-competent RISC differ from Ago2 at key positions that do not include the putative metal-coordinating residues themselves. However, it is not yet possible, based either on biochemical or structural studies, to provide a precise explanation for the catalytic defects in these proteins. It is conceivable that Ago1 and Ago3 fail to coordinate the catalytic metal or that the structure of the active site is distorted sufficiently that a bound metal is unable to access the scissile phosphate. Alternatively, catalytic mechanisms with two metal ions have been proposed for RNase H, which leaves open the possibility that catalytically inert Ago family members might lack structures essential to bind the second metal ion (Liu, 2004).

The relationship between the nuclease domain in PIWI and conserved nuclease domains in viral reverse transcriptases, transposases, and viral integrases has potential evolutionary implications. In Drosophila, plants, and C. elegans, the RNAi pathway has a major role in controlling parasitic nucleic acids such as viruses and transposons. The fact that the RNAi machinery shares a core structural domain with viruses and transposons suggests that this nucleic acid immune system may have arisen in part by pirating components from the replication and movement machineries of the very elements that RNAi protects against. This hypothesis is made even more poignant by considering the role of RNA-dependent RNA polymerases in RNAi, their functional relationship to viral replicases, and the possibility that the siRNAs themselves might first have served as primers that enable such replicases to duplicate primordial genomes (Liu, 2004).

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)

Small interfering RNAs (siRNAs) are the mediators of mRNA degradation in the process of RNA interference (RNAi). A human biochemical system is described that recapitulates siRNA-mediated target RNA degradation. By using affinity-tagged siRNAs, it has been demonstrated that a single-stranded siRNA resides in the RNA-induced silencing complex (RISC) together with eIF2C1 and/or eIF2C2 (human GERp95) Argonaute proteins. RISC is rapidly formed in HeLa cell cytoplasmic extract supplemented with 21 nt siRNA duplexes, but also by adding single-stranded antisense RNAs, which range in size between 19 and 29 nucleotides. Single-stranded antisense siRNAs are also effectively silencing genes in HeLa cells, especially when 5'-phosphorylated, and expand the repertoire of RNA reagents suitable for gene targeting (Martinez, 2002).

It has been proposed that siRNAs act as primers for target RNA-templated dsRNA synthesis, even though homologs of the RNA-dependent RNA polymerases known to participate in gene silencing in other systems are apparently not encoded in D. melanogaster or mammalian genomes. Analysis of the fate of siRNA duplexes in the HeLa cell system does not provide evidence for such a siRNA-primed activity, but indicates that the predominant pathway for siRNA-mediated gene silencing is sequence-specific endonucleolytic target RNA degradation. Further evidence against siRNA-induced propagation of gene silencing in mammalian systems is that (1) the silenced gene returns to normal levels between 5 to 9 days posttransfection; (2) simultaneously expressed isoforms can be selectively targeted by siRNA duplexes (Martinez, 2002 and references therein).

RNA interference (RNAi) is an important means of eliminating mRNAs, but the intracellular location of RNA-induced silencing complex (RISC) remains unknown. Argonaute 2, a key component of RISC, is not randomly distributed but concentrates in mRNA decay centers that are known as cytoplasmic bodies. The localization of Argonaute 2 in decay centers is not altered by the presence or absence of small interfering RNAs or their targeted mRNAs. However, RNA is required for the integrity of cytoplasmic bodies because RNase eliminates Argonaute 2 localization. In addition, Argonaute 1, another Argonaute family member, is concentrated in cytoplasmic bodies. These results provide new insight into the mechanism of RNAi function (Sen, 2005).

MicroRNAs (miRNAs) are approximately 21-nucleotide-long RNA molecules regulating gene expression in multicellular eukaryotes. In metazoa, miRNAs act by imperfectly base-pairing with the 3' untranslated region of target messenger RNAs (mRNAs) and repressing protein accumulation by an unknown mechanism. Endogenous let-7 microribonucleoproteins (miRNPs) or the tethering of Argonaute (Ago) proteins to reporter mRNAs in human cells inhibit translation initiation. M(7)G-cap-independent translation is not subject to repression, suggesting that miRNPs interfere with recognition of the cap. Repressed mRNAs, Ago proteins, and miRNAs were all found to accumulate in processing bodies. It is proposed that localization of mRNAs to these structures is a consequence of translational repression (Pillai, 2005).

microRNAs (miRNAs) bind to Argonaute (Ago) proteins and inhibit translation or promote degradation of mRNA targets. Human let-7 miRNA inhibits translation initiation of mRNA targets in an m⁷G cap-dependent manner and also appears to block protein production, but the molecular mechanism(s) involved is unknown and the role of Ago proteins in translational regulation remains elusive. This study identified a motif (MC) within the Mid domain of Ago proteins, which bears significant similarity to the m⁷G cap-binding domain of eIF4E, an essential translation initiation factor. Conserved aromatic residues were identified within the MC motif of human Ago2 that are required for binding to the m⁷G cap and for translational repression but do not affect the assembly of Ago2 with miRNA or its catalytic activity. It is proposed that Ago2 represses the initiation of mRNA translation by binding to the m⁷G cap of mRNA targets, thus likely precluding the recruitment of eIF4E (Kiriakidou, 2007).

An important feature of miRNA-directed translational repression is its apparent cooperativity: increasing the number of miRNA recognition elements (MREs) in the 3′-UTR of an mRNA target enhances translational repression. Cooperativity is also seen when multiple MREs for different miRNAs are found in the 3′-UTR of the same mRNA target, arguing that common factors, notably Ago proteins, bound to all miRNAs are responsible for the enhanced translational repression. Indeed, this cooperativity is accurately recapitulated in experiments with tethered Ago2; increasing the number of BoxB sites in the 3′-UTR of the reporter leads to enhancement of the translational repression by λN-HA-Ago2. It is proposed that multiple MREs, within the same mRNA target, increase the number of Ago2 molecules bound to the mRNA, thus increasing the probability that they will interact with the m⁷G cap and augment translational repression by limiting availability of the m⁷G cap to eIF4E. In this model, Ago2 binds to m⁷G cap less efficiently than eIF4E. Therefore, optimal repression by Ago2 and thus optimal eIF4E competition would require multiple Ago2 molecules. Weak Ago2 binding to the m⁷G cap also makes biological sense, since an Ago2 protein with high affinity to the m⁷G cap would lead to generalized and strong translational inhibition. This model is also consistent with weak translational repression of mRNA targets that bear single MREs. Indeed, the vast majority of mRNA targets contain a single MRE for any given miRNA and the level of translational repression is typically modest (usually 1.5- to 2-fold repression). Such modest and noncomplete repression may also explain why many miRNAs cosediment with actively translating, endogenous, mRNAs in polysomes. Lastly, these findings do not exclude additional mechanisms of miRNA and Ago regulation, perhaps in the presence of additional factors such as inhibition of protein production on actively translating ribosomes during elongation or degradation of mRNAs (Kiriakidou, 2007).

An important observation is that the MC motif is not detected in Ago proteins from organisms that do not contain miRNAs, or do not use miRNAs for translational repression. Specifically, all mammalian Ago proteins and certain Ago proteins from nematodes and flies, where translational repression by miRNAs has been demonstrated, contain the MC domain, and thus these Ago proteins may be capable of repressing translation. The MC domain is present in Drosophila AGO1, which is required for miRNA function, but not in Drosophila AGO2, which functions predominantly in siRNA pathways, although more recent studies show overlapping functions of Ago1 and Ago2 pathways in flies. The MC domain is present in C.elegans ALG-1 and ALG-2 Ago proteins but absent from the remaining 25 members of the C.elegans Argonaute protein family, consistent with the finding that there are distinct RNAi-related pathways in nematodes, with ALG-1 and ALG-2 proteins participating in the microRNA pathway and all other nematode Argonaute proteins being associated with exo- or endo-RNAi pathways. Finally, the MC domain is absent from Ago proteins in organisms that do not have miRNAs such as fission yeast and Archaea. Although the MC motif is not found in Archaeal Agos, the structures of the P. furiosus and A. aeolicus Ago proteins show that a major portion of the Mid domain is accessible and thus may be capable of interacting with other factors. The MC domain is also not present in PIWI proteins, which are almost exclusively expressed in the germline. Notably, tethering of HIWI, a human PIWI protein, in the 3'-UTR of RL-5BoxB, is unable to repress RL translation. In contrast, tethering of all human Ago proteins (Ago1-4) in the 3'-UTR of RL-5BoxB results in strong repression of RL translation. These studies along with the finding that translational repression is unaffected in Ago2 null mouse embryonic fibroblasts also show that the endonuclease activity of mammalian Ago proteins is not required for translational repression. In flies, PIWI proteins associate with repeat-associated siRNAs. Mammalian PIWI proteins do not assemble with miRNAs or siRNAs but bind to slightly larger RNAs termed piRNAs. The mouse MIWI protein can associate with m⁷GTP sepharose, suggesting that MIWI proteins may also function in translation. However, since the MC domain is absent from the MIWI protein, it is possible that MIWI contains another cap-binding motif or associates with the cap-analog resin indirectly, via interactions with another cap-binding protein. However, the biochemical function of MIWI proteins and of piRNAs is unknown, and it is difficult to ascertain the functional consequences of this interaction at this point. Finally, the absence of the MC motif from plant Agos is intriguing and suggests that plant miRNAs may not be capable of repressing translation through interactions with the cap (but other mechanisms cannot be excluded). So far translational repression by miRNAs in plants has only been implicated for the control of very few mRNA targets, while most known plant miRNAs show extensive complementarity with their targets, directing target mRNA cleavage (Kiriakidou, 2007).

The RNA-Induced Silencing Complex (RISC) is a ribonucleoprotein particle composed of a single-stranded short interfering RNA (siRNA) and an endonucleolytically active Argonaute protein, capable of cleaving mRNAs complementary to the siRNA. The mechanism by which RISC cleaves a target RNA is well understood, however it remains enigmatic how RISC finds its target RNA. This study shows, both in vitro and in vivo, that the accessibility of the target site correlates directly with the efficiency of cleavage, demonstrating that RISC is unable to unfold structured RNA. In the course of target recognition, RISC transiently contacts single-stranded RNA nonspecifically and promotes siRNA-target RNA annealing. Furthermore, the 5' part of the siRNA within RISC creates a thermodynamic threshold that determines the stable association of RISC and the target RNA. RISC exhibits annealing activity which promotes siRNA-target RNA interactions and therefore renders nucleic-acid hybridization more efficient compared to the annealing of a single-stranded siRNA to a target in the absence of hAgo2. This activity might rely on (1) the siRNA-organization, with a helical geometry of the 5'-region favoring duplex formation, or (2) a nonspecific affinity of RISC toward RNA substrates, that might relate to the overall basicity of hAgo proteins. This study therefore provide mechanistic insights by revealing features of RISC and target RNAs that are crucial to achieve efficiency and specificity in RNA interference (Ameres, 2007).

RNA interference is triggered by small interfering RNA and microRNA, and is a potent mechanism in post-transcriptional regulation for gene expression. GW182 (also known as TNRC6A), an 182-kDa protein encoded by TNRC6A, is important for this process, although details of its function remain unclear. This study reports a novel 210-kDa isoform of human GW182, provisionally named trinucleotide GW1 (TNGW1) because it contains trinucleotide repeats in its mRNA sequence. TNGW1 was expressed independently of GW182 and was present in human testis and various human cancer cells. Using polyclonal and monoclonal antibodies, TNGW1 was detected in only approximately 30% of GW bodies. Expression of EGFP-tagged TNGW1 in HeLa cells was colocalized to cytoplasmic foci enriched in Ago2 (also known as EIF2C2) and RNA decay factors. Tethering TNGW1 or GW182 to the 3'-UTR of a luciferase-reporter mRNA led to strong repression activity independent of Ago2, whereas the tethered Ago2-mediated suppression was completely dependent on TNGW1 and/or GW182. These data demonstrated that GW182 and, probably, TNGW1 acted as a repressor in Ago2-mediated translational silencing. Furthermore, TNGW1 might contribute to diversity in the formation and function of GW and/or P bodies (Li, 2008).

In the mouse neocortex, neural progenitor cells generate both differentiating neurons and daughter cells that maintain progenitor fate. This study shows that the TRIM-NHL protein TRIM32 regulates protein degradation and microRNA activity to control the balance between those two daughter cell types. In both horizontally and vertically dividing progenitors, TRIM32 becomes polarized in mitosis and is concentrated in one of the two daughter cells. TRIM32 overexpression induces neuronal differentiation while inhibition of TRIM32 causes both daughter cells to retain progenitor cell fate. TRIM32 ubiquitinates and degrades the transcription factor c-Myc but also binds Argonaute-1 and thereby increases the activity of specific microRNAs. Let-7 is one of the TRIM32 targets and is required and sufficient for neuronal differentiation. TRIM32 is the mouse ortholog of Drosophila Brat and Mei-P26 and might be part of a protein family that regulates the balance between differentiation and proliferation in stem cell lineages (Schwamborn, 2009).

The data suggest that the increased levels of TRIM32 in one of the two daughter cells contribute to the decision of this cell to undergo neuronal differentiation. Like Brat, TRIM32 localizes asymmetrically in mitosis. Brat is localized by binding to Miranda, which, in turn, is recruited to the basal side by the protein Lgl and excluded from the apical side by aPKC (Knoblich, 2008). In fly neuroblasts, aPKC promotes self-renewal whereas Lgl inhibits proliferation. Although Miranda is not conserved, mouse Lgl and aPKC have similar effects on neural progenitor proliferation. In Lgl knockout mice, neural precursors overproliferate and eventually die by apoptosis. Removing one of the two aPKC mouse homologs does not affect the rate of neurogenesis, but depletion of its binding partner Par-3 results in premature cell-cycle exit of cortical progenitors. Despite these similarities, the precise mechanism by which TRIM32 localizes may be quite distinct. In Drosophila, the apical Par-3/6/aPKC complex directs the basal localization of Brat and Miranda but also orients the mitotic spindle along the apical-basal axis. In mice, however, the vast majority of progenitor divisions do not occur along the apical-basal axis. TRIM32 is asymmetric even in those planar divisions and provides a suitable explanation for how unequal fates can be generated independently of cleavage plane orientation. Therefore, the relevance of TRIM32 segregation is independent of the somewhat conflicting results that have been reported for the fraction of horizontal versus vertical divisions. Since TRIM32 asymmetry does not follow the polarity set up by Par-3/6/aPKC, however, it is likely that it is established by mechanisms distinct from Drosophila (Schwamborn, 2009).

What could those mechanisms be? TRIM32 often concentrates in the retracting basal fiber, a structure that is not present in Drosophila neuroblasts. TRIM32 might be present in the cytoplasm of the fiber and could be retained in the basal part of the cell during mitosis, when the fiber becomes extremely thin and its cytoplasm flows into the dividing progenitor. This would explain why TRIM32 is asymmetric even when the spindle is not oriented along the apical-basal axis. Since TRIM32-GFP expression prevents mitosis even at low levels, this observation cannot be verified by live imaging. The model would predict that the cell inheriting the basal fiber preferentially undergoes neuronal differentiation. This is in good agreement with some previous live-imaging studies, but other studies have actually proposed that the fiber is maintained in mitosis and serves as a guide for migration of the newly formed neuron. At the moment, it cannot be excluded that other mechanisms contribute to the asymmetric localization of TRIM32 (Schwamborn, 2009).

How does TRIM32 affect proliferation and differentiation? The data suggest that TRIM32 acts through two distinct pathways. Through its N-terminal RING finger, TRIM32 ubiquitinylates c-Myc and targets it for proteasome-mediated degradation. High levels of c-Myc are important for the ability of NSCs to self-renew and make NSCs relatively easy targets for reprogramming into ES cells. Furthermore, the bFGF–SHP2–ERK–c-Myc–Bmi-1 pathway is critical for the self-renewal capacity of neural progenitor cells, and Myc overexpression is known to promote neural progenitor proliferation in the mouse CNS. Therefore, a TRIM32-mediated reduction in the levels of c-Myc may well serve as a first step to induce neuronal differentiation. In agreement with this, overexpression of c-Myc in GFAP-positive astrocytes promotes formation of less differentiated Nestin-positive progenitor-like cells while a conditional ablation of the c-Myc ortholog N-Myc in mouse neuronal progenitor cells dramatically increases neuronal differentiation (Schwamborn, 2009).

Through its C-terminal NHL domain, TRIM32 acts as a potent activator of certain microRNAs. Although Drosophila Mei-P26 also binds Ago1, it inhibits rather than enhances microRNAs, and the mechanisms by which TRIM32 and its invertebrate homologs regulate microRNAs may actually be quite distinct. This is consistent with the observation that microRNAs support self-renewal in Drosophila stem cells while they potentiate differentiation in mammalian stem cells. In particular, Let-7a has an antiproliferative effect, and its expression reduces tumor growth and can prevent self-renewal in breast cancer cells. In NSCs, Let-7a is expressed and upregulated during differentiation. It is interesting to note that one of the targets for Let-7a is Myc. Protein degradation and concomitant translational inhibition through microRNAs might be the key strategy through which TRIM32 induces differentiation in NSCs (Schwamborn, 2009).

Although brat and mei-P26 mutant flies develop tumors, TRIM32 has not been described as a tumor suppressor. In fact, several reports have even suggested that TRIM32 might induce rather than prevent tumor formation. TRIM32 is mutated in patients carrying limb girdle muscular dystrophy type 2H. Since TRIM32 expression is upregulated during myogenic differentiation, the muscular dystrophy in these patients could be explained by a differentiation defect in the satellite cell lineage analogous to the one found in NSC lineages. TRIM32 has also been described as a gene potentially responsible for Bardet-Biedl syndrome and therefore has also been named BBS11. Distinct TRIM32 mutations are responsible for the two diseases, but none of them seems to cause cancer since an increase in tumor formation is not described for any of the two diseases. Since TRIM32 is a bifunctional molecule, mutating only the RING or the NHL domain might not be sufficient to prevent the antiproliferative function of TRIM32. In Drosophila, tumors only form in a small subset of brat mutant neuroblasts (Bowman, 2008). In other neuroblasts, redundancy with other tumor suppressors prevents overproliferation. Should a similar degree of redundancy exist in vertebrates, this might explain why TRIM32 is not a common target for oncogenic mutations. A similar lack of a human tumor phenotype has been shown for the Drosophila tumor suppressor Lgl. In Drosophila, lgl mutant neuroblasts overproliferate and form brain tumors. In mice, however, lgl mutant neural progenitors overproliferate initially but then die by apoptosis. A vertebrate-specific mechanism that prevents tumorigenesis in response to stem cell overproliferation could provide an alternative explanation for the lack of tumor formation when TRIM32 function is compromised. Although such a mechanism has been suggested previously the underlying mechanism remains unclear (Schwamborn, 2009).

These data establish TRIM-NHL proteins as a family of conserved stem cell regulators. The fact that Mei-P26 regulates stem cell proliferation in Drosophila ovaries (Neumuller, 2008) suggests that the function of this protein family might extend way beyond the brain. If this is the case, the presence of a catalytically active RING finger domain that could be inhibited by pharmaceutical compounds might make these proteins attractive targets for the manipulation of stem cell proliferation and the stimulation of regeneration in vivo (Schwamborn, 2009).

Small-RNA (sRNA)-guided transcriptional gene silencing by Argonaute (Ago)-containing complexes is fundamental to genome integrity and epigenetic inheritance. The RNA cleavage ("Slicer") activity of Argonaute has been implicated in both sRNA maturation and target RNA cleavage. Typically, Argonaute slices and releases the passenger strand of duplex sRNA to generate active silencing complexes, but it remains unclear whether slicing of target nascent RNAs, or other RNAi components, also contributes to downstream transcriptional silencing. This study developd a strategy for loading the fission yeast Ago1 with a single-stranded sRNA guide, which bypasses the requirement for slicer activity in generation of active silencing complexes. Slicer-defective Ago1 was shown to mediate secondary sRNA generation, H3K9 methylation, and silencing similar to or better than wild-type and associates with chromatin more efficiently. The results define an ancient and minimal sRNA-mediated chromatin silencing mechanism, which resembles the germline-specific sRNA-dependent transcriptional silencing pathways in Drosophila and mammals (Jain, 2016).

date revised: 25 April 2024

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