InteractiveFly: GeneBrief

let-7 : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

In Caenorhabditis elegans, the heterochronic pathway controls the timing of developmental events during the larval stages. A component of this pathway, the let-7 small regulatory RNA (Lagos-Quintana, 2001) is expressed at the late stages of development and promotes the transition from larval to adult (L/A) stages. The stage-specificity of let-7 expression, which is crucial for the proper timing of the worm L/A transition, is conserved in Drosophila and other invertebrates. In Drosophila, pulses of the steroid hormone 20-hydroxyecdysone (ecdysone) control the timing of the transition from larval to pupal to adult stages. To test whether Drosophila let-7 expression is regulated by ecdysone, Northern blot analysis was used to examine the effect of altered ecdysone levels on let-7 expression in mutant animals, organ cultures, and S2 cultured cells. Experiments were conducted to test the role of Broad-Complex (BR-C), an essential component in the ecdysone pathway, in let-7 expression. Ecdysone and BR-C are required for let-7 expression, indicating that the ecdysone pathway regulates the temporal expression of let-7 in Drosophila. These results demonstrate an interaction between steroid hormone signaling and the heterochronic pathway in insects (Sempere, 2002).

The temporal coordination of cell proliferation, differentiation, and apoptosis during development is essential for the correct morphogenesis of an adult animal. In the fruit fly Drosophila melanogaster and the nematode Caenorhabditis elegans, genetic regulatory circuits control the timing of the transition from larval to adult stages. In Drosophila, larvae pupariate and initiate metamorphosis after the third larval instar. In C. elegans, the adult stage follows immediately after the fourth (final) larval molt. Studies on metamorphosis in Drosophila have revealed a pivotal role for the steroid hormone 20-hydroxyecdysone (ecdysone) and ecdysone-regulated gene expression. Ecdysone orchestrates a complex, hierarchical gene expression cascade that transforms the larva into a highly motile reproductive adult fly. In C. elegans, the heterochronic gene pathway generates the temporal contexts in which the appropriate developmental programs are executed throughout development of the larva to the adult. Although nematodes do not undergo an overt metamorphosis, as does Drosophila, the life cycle of the two animals is similar in that both undergo stages of molting development prior to the adult stage. This common developmental strategy groups them together as ecdysozoans, in recognition that a common ancestor underwent a series of larval stages punctuated by cuticular molts (Sempere, 2002 and references therein).

Dynamic changes in ecdysone levels regulate progression through the larval stages of holometabolous insects. During the third (final) instar of Drosophila, a series of low-level ecdysone pulses signal the transition from feeding to wandering, in preparation for pupariation. Following a high-level ecdysone pulse, the white prepupa forms and larval tissues begin to either remodel or histolize. Meanwhile, precursors of adult structures and tissues, which include the imaginal discs, histoblasts, and imaginal cell nests along the midgut, initiate their proliferation and differentiation programs. Some 10-12 h after puparium formation, a second ecdysone pulse leads to head eversion and pupa formation. A broad and high-level peak of ecdysone secretion during the pupal stage triggers the terminal differentiation of the adult structures (Sempere, 2002).

Ecdysone pulses trigger each developmental transition by initiating a program of downstream gene expression. Ecdysone binds a heterodimeric protein receptor, composed of an Ecdysone receptor subunit (EcR) and an RXR-like subunit encoded by the ultraspiracle (usp) gene. The ecdysone-receptor complex binds to a cis-acting regulatory element, known as the ecdysone response element (EcRE) in the enhancers of specific target genes, thereby causing an increase in target gene transcription. According to the Ashburner model (Ashburner, 1974), postlarval development begins by the hormone-dependent activation of a small set of 'early' genes that include Broad-Complex (BR-C), E74, and E75. Each of these genes encodes a complex set of protein isoforms that function as sequence-specific DNA binding proteins and transcriptional regulators. The protein products of early genes activate a second cascade of gene expression, the 'late' genes, and inhibit early gene expression by feedback. The outcome of this unfolding genetic cascade is manifest at the cell and tissue levels as biochemical and morphological differentiation (Sempere, 2002 and references therein).

In C. elegans, a newly hatched larva develops through four larval stages (L1 to L4), punctuated by molts, to a reproductive adult. Blast cells, set aside during embryogenesis, divide during the larval stages and give rise to stage-specific larval features, and the adult-specific reproductive structures. The heterochronic genes lin-4 and let-7 are crucial for promoting the transitions from early to late developmental programs (Lee, 1993; Reinhart, 2000). lin-4 and let-7 are small regulatory RNAs (22 and 21 nucleotides, respectively), which act as translational repressors by base-pairing with the 3'-UTRs of their target gene mRNAs (Lee, 1993; Reinhart, 2000). The accumulation of lin-4 RNA at the beginning of the L2 stage downregulates the protein levels of its target genes, lin-14 and lin-28, and permits the coordinated transition from L1 to later programs. Similarly, the accumulation of let-7 RNA at the beginning of the L4 stage downregulates lin-41 (Slack, 2000) and possibly lin-57 (A. Rougvie, personal communication to Sempere, 2002) and promotes the larval to adult (L/A) transition from L4 to adult programs (Sempere, 2002 and references therein).

The participation of two small regulatory RNAs in the heterochronic pathway raises the question of whether similar regulatory RNAs could be involved in the control of postembryonic development in other animals. Indeed, let-7 is conserved across the bilaterian clade, including flies and humans (Pasquinelli, 2000). In invertebrates, let-7 RNA expression coincides with the onset of the L/A transition. Similarly, let-7 expression is upregulated during vertebrate development, although at somewhat different stages depending on the species (Pasquinelli, 2000). Studies in human (Pasquinelli, 2000) and murine cell lines demonstrate the presence of high levels of let-7 RNA in mature cell types (e.g., brain and lung) and marginal let-7 expression in immature or totipotent cell types (bone marrow and murine embryonic stem cell). Taken together, these observations suggest a general role for let-7 in the terminal differentiation of bilaterians (Pasquinelli, 2000, Sempere, 2002).

In Drosophila, let-7 RNA first appears at the end of the third larval instar, a few hours before puparium formation, and reaches high levels during pupal development (Pasquinelli, 2000; Hutvagner, 2001). Given the leading role of ecdysone in the temporal coordination of metamorphosis, investigations were carried out to see whether the expression of let-7 in Drosophila is dependent on the ecdysone gene pathway. Indeed, ecdysone and the early ecdysone-inducible gene BR-C are required for let-7 expression in intact animals, organ cultures, and S2 cells. These results suggest that hormone-induced expression of let-7 could control developmental stage transitions in animals (Sempere, 2002).

Several lines of evidence are presented that both ecdysone and the early ecdysone-inducible gene BR-C are required for the expression of let-7 RNA in Drosophila. This indicates that the ecdysone pathway regulates the temporal expression of let-7 in the fly. Previously identified ecdysone-inducible genes include a series of transcriptional factors that are organized in a hierarchical network to control metamorphic processes. In C. elegans, let-7 is a translational repressor (Slack, 2000), and so Drosophila let-7 may mediate aspects of the hormonal control of metamorphosis by regulating gene expression post-transcriptionally (Sempere, 2002).

The primary evidence that let-7 expression is triggered by the ecdysone pathway comes from two lines of experiments: (1) mutant animals defective in ecdysone biosynthesis or in BR-C activity display reduced or absent let-7 RNA levels; (2) sustained expression of let-7 RNA in organ culture requires the application of exogenous ecdysone. These experiments do not rule out the possibility that let-7 expression could be a collateral consequence of ecdysone signals, for example, as a consequence of pupariation or progression through metamorphosis. However, if this were the case, then ecdysone-induced let-7 expression would not be expected in S2 cells, since S2 cells do not undergo morphogenesis. The finding that ecdysone induces let-7 expression in S2 cells strongly suggests that ecdysone pathway triggers let-7 expression within the cells exposed to the hormone, and independently of overt metamorphosis. The requirement of BR-C activity in animals and S2 cells for let-7 RNA induction indicates that BR-C is an intermediate player between the ecdysone signal and activation of let-7, and that the let-7 response to ecdysone exhibited in S2 cells likely reflects the same process as in vivo. One difference between the response to ecdysone in S2 cells and animals is that, in S2 cells, let-7 expression begins about 24 h after the addition of ecdysone, while in animals, let-7 expression begins about 4 h after the pulse of ecdysone at the end of the third larval stage. Components of the pathway mediating let-7 activation by ecdysone in S2 cells may be relatively limiting compared to intact animals, and perhaps the concentration of ecdysone needed to activate let-7 expression when applied to S2 cells may not be as effective as that in vivo. Although a role for RNA stability or processing in the induction of let-7 by ecdysone cannot be ruled out, the simplest interpretation is that let-7 induction occurs at the transcriptional level, since levels of let-7 RNA and its precursor let-7L (Grishok, 2001; Hutvagner, 2001) increase or decrease coordinately, depending on the status of ecdysone signaling (Sempere, 2002).

The timing of let-7 expression is conserved in invertebrates: let-7 RNA accumulates toward the end of larval development in flies, worms, and mollusks, coinciding with the specification of adult programs (Pasquinelli, 2000). Although the results indicate that in dipterans let-7 is induced by ecdysone, let-7 expression could be coupled to other signals in other animals. In C. elegans, the timing of let-7 expression is controlled partly by upstream components of the heterochronic pathway in conjunction with other regulatory signals. daf-12, an orphan nuclear hormone receptor (NHR), is an upstream component of the heterochronic pathway implicated in the regulation of let-7 expression. DHR96, the closest ortholog of daf-12 in Drosophila, also encodes an orphan NHR. DHR96 is one of the eight NHRs whose expression is regulated by ecdysone during metamorphosis. daf-12 and dhr96 could represent an evolutionarily conserved point of convergence in C. elegans and Drosophila let-7 regulatory pathways. Whether C. elegans utilizes other components of the ecdysone pathway during its development remains an open question. More than 200 NHRs have been predicted in C. elegans, and some of these are clear orthologs of Drosophila NHRs involved in metamorphosis, suggesting that they could play similar roles in C. elegans (Sempere, 2002).

The increase in let-7 RNA at pupariation in response to ecdysone/BR-C activity has suggested a potential role for let-7 in Drosophila metamorphosis. In C. elegans, let-7 promotes the larval to adult transition by downregulating lin-41 protein levels. There are several complementary sites to let-7 in the 3'-UTR of lin-41 mRNA to which let-7 could bind to repress translation (Slack, 2000). lin-41 codes for a RBCC protein (Slack, 2000) and is a founding member of the NHL domain family. In Drosophila, there are three orthologs of lin-41: dappled, brat, and mei-P26. These lin-41 orthologs appear to be involved somehow in growth suppression in flies; mutations in dappled and brat result in melanotic tumors, and mutations in mei-P26 result in ovarian tumors, dappled and brat are expressed at the end of the third instar in fat bodies and ring gland (brain), and brain and wing imaginal discs, respectively. mei-P26 is expressed in the germ line. These expression patterns overlap with that of let-7 RNA, suggesting that the lin-41 orthologs could be let-7 targets. Indeed, sequences in the 3'-UTR of dappled, brat, and mei-P26 resemble let-7 complementary sites to which let-7 could bind. Future work is required to determine whether let-7 is a regulator of these and/or other genes and to assess the implications of this regulation in metamorphic processes, such as apoptosis, differentiation, and morphogenesis (Sempere, 2002 and references therein).

The let-7-Imp axis regulates aging of the Drosophila testis stem-cell niche

Adult stem cells support tissue homeostasis and repair throughout the life of an individual. During ageing, numerous intrinsic and extrinsic changes occur that result in altered stem-cell behaviour and reduced tissue maintenance and regeneration. In the Drosophila testis, ageing results in a marked decrease in the self-renewal factor Unpaired (Upd), leading to a concomitant loss of germline stem cells. This study demonstrates that IGF-II messenger RNA binding protein (Imp) counteracts endogenous small interfering RNAs to stabilize upd (also known as os) RNA. However, similar to upd, Imp expression decreases in the hub cells of older males, which is due to the targeting of Imp by the heterochronic microRNA let-7. In the absence of Imp, upd mRNA therefore becomes unprotected and susceptible to degradation. Understanding the mechanistic basis for ageing-related changes in stem-cell behaviour will lead to the development of strategies to treat age-onset diseases and facilitate stem-cell-based therapies in older individuals (Toledano, 2012).

Many stem cells lose the capacity for self-renewal when removed from their local microenvironment (or niche), indicating that the niche has a major role in controlling stem-cell fate. Changes to the local and systemic environments occur with age that result in altered stem-cell behaviour and reduced tissue maintenance and regeneration. The stem-cell niche in the Drosophila testis is located at the apical tip, where both germline stem cells (GSCs) and somatic cyst stem cells are in direct contact with hub cells. Hub cells express the self-renewal factor Upd, which activates the JAK-STAT signalling pathway to regulate the behaviour of adjacent stem cells. Ageing results in a progressive and significant decrease in the levels of upd in hub cells. However, constitutive expression of upd in hub cells was sufficient to block the age-related loss of GSCs, suggesting that mechanisms might be in place to regulate upd and maintain an active stem-cell niche (Toledano, 2012).

To identify potential regulators of upd, a collection of transgenic flies carrying green fluorescent protein (GFP)-tagged proteins was screened for expression in hub cells. The Drosophila homologue of Imp protein is expressed throughout the testis tip in young flies (Fabrizio, 2008); however, antibody staining revealed a decrease (~50%) in Imp expression in the hub cells of aged males. Imp is a member of a conserved family of RNA-binding proteins that regulate RNA stability, translation and localization (Yisraeli, 2005). Given the similarity in the ageing-related decline in Imp protein and upd mRNA in hub cells, it is proposed that Imp could be a new regulator of upd (Toledano, 2012).

To address whether Imp acts in hub cells to regulate upd, the bipartite GAL4-UAS system was used in combination with RNA-mediated interference (RNAi) to reduce Imp expression exclusively in hub cells. Fluorescence in situ hybridization (FISH) to detect upd mRNA was used in combination with immunofluorescence microscopy to determine whether the loss of Imp expression affects upd levels. The loss of Imp specifically in hub cells resulted in reduced expression of upd, as well as a significant reduction in GSCs and hub cell), when compared with controls. Consistent with a reduction in JAK-STAT signalling, decreased accumulation of STAT was observed when Imp levels were reduced by RNAi in hub cells (Toledano, 2012).

RNA-binding proteins characteristically target several RNAs; therefore, it was of interest to determine whether upd is a physiologically relevant target of Imp. Expression of upd together with an Imp RNAi construct was sufficient to completely rescue the defects caused by reduced Imp expression in hub cells, suggesting that Upd acts downstream of Imp to maintain GSCs and niche integrity. Importantly, the constitutive expression of upd alone in hub cells did not lead to an increase in GSCs in testes from 1-day-old males. These data suggest that Imp acts in hub cells to promote niche integrity and GSC maintenance, at least in part, by positively regulating upd (Toledano, 2012).

If Imp acts in hub cells in adult testes to regulate upd mRNA, it is speculated that the loss of Imp function during development might lead to a decrease in upd and a subsequent reduction in GSCs. Null mutations in Imp result in lethality at the pharate adult stage; therefore, testes from third instar larvae (L3) carrying Imp null alleles, Imp⁷ and Imp⁸, were examined. Deletion of the Imp locus was verified by PCR of genomic DNA. Combined immunofluorescence and FISH showed that although Fas3⁺ hub cells were easily detected, the expression of upd was significantly reduced: 24% of Imp⁷ mutants and 15% of Imp⁸ mutants had no detectable upd at this stage. In addition, the average number of GSCs and hub cells in testes from Imp mutants was significantly reduced when compared with control L3 testes. Notably, the re-expression of Imp in somatic niche cells was sufficient to rescue upd expression in Imp mutants to comparable levels to controls, and the reduction in the average number of GSCs and hub cells in Imp mutants was also reversed (Toledano, 2012).

Imp family members contain conserved KH domains that mediate direct binding to RNA targets. To determine whether Imp could associate directly with upd mRNA in vivo, testes were dissected from young flies expressing GFP-tagged Imp. Immunoprecipitation of Imp with anti-GFP antibodies, followed by quantitative reverse transcriptase PCR (qRT-PCR) analysis, showed a significant enrichment (~208-fold) of associated upd mRNA relative to control antibodies. Minimal enrichment for the ubiquitously expressed RNAs rp49 (also known as RpL32; ~4-fold) and GapDH (also known as Gapdh1; ~8 fold) or for the negative control med23 (~4-fold), was observed after Imp immunoprecipitation, indicating that the interaction between Imp and upd mRNA in hub cells is specific. Consistent with these observations, Imp protein and upd RNA co-localized in hub cells within perinuclear foci, probably ribonucleoprotein particles (Toledano, 2012).

An in vitro protein-RNA binding assay showed that Imp associates with the upd 3' untranslated region (UTR), specifically the first 250 base pairs (region 1), as no substantial binding to other portions of the upd 3'UTR was detected. Moreover, Imp did not bind the 5' untranslated or coding regions of upd or to the med23 3'UTR. Notably, a putative consensus binding sequence CAUH (in which H denotes A, U or C) for the mammalian IMP homologues (IGF2BP1- 3) occurs 22 times within the upd 3'UTR, including a cluster of four tandem repeats within the first 35 nucleotides of region 1. To test whether this motif mediates binding between Imp and upd, the first 33 nucleotides were removed to generate a sequence excluding the CAUH repeats, which resulted in a reduction in binding, compare domain 1 with domain 2. Point mutations in the third nucleotide of each motif (U = G) did not abolish the binding; however, point mutations in the consensus motif of MRPL9 RNA, a target of mammalian IGF2BPs, also did not abolish binding, suggesting that secondary structures probably mediate the association between IGFBP family members and their target RNAs. Altogether, the data identify the first 33 base pairs of the upd 3'UTR as a putative target sequence for Imp, and support observations that Imp associates specifically with upd in vivo (Toledano, 2012).

To gain further insight into the mechanism by which Imp regulates upd, a GFP reporter was constructed that contained the 3'UTR from either upd or med23. Transcript levels for gfp were fivefold higher in Drosophila Schneider (S2) cells that co-expressed Imp with the gfp-upd-3'UTR reporter than in cells that co-expressed Imp with the gfp-med23-3'UTR reporter. The significant increase in reporter mRNA levels indicates that it is likely that Imp regulates upd mRNA stability (Toledano, 2012).

RNA-binding proteins, including mammalian IGF2BP1, have been shown to counter microRNA (miRNA)-mediated targeting of mRNAs. However, no consensus miRNA seeds were located within the first 34 base pairs of domain 1 of the upd 3'UTR. It is speculated that if Imp binding blocks small RNA-mediated degradation of upd, polyadenylated, cleaved upd degradation intermediates would be detected in the testes of older males, when Imp expression in hub cells is reduced. Using a modified rapid amplification of complementary DNA ends (RACE) technique, a specific cleavage product was identifed starting at nucleotide 33 of the upd 3'UTR in the testes of 30-day-old flies, but not in RNA extracts from the testes of 1-day-old males. Importantly, the same degradation product of upd was also detected in the testes of young flies when Imp was specifically depleted from hub cells using RNAi-mediated knockdown. As a positive control, the esi-2-mediated cleavage product of mus308 was detected in testes from both 1- and 30-day-old flies (Toledano, 2012).

To test whether small RNAs might mediate upd cleavage, small RNA libraries generated from the testes of 1- and 30-day-old flies were cloned and deep-sequenced. Although no small RNAs with exact pairing to the upd degradation product were identified, two short interfering RNAs (siRNAs; termed siRNA1 and siRNA2) with high sequence complementarity to the predicted target site in the upd 3'UTR were present in the testis library generated from 30-day-old males. Using qRT- PCR for mature small RNAs, it was found that the siRNA2 levels in the testes, relative to the levels of the control small RNAs bantam and mir-184, were similar in young and old males (deep sequencing analysis demonstrated that expression of these two control miRNAs did not change with age). The source of siRNA2 is the gypsy5 transposon, which is inserted at several loci throughout the fly genome and is conserved in numerous Drosophila species (Toledano, 2012).

To gain further insight into the mechanism by which Imp and siRNA2 regulate upd, the levels of the upd GFP reporter (gfp-upd-3'UTR) in the presence or absence of Imp and siRNA2 was investigated in S2 cells. To generate a reporter that should not be susceptible to siRNA-mediated degradation, the cleavage site in the upd 3'UTR that was identified by RACE (³²AUU = CGG; gfp-upd-3'UTR^mut) was mutated. Cells were transfected with either of the GFP reporter constructs, with or without haemagglutinin-tagged Imp (Imp- HA), and subsequently transfected with siRNA2; gfp expression was quantified by qRT- PCR (Toledano, 2012).

The co-expression of siRNA2 and the gfp-upd-3'UTR reporter resulted in a significant decrease in gfp transcript levels. Conversely, the co-expression of Imp blocked siRNA2-mediated reduction of gfp mRNA such that gfp levels were higher than in control cells. Furthermore, mutation of the putative cleavage site rendered the upd 3'UTR resistant to siRNA2-mediated degradation. These data, in combination with the in vitro binding data, suggest that Imp binds to and protects the upd 3'UTR from endogenous and exogenous siRNA2 in S2 cells. Thus, endo-siRNA2 is a bona fide candidate that could direct upd degradation when Imp is absent or its levels are reduced, although targeting by other small RNAs cannot be excluded (Toledano, 2012).

In Drosophila, Argonaute-1 (AGO1) is the principle acceptor of miRNAs and primarily regulates targets in a cleavage-independent mode, whereas AGO2 is preferentially loaded with siRNAs and typically regulates targets by mRNA cleavage. AGO2 expression was detected throughout the tip of the testis, as verified by immunostaining of testes from transgenic flies expressing 3×Flag-HA-tagged AGO2. To test whether AGO2 binds to upd mRNA in vivo, thereby potentially regulating upd levels directly, testes were dissected from aged (30-day-old) 3×Flag- HA- AGO2 males. Immunoprecipitation of AGO2, followed by qRT- PCR, showed significant enrichment (~102-fold) of upd mRNA bound to AGO2. Negligible binding of a negative control, rp49, to AGO2 was detected, suggesting specific association of AGO2 with upd mRNA in vivo and supporting a previous findings that upd is probably targeted by the siRNA pathway (Toledano, 2012).

To test whether Imp can impede the binding of AGO2 to the upd 3'UTR, S2 cells stably expressing Flag-tagged AGO2 were transfected with the gfp-upd-3'UTR reporter. Consistent with our previous observations, transcript levels of gfp-upd-3'UTR increased ~18-fold when Imp was co-expressed. Despite increases in the overall levels of gfp mRNA, the presence of Imp markedly reduced the association of AGO2 with the upd 3'UTR, indicating that Imp antagonizes the ability of AGO2 to bind the upd 3'UTR (Toledano, 2012).

Similar to the AGO family, Drosophila encodes two Dicer proteins that seem to have distinct roles in small RNA biogenesis. Dicer-1 (Dcr-1) is essential for the generation of miRNAs, and Dcr-2 is required for siRNA production from exogenous and endogenous sources. If siRNAs were involved in upd degradation in older males, it would be predicted that the loss of Dcr-2 would suppress the ageing-related decline in upd and GSCs. Consistent with a role for Dcr-2 in the generation of siRNAs, siRNA2 levels were significantly reduced in Dcr-2 homozygous mutants relative to heterozygous controls. Testes from 30- and 45-day-old Dcr-2 mutant flies showed increased levels of upd by qRT- PCR when compared with controls. Whereas a ~90% reduction of upd is observed in the testes from aged Dcr-2 heterozygous controls, only a ~45% reduction in upd was observed in testes from age-matched, Dcr-2 homozygous mutants, indicating that upd levels are higher when Dcr-2 function is compromised. Furthermore, the testes from aged Dcr-2 mutants contained more GSCs, on average, when compared with controls. Conversely, the forced expression of Dcr-2 in hub cells resulted in a reduction in the average number of GSCs and led to a significant reduction in upd levels, as detected using qRT- PCR and combined immunofluorescence and FISH, which seemed to be specific, as no significant change in Imp transcript levels was observed. Expression of Imp in combination with Dcr-2 resulted in a significant increase in upd levels. These observations indicate that Imp can counter the decrease in upd levels resulting from forced Dcr-2 expression, providing further evidence that Imp protects upd from targeted degradation by the siRNA pathway (Toledano, 2012).

The data suggest that Imp has a role in stabilizing upd in hub cells; therefore, the ageing-related decline in Imp would be a major contributing factor to the decrease in upd mRNA in the hub cells of aged males. To investigate the mechanism that leads to the decline in Imp expression with age, the Imp 3'UTR was examined for potential instability elements. Within the first 160 base pairs there is a canonical seed sequence for the heterochronic miRNA let-7. Expression of a reporter gene under the control of the let-7 promoter showed that let-7 expression increases in hub cells of ageing male, which was confirmed by let-7 FISH of testes from aged males. In addition, mature let-7 miRNA was enriched twofold in the testes from 30-day-old flies, relative to 1-day-old males. Therefore, an age-related increase in let-7 is one mechanism by which Imp expression could be regulated in an ageing-dependent manner in testes from older males (Toledano, 2012).

Consistent with these observations, the forced expression of let-7 specifically in hub cells led to a decrease in Imp. In addition, let-7 expression in S2 cells reduced the levels of a heterologous gfp-Imp-3'UTR^WT reporter. S2 cells were transfected with a let-7 mimic or with negative control miRNA, and gfp expression was quantified by qRT- PCR. There was a 70% reduction in gfp-Imp-3'UTR^WT expression in the presence of let-7, relative to control miRNA. A gfp-Imp-3'UTR^mut reporter with mutations in the canonical seed for let-7 (at nucleotide 137) was unaffected by let-7 expression, indicating that mutation of the let-7 seed rendered the RNA resistant to degradation. These data confirm that let-7 can destabilize Imp through sequences in the 3'UTR. However, further increasing the levels of let-7 resulted in a decrease in gfp expression from the mutated 3'UTR, indicating that other, putative let-7 seeds in the Imp 3'UTR can be targeted by let-7 (Toledano, 2012)

If the age-related decrease in Imp contributes to a decline in upd and subsequent loss of GSCs, it is proposed that re-expression of Imp in hub cells would rescue the ageing-related decrease in upd. Therefore, flies in which Imp was constitutively expressed in hub cells were aged, and upd levels were quantified by qRT- PCR. The expression of an Imp construct containing a truncated 3'UTR (Imp-KH- HA) lacking let-7 target sequences specifically in hub cells was sufficient to suppress the ageing-related decline in upd, with concomitant maintenance of GSCs, similar to what was observed by re-expressing upd in the hub cells of aged males. Maintenance of Imp-KH- HA expression in aged males was verified by staining with an anti-HA antibody. Conversely, the expression of an Imp construct that is susceptible to degradation by let-7 (Imp^T21) did not lead to an accumulation of Imp in the testes of 30- and 50-day-old flies, as levels were similar to the levels of endogenous Imp at later time points. Consequently, the expression of this construct was not sufficient to block the ageing-related decline in GSCs. These data indicate that let-7-mediated regulation of Imp contributes to the decline in Imp protein in older flies, and supports a model in which an ageing-related decline in Imp, mediated by let-7, exposes upd to degradation by siRNAs. Thus, both the miRNA and siRNA pathways act upstream to regulate the ageing of the testis stem-cell niche by generating let-7 and siRNA2, which target Imp and upd, respectively (Toledano, 2012).

Drosophila has proven to be a valuable model system for investigating ageing-related changes in stem-cell behaviour. Cell autonomous and extrinsic changes contribute to altered stem-cell activity; thus, determining the mechanisms underlying the ageing-related decline of self-renewal factors, such as the cytokine-like factor Upd, may provide insight into strategies to maintain optimal niche function (Toledano, 2012).

The data indicate that Imp can regulate gene expression by promoting the stability of selected RNA targets by countering inhibitory small RNAs. Therefore, rescue of the aged niche by Imp expression may be a consequence of effects on Imp targets, in addition to upd, in somatic niche cells. Furthermore, as Imp is expressed in germ cells, it could also act in an autonomous manner to regulate the maintenance of GSCs. The canonical let-7 seed in the Imp 3'UTR is conserved in closely related species, and reports have predicted that the let-7 family of miRNAs target mammalian Imp homologues (IGF2BP1- 3). Given the broad role of the let-7 family in ageing, stem cells, cancer and metabolism, the regulation of Imp by let-7 may be an important, conserved mechanism in numerous physiological processes (Toledano, 2012).

Non-coding RNAs can ensure biological robustness and provide a buffer against relatively small fluctuations in a system. However, after a considerable change, a molecular switch is flipped, which allows a biological event to proceed unimpeded. In the current model, Imp preserves niche function in young flies until a time at which miRNAs and siRNAs act together to trigger an 'ageing' switch that leads to a definitive decline in upd and, ultimately, in stem-cell maintenance. Therefore, targeting signalling pathways at several levels using RNA-based mechanisms will probably prove to be a prevalent theme to ensure robustness in complex biological systems (Toledano, 2012).

An intrinsic tumour eviction mechanism in Drosophila mediated by steroid hormone signalling

Polycomb group proteins are epigenetic regulators maintaining transcriptional memory during cellular proliferation. In Drosophila larvae, malfunction of Polyhomeotic (Ph), a member of the PRC1 silencing complex, results in neoplastic growth. This study reports an intrinsic tumour suppression mechanism mediated by the steroid hormone ecdysone during metamorphosis. Ecdysone alters neoplastic growth into a nontumorigenic state of the mutant ph cells which then become eliminated during adult stage. This study demonstrates that ecdysone exerts this function by inducing a heterochronic network encompassing the activation of the microRNA lethal-7, which suppresses its target gene chronologically inappropriate morphogenesis (chinmo). This pathway can also promote remission of brain tumours formed in brain tumour mutants, revealing a restraining of neoplastic growth in different tumour types. Given the conserved role of let-7, the identification and molecular characterization of this innate tumour eviction mechanism in flies might provide important clues towards the exploitation of related pathways for human tumour therapy (Jiang, 2018).

Deregulation of PcG gene expression has been associated with various types of human cancer. For example, loss of expression of the human ph homologue has been linked to the formation of osteosarcomas. As the molecular mechanisms of PcG proteins in human cancers are largely unknown, understanding the tumour-suppressor function of PcG genes in Drosophila therefore could provide insights in human cancer biology. During the past decades imaginal discs have been used as a powerful paradigm to investigate mechanisms underlying the formation and progression of several types of tumour, including Ras-, PcG-, or Hippo pathway-induced tumours. In addition, it is worth noting that the let-7 consensus sequence is identical from Caenorhabditis elegans to humans, suggesting that let-7 may control functionally conserved targets in regulating proliferation and differentiation during development. In various types of human cancer, downregulation of one or more let-7 members has been observed. Moreover, induced expression of let-7 in cancer cell lines can suppress cell proliferation and tumour growth. In the human genome, the let-7 family consists of more than ten members. However, the transcriptional regulation, spatial and temporal expression, and their tissue-specific and/or redundant functions of the let-7 family in human are far more complicated and still remain elusive (Jiang, 2018).

This study identified an intrinsic mechanism reprogramming tumorigenic to nontumorigenic cells of at least two different tumour types, by marking the cells for destruction in adult Drosophila. The steroid hormone-induced miRNA let-7 is a key mediator of this mechanism. Interestingly, let-7 and its target genes, including chinmo have been shown to act as heterochronic genes that regulate developmental transitions. Other findings from this lab indicate that ph⁵⁰⁵ tumour cells are reprogrammed from the original larval imaginal disc identity to an early embryonic state. By artificially overexpressing a differentiation factor, these cancer cells can be induced to lose their neoplastic state, however. It appears as if these tumour cells are trapped in an immature condition, unable to differentiate. Pulses of ecdysone are a major timer of developmental transitions in flies and their target, let-7, is for example required for enforcing the terminal cell cycle arrest in pupal stage wing discs. These findings suggest that flies have evolved tumour suppressive mechanisms by inducing let-7-controlled heterochronic gene networks to enforce cellular differentiation in epigenetically derailed tumours. Indeed, differentiation therapy is considered a promising approach for curing human cancers. However, the strategy has been applied only in limited cases. As such, identification of an innate tumour eviction mechanism in flies based on these principles may provide new ideas how such cancer treatments could be further improved in human patients (Jiang, 2018).

REGULATION

Transcriptional Regulation

ecd¹ is a temperature-sensitive mutation in the ecdysoneless gene (Garen, 1977) that impairs the biosynthesis of ecdysone at restrictive temperature (29°C). To test whether reduced ecdysone at the end of the L3 stage affects the expression of let-7 RNA, ecd¹ animals were transferred to 29°C at various times during the L3 stage to interfere with the generation of this ecdysone pulse. At the permissive temperature (20°C), ecd¹ animals pupariate around 230 h after egg laying. This time of PF was used as a reference for defining the duration of the L3 stage of ecd¹ animals. ecd¹ animals were synchronized at egg laying and reared at 20°C until they were transferred to 29°C in the early and mid L3 stage. These developmental stages are approximate, because ecd¹ animals grow more slowly and less synchronously than the wild type. The majority of ecd¹ animals that were upshifted in the early L3 stage remained as larvae (98%), and these ecd¹ retarded larvae were harvested at various times relative to PF of ecd¹ animals maintained at 20°C. ecd¹ animals upshifted in the mid-L3 stage produced a mixture of pupariating (40%) and nonpupariating (60%) individuals, and these were harvested separately. Wild-type animals reared at 20°C were upshifted as late L3 larvae for comparison. Using Northern analysis of total RNA, it was found that let-7 RNA is marginally expressed in ecd¹ animals; these animals fail to pupariate due to the absence of ecdysone. In contrast, let-7 is expressed at much higher levels in pupariating ecd¹ animals. This correlation between PF and let-7 expression in ecd¹ animals suggests that these two events are triggered by the same pulse of ecdysone. This could reflect that the induction of let-7 is mediated by the ecdysone signaling pathway. Alternatively, let-7 expression could be activated by another developmental signal associated with PF and/or progression through metamorphosis (Sempere, 2002).

In ecd¹ pupae maintained at 29°C for more than 6 h after PF, levels of let-7 RNA were reduced compared with the wild type, suggesting that a prolonged pulse of high ecdysone titer throughout pupal development may be required to sustain let-7 expression. A similar reduction of let-7 RNA levels was also observed in ecd¹ pupae transferred at different times after PF (Sempere, 2002).

It is inferred from the absence of let-7 RNA in nonpupariating ecd¹ mutants that ecdysone is required for let-7 expression. To test this supposition, organ cultures from third instar larvae were incubated with ecdysone. Late L3 larvae were dissected to expose the internal organs to the medium. After washes to remove ecdysone circulating in the hemolymph, organs were cultured in Schneider's medium with or without 5 microM ecdysone, for various lengths of time (0-20 h). Levels of let-7 RNA were examined by Northern analysis of total RNA recovered from these harvested organs. let-7 is already expressed at relatively low levels at the time of dissection (0 h), presumably due to the rising titer of ecdysone. let-7 expression remains at this low level in organs incubated without ecdysone during the first 12 h, and levels of let-7S and its precursor form (let-7L) decrease substantially at later times. In contrast, levels of let-7 RNAs increase after 12 h in organs incubated with ecdysone. This increased accumulation likely results from increased transcription since levels of let-7S and let-7L increase coordinately (Sempere, 2002).

The early gene, Broad-Complex (BR-C), is located at the top of the regulatory hierarchy in the ecdysone pathway and plays an essential role in regulating the expression of downstream targets. BR-C encodes four isoforms of a zinc finger transcription factor (Z1-Z4) that not only control directly the expression of late genes, but that are also required for full expression of other early genes. To test whether BR-C is involved in mediating the response of let-7 expression to ecdysone pulses, the levels of let-7 RNA were determined in animals homozygous for a BR-C null mutation. Homozygous npr⁶ animals are deficient in all four BR-C isoforms (Z1-Z4), rendering them unresponsive to the ecdysone pulse at the end of the L3, and hence they fail to pupariate. npr⁶ animals remain as larvae for about 5-10 days after the normal time of pupariation and then die. npr⁶/npr⁶ and npr⁶/+ animals were synchronized and harvested at various times relative to the time of PF (of the npr⁶/+ animals). let-7 RNA was detected at very low levels in npr⁶/npr⁶ animals, compared with npr⁶/+ siblings, indicating that BR-C is required in vivo to activate let-7 expression in response to ecdysone. Interestingly, there is a slight increase of let-7 expression in npr⁶/npr⁶ animals 48 h after 'PF', possibly corresponding to a rise of ecdysone titer. This suggests that other components of the ecdysone pathway could play a secondary role in let-7 expression, independent of BR-C (Sempere, 2002).

The above in vivo experiments suggest that ecdysone signaling is required for let-7 expression, and that there is at least one intermediate player between ecdysone and let-7 expression, the BR-C transcription factor. Next it was asked whether ecdysone directly activates let-7, that is, does ecdysone act on a tissue and trigger the expression of let-7 in that tissue, instead of initiating a signaling cascade resulting in the expression of let-7 in another tissue? To address this, the ability of ecdysone to initiate let-7 expression was examined in cultured S2 cells, which are known to be responsive to ecdysone. S2 cells were incubated in the presence of ecdysone at a final concentration of 5 microM for 6-62 h, and harvested every 6 h. let-7 RNA was first detected at approximately 18 h after incubation with ecdysone, increased in level from 24 to 42 h, and reaching a plateau at 42-54 h, and decreasing thereafter. let-7 RNA was not detected in untreated S2 cells. This result indicates that ecdysone induces let-7 expression directly in cells to which it is applied. The long delay between the ecdysone primary action and let-7 activation in S2 cells is consistent with the participation of intermediate regulators (Sempere, 2002).

To test whether ecdysone is required to sustain let-7 expression in S2 cells, a pulse-chase experiment was carried out. S2 cells were incubated for 24 h in the presence of 5 microM ecdysone, and then divided into two cultures. From one culture, the medium was removed and replaced by an ecdysone-free medium. The other culture was kept in 5 microM ecdysone for the remainder of the experiment. Northern blot analysis of total RNA showed a decrease in let-7 RNA levels in S2 cells after removal of ecdysone from the medium, compared with the cells maintained with ecdysone. The levels of let-7S and let-7L coordinately decreased. These results indicate that ecdysone is required for both the initiation and maintenance of let-7 transcription in S2 cells (Sempere, 2002).

BR-C is involved in relaying the ecdysone signal to trigger let-7 expression in vivo. To determine whether a similar BR-C-dependent pathway mediates let-7 activation in S2 cells, BR-C activity was inhibited by RNA interference (RNAi). S2 cells were transfected with a dsRNA corresponding to a conserved sequence in all four BR-C transcript isoforms. Control cells were transfected with a nonspecific dsRNA (from an unrelated C. elegans sequence). After 30 min of incubation with dsRNA, cells were treated with 5 microM ecdysone. In parallel, non-transfected cells were also treated with 5 microM ecdysone. S2 cells were harvested for 32, 40, and 48 h after the addition of ecdysone, and total RNA was analyzed by Northern blotting. The profile of let-7 expression was very similar in nontransfected cells and in cells transfected with nonspecific dsRNA. However, let-7 expression was dramatically reduced in cells transfected with dsRNA against BR-C. This indicates that BR-C is required for ecdysone-dependent let-7 expression in S2 cells. Since levels of let-7S and let-7L RNA coordinately decrease when BR-C activity is reduced by RNAi, BR-C likely affects transcription of let-7 in response to ecdysone. The residual let-7 expression in BR-C RNAi cells could be due to the ineffecient uptake of the dsRNA or the incomplete inhibition of BR-C activity. Alternatively, other components of the ecdysone pathway could contribute to let-7 expression, consistent with the residual let-7 expression observed in npr⁶ mutant animals (Sempere, 2002).

The lin-4 and let-7 small temporal RNAs play a central role in controlling the timing of C. elegans cell fate decisions. let-7 has been conserved through evolution, and its expression correlates with adult development in bilateral animals, including Drosophila. The best match for lin-4 in Drosophila, miR-125, is also expressed during pupal and adult stages of Drosophila development. This study asks whether the steroid hormone ecdysone induces let-7 or miR-125 expression at the onset of metamorphosis, attempting to link a known temporal regulator in Drosophila with the heterochronic pathway defined in C. elegans. let-7 and miR-125 are coordinately expressed in late larvae and prepupae, in synchrony with the high titer ecdysone pulses that initiate metamorphosis. Unexpectedly, however, their expression is neither dependent on the EcR ecdysone receptor nor inducible by ecdysone in cultured larval organs. Although let-7 and miR-125 can be induced by ecdysone in Kc tissue culture cells, their expression is significantly delayed relative to that seen in the animal. let-7 and miR-125 are encoded adjacent to one another in the genome, and their induction correlates with the transient appearance of an ~500-nt RNA transcribed from this region, providing a mechanism to explain their precise coordinate regulation. It is concluded that a common precursor RNA containing both let-7 and miR-125 is induced independently of ecdysone in Drosophila, raising the possibility of a temporal signal that is distinct from the well-characterized ecdysone-EcR pathway (Bashirullah, 2003).

Mapping of the miR-125 sequence to the Drosophila genome reveals that it is encoded adjacent to let-7, with the precursor sequences located ~300 bp from one another at position 36F in the polytene chromosomes. This close physical proximity combined with the precise temporal coordination of let-7 and miR-125 expression raises the interesting possibility that they might be expressed from a common precursor RNA. To test this possibility, a fragment encompassing this region was used as a probe for Northern blot hybridization using RNA samples from staged Drosophila as well as RNA samples from Kc cells treated with 20E. An ~500-nt RNA can be detected in synchrony with the appearance of let-7 and miR-125 RNA in late third instar larvae, early pupae, and Kc cells treated with 20E for 25 h. This expression, however, is transient, as the ~500-nt RNA is not detected at later stages during Drosophila development. The size and temporal expression pattern of this RNA is consistent with the proposal that it acts as an initial common precursor for the synthesis of both let-7 and miR-125. Moreover, the transient expression of this precursor indicates that the let-7/miR-125 gene cluster is only expressed during early Drosophila metamorphosis, while the more stable 21- to 22-nt products persist through adult stages (Bashirullah, 2003).

Thus, let-7 and miR-125 are induced in late third instar larvae and prepupae in a temporal pattern that mirrors that of a known ecdysone primary-response mRNA, E74A. In spite of this tight temporal correlation, however, the data argue against a role for ecdysone signaling in controlling the timing of let-7 and miR-125 induction in Drosophila. Little effect on their induction is seen when EcR function is blocked by RNAi, and no induction is seen of let-7 or miR-125 by the physiologically active form of the hormone, 20E, in cultured larval organs under conditions where E74A is abundantly expressed. let-7 and miR-125 induction by 20E in Kc cells is delayed by at least 10 h relative to that of the primary-response E74A mRNA. This delay suggests that these microRNAs are expressed as either a secondary-response to the hormone or as an indirect consequence of the 20E-induced differentiation program in these cells. Regardless, this pattern of expression is distinct from the precise coordinate expression of E74A, let-7, and miR-125 seen in vivo, indicating that a different mechanism is responsible for miRNA induction in the animal (Bashirullah, 2003).

One study [Sempere (2002)] of let-7 regulation in Drosophila arrives at a different conclusion from that reached in this study. Much of their data, however, is consistent with the results presented in this study. Sempere presents a time course of let-7 induction by 20E in S2 tissue culture cells that is very similar to that reported in this study in Kc cells, although Sempere does not use a primary-response gene as a temporal marker for direct induction by the ecdysone-receptor complex. As they point out, their results with the ecd1 ecdysone-deficient mutant and npr6 mutant are difficult to interpret because the lack of let-7 expression in these mutants could simply be attributed to their developmental arrest as third instar larvae and inability to initiate puparium formation. The most significant contradiction with their data is their results with cultured larval organs; these show increased levels of let-7 RNA in the presence of 20E. The Sempere experiment is, however, difficult to interpret. The 0-h time point has let-7 RNA present, as do the 8- and 12-h time points in the absence of 20E, indicating that the organs used for this study were taken at a time when let-7 was already expressed. It is possible that 20E could be stabilizing or maintaining let-7 expression in these organs. The authors provide no marker for a known primary-response to ecdysone, so the timing of their induction cannot be interpreted. The authors also take their earliest collection (8 h) at the time when primary-response genes are starting to be repressed, several hours after initial gene induction (Bashirullah, 2003).

Some miRNAs map within close proximity to one another, forming apparent gene clusters in C. elegans, Drosophila, and humans. A similar arrangement is found for let-7 and miR-125 in Drosophila, that map within ~300 bp of one another on the left arm of the second chromosome. In addition, a precursor RNA is detected that could encode both miRNAs, providing a means of explaining their tight temporal coordination. This observation is consistent with the recent identification in HeLa cells of relatively long primary transcripts (pri-miRNAs) that contain multiple ~70-nt stem-loop microRNA precursors, and indicates that similar precursors can be expressed in a developing organism. Although expression of some miRNAs may be regulated by multiple processing steps, the low abundance and transient appearance of the let-7/miR-125 relatively long primary transcripts (pri-miRNA) and shorter pre-miRNA forms indicate that transcription of the primary transcript could be largely responsible for the precise temporal appearance of the mature miRNAs (Bashirullah, 2003).

Interestingly, miR-125 provides the best match in the Drosophila genome to a second small heterochronic RNA in C. elegans, lin-4. The two mismatches between lin-4 and miR-125, however, are sufficient to render miR-125 undetectable on Northern blots of Drosophila RNA using lin-4 sequences as a probe. Moreover, it is likely that these sequence differences have an impact on the specificity of miR-125 interactions with possible target sequences in 3'UTRs. The functional significance of this sequence similarity thus remains to be determined (Bashirullah, 2003).

These studies leave unanswered the question of what induces let-7 and miR-125 expression at the onset of Drosophila metamorphosis. The timing of the upregulation of these miRNAs in staged animals argues that it is responding to a temporal signal that occurs in parallel with the well-characterized 20E/EcR/USP signaling pathway. Because of this tight correlation, and the widespread expression of let-7 RNA in Drosophila (Sempere, 2002), the most likely candidate regulator would be a hormone that acts through a receptor other than EcR. This conclusion is consistent with several recent studies that have provided indirect evidence of other hormone signaling pathways in Drosophila (Bashirullah, 2003).

Many genes are coordinately upregulated in mid-third instar larvae, when the Adh gene switches from its larval to adult promoter, and the larval salivary gland switches its genetic program from the ng/Pig genes to the Sgs glue genes. The temporal signal for this mid-third instar transition remains undefined, but could comprise a 20E/EcR-independent pathway. Similarly, the E74A and E75A early mRNAs are coordinately induced at some times during development when the ecdysteroid titer is thought to be low, arguing that another temporal signal may be responsible for this response. Finally, alpha-ecdysone, the precursor to 20E, is sufficient to drive furrow progression in Manduca eye primordia as well as stimulate optic lobe neuroblast proliferation, suggesting that this hormone, which is a poor activator of the EcR/USP complex, can act as temporal signal in this lepidopteran insect. Further studies should help to elucidate the roles of other ecdysteroids during insect development and provide a foundation for better understanding the temporal regulation of let-7 and miR-125 in Drosophila (Bashirullah, 2003).

It is interesting to note that the DAF-12 orphan nuclear receptor is required for the proper timing of let-7 induction in C. elegans. Genetic evidence suggests that DAF-12 is regulated by a steroid hormone under the control of the daf-9 cytochrome P450 gene. Perhaps more interesting from the perspective of this study, the Drosophila genome encodes an ortholog of DAF-12: DHR96. It is possible that functional studies of DHR96 in Drosophila will shed light on the regulation of the let-7/miR-125 gene cluster in this insect model system (Bashirullah, 2003).

lin-4 and let-7 are founding members of an extensive family of genes that produce small transcripts, termed microRNAs (miRNAs). In Caenorhabditis elegans, lin-4 and let-7 control the timing of postembryonic events by translational repression of target genes, permitting progression from early to late developmental programs. To identify Drosophila melanogaster miRNAs that could play similar roles in the control of developmental timing, the developmental expression profile of 24 Drosophila miRNAs were characterized; seven miRNAs are either upregulated or downregulated in conjunction with metamorphosis. The upregulation of three of these miRNAs (mir-100, mir-125, and let-7), and the downregulation of a fourth (mir-34) requires the hormone ecdysone (Ecd) and the activity of the Ecd-inducible gene Broad-Complex. Interestingly, mir-125 is a putative homolog of lin-4. mir-100, -125, and let-7 are clustered within an 800-bp region on chromosome 2L, suggesting that these three miRNAs may be coordinately regulated via common cis-acting elements during metamorphosis. In S2 cells, Ecd and the juvenile hormone analog methoprene exert opposite effects on the expression of these four miRNAs, indicating the participation of both these hormones in the temporal regulation of mir-34, -100, -125, and let-7 expression in vivo (Sempere, 2003).

The 24-h lag between the addition of Ecd to cultured S2 cells and the expression of mir-100 and mir-125 suggest that the initial Ecd signal activates mir-100 and mir-125 expression indirectly via intermediate regulators. One such intermediate could be BR-C, which is required for mir-100 and mir-125 expression in animals. To test whether BR-C activity is required for the Ecd-induced expression of mir-100 and mir-125 in S2 cells, BR-C activity was inhibited by RNAi using a 700-nucleotide dsRNA corresponding to a common region of all BR-C isoforms. S2 cells were incubated for 30 min with BR-C dsRNA or mock dsRNA, corresponding to unrelated C. elegans sequence. Then, the transfected and nontransfected cultures were treated with Ecd and harvested 32, 40, and 48 h later. The levels of mir-100 and mir-125 RNAs were considerably lower in BR-C RNAi cells as compared with nontransfected or mock RNAi cells. This result further supports the conclusion that BR-C is required to mediate the activation of mir-100 and mir-125 by an Ecd signal in vivo. This result also argues against the possibility that mir-100, mir-125, and let-7 RNAs were detected at very low levels in nonpupariating ecd¹ and npr⁶ mutants simply because these mutant animals were arrested at a stage before mir-100, mir-125, and let-7 are normally upregulated (Sempere, 2003).

It should be noted that BR-C RNAi does not result in complete loss of mir-100 and mir-125 expression, suggesting that RNAi treatment is not fully effective. Consistent with an incomplete knockdown of BR-C by RNAi, miR-34 levels are unaffected by BR-C RNAi in Ecd-treated cells. Based on results with npr⁶ mutant animals, one would have expected that mir-34 expression would be derepressed by BR-C RNAi in Ecd-treated cells. Since BR-C activity may not have been completely eliminated by RNAi, the requirement for BR-C activity in the repression of mir-34 could not be assesssed in S2 cells (Sempere, 2003).

Interestingly, the sequence of mir-125 is quite similar to the sequence of lin-4, suggesting that they may be homologs. The evolutionary conservation of microRNAs such as mir-125/lin-4, mir-100, and let-7 implies the conservation of multiple complementary target sequences for each of the miRNAs. In C. elegans, lin-4 translationally downregulates LIN-14 and LIN-28 protein expression by base-pairing to partially complementary sites in the 3' UTR of their mRNAs. Translational repression of lin-14 and lin-28 activities by lin-4 are required for the temporal transition from early to late developmental programs, eventually leading to the adult differentiation of hypodermis and vulva. Although lin-28 is a member of an evolutionary conserved gene family, no obvious Drosophila or human homologs of lin-14 have been found and the targets of mir-125 in flies and vertebrates are yet to be identified (Sempere, 2003).

The finding that the mir-100, -125, and let-7 gene cluster is coregulated in Drosophila suggests that these three genes may act in concert to control the translation of target genes involved in adult morphogenesis and differentiation. mir-100, -125, and let-7 are quite distinct in sequence, and so they probably base-pair to distinct cognate binding sites in their target mRNAs. These three miRNAs could have unique target mRNAs that they repress in parallel, and/or they could act together to repress particular targets that contain the appropriate combination of cognate sites (Sempere, 2003).

With the exception of C. elegans, the chromosomal clustering of mir-100, mir-125/lin-4, and let-7 appears to be widely conserved in animal phylogeny. In the mosquito Anopheles gambiae, mir-100, -125, and let-7 are clustered within 5000 bp and oriented in the same direction. In vertebrates, mir-100, -125, and let-7 are similarly clustered, although with somewhat greater spacing. Remarkably, mir-100 and let-7 are located together in two distinct chromosomal locations within 500 and 2000 bp in the Puffer fish Takifugu rubripes, and within 700 bp and 5000 bp in humans. The conservation of the clustered arrangement of mir-100, mir-125, and let-7 suggests that important aspects of their regulation may also be evolutionary conserved (Sempere, 2003).

Hormonal regulation of Drosophila microRNA let-7 and miR-125 that target innate immunity

The steroid 20-hydroxy-ecdysone (20-HE) and the sesquiterpenoid Juvenile Hormone (JH) coordinate insect life stage transitions. 20-HE exerts these effects by the sequential induction of response genes. In the nematode C. elegans hormones also play a role in such transitions, but notably, microRNA such as let-7 and lin-4 have likewise been found to help order developmental steps. Little is known about the corresponding function of homologous microRNA in Drosophila, and the way microRNA might be regulated by 20-HE in the fly is ambiguous. This study used Drosophila S2 cells to analyze the effects of 20-HE on Drosophila microRNA let-7 and miR-125, the homolog of lin-4. The induction by 20-HE of let-7 and miR-125 in S2 cells is inhibited by RNAi knockdown of the ecdysone receptor and, as previously shown, by knockdown of its cofactor broad-complex C. To help resolve the currently ambiguous role of 20-HE in the control of microRNA, it was shown that nanomolar concentrations of 20-HE primes cells to subsequently express microRNA when exposed to micromolar levels of 20-HE. The role microRNA plays in the established relationship between 20-HE and the induction of innate immunity was examined. The 3'UTR of the antimicrobial peptide diptericin was found to have a let-7 binding site and let-7 was found to represses translation from this site. It is concluded that 20-HE facilitates the initial expression of innate immunity while it simultaneously induces negative regulation via microRNA control of antimicrobial peptide translation (Garbuzov, 2010).

Hormonal activation of let-7-C microRNAs via EcR is required for adult Drosophila melanogaster morphology and function

Steroid hormones and their nuclear receptors drive developmental transitions in diverse organisms, including mammals. This study shows that the Drosophila steroid hormone 20-hydroxyecdysone (20E) and its nuclear receptor directly activate transcription of the evolutionarily conserved let-7-complex (let-7-C) locus, which encodes the co-transcribed microRNAs miR-100, let-7 and miR-125. These small RNAs post-transcriptionally regulate the expression of target genes, and are required for the remodeling of the Drosophila neuromusculature during the larval-to-adult transition. Deletion of three 20E responsive elements located in the let-7-C locus results in reduced levels of let-7-C microRNAs, leading to neuromuscular and behavioral defects in adults. Given the evolutionary conservation of let-7-C microRNA sequences and temporal expression profiles, these findings indicate that steroid hormone-coupled control of let-7-C microRNAs is part of an ancestral pathway controlling the transition from larval-to-reproductive animal forms (Chawla, 2012).

This study presents a series of data indicating that the let-7-C locus is a direct transcriptional target of EcR in vivo. The ~2.5 kb primary let-7-C transcript (pri-let-7-C) is detected during the mid-third larval transition, a developmental stage when pulses of 20E activate the transcription of inducible genes through the EcR/Usp nuclear hormone heterodimer. It was found that pri-let-7-C is rapidly induced in cultured Drosophila cells by 20E, and that the 20E responsiveness of the let-7-C locus requires EcR and is mediated by three 13-nucleotide EcR/Usp-binding sites. The deletion of these three EcREs eliminates let-7-C locus expression in larval and pupal tissues, including salivary glands and imaginal discs. EcRE deletion also causes a delay in let-7-C miRNA expression, as well as a reduction of let-7-C miRNA levels in adult flies, and is associated with known let-7-C mutant phenotypes. Taken together, these data strongly suggests that EcR binds to the endogenous let-7-C locus and activates its transcription in response to 20E, and that this transcriptional regulation is required for let-7-C miRNA function (Chawla, 2012).

This work describes a clear convergence in the molecular mechanisms that control developmental timing in Drosophila and C. elegans. Members of the let-7 and miR-125 families of miRNAs were originally identified in C. elegans as part of a pathway of heterochronic genes that promote stage-specific cell fate decisions. This study has directly linked the fly orthologs of these heterochronic genes to the 20E/EcR pathway, which triggers stage-specific transcriptional cascades that direct major developmental transitions, including molting and metamorphosis. One essential function of the 20E/EcR pathway is to activate the expression of let-7-C miRNAs at the end of larval development to promote the formation of adult morphologies required for adult function (Chawla, 2012).

Previous studies showing the slow onset of let-7 and miR-125 expression in tissue culture cells treated with Ecdysone had suggested that hormonal regulation of let-7-C expression may be indirect and not involve the 20E/EcR pathway. This study resolved this issue, showing that pri-let-7-C is detected within 30 minutes of 20E treatment in an EcR-dependent fashion and thus is a likely direct target of 20E/EcR. The delayed expression of processed miRNAs previously observed might be due to the effects of Ecdysone on factors involved in the processing of pri-let-7-C, a possibility that has not been investigated in this study. An unresolved question, however, is why EcR knockdown initiated approximately 18 hours before puparium formation had no effect, in a previous study, on the onset of let-7 and miR-125 expression. It is now thought that let-7 and miR-125 were processed from pri-let-7-C transcripts that were already present when EcR levels were depleted in that experiment. Experiments presented in this study, including analysis of EcR-deficient cell lines and EcR-dominant negative transgenes, indicate that EcR is required for let-7-C expression in vivo (Chawla, 2012).

Some nuclear hormone receptors both activate and repress the expression of direct targets. The C. elegans DAF-12 hormone receptor, for example, activates the miR-241 promoter in the presence of DA steroid hormone, whereas it represses the miR-241 promoter in the absence of DA. Evidence supporting an analogous repressor function has been reported for EcR. The current results suggest that EcR solely functions to activate let-7-C expression in salivary gland and imaginal discs, as removal of EcREs 1-3 results in complete elimination, rather than derepression, of reporter expression in those tissues. The situation is a little less clear in the late larval CNS, as removal of the EcREs reduces but does not eliminate lacZ expression there. This EcRE-deleted reporter is not precociously expressed, though, suggesting that the EcRE sites are not required for reporter repression. It is therefore suspected that let-7-C is co-activated in the CNS by EcR and at least one additional 20E-dependent factor. Indeed, the Sgs3 and Sgs4 salivary glue genes are controlled by complex ecdysone response units, which contain binding sites for other tissue-specific transcription factors such as the homeotic forkhead gene and potential binding sites for Broad-Complex gene (Chawla, 2012).

A growing number of papers have suggested the functional orthology between the 20E/EcR and DA/Daf-12 pathways, as they play similar roles in regulating developmental timing, physiology, reproductive maturation and longevity in flies and worms, respectively. These pathways not only play analogous functions but also share upstream components: the production of 20E as well as DA involves the TGFβ and IGF pathways. The work presented here suggests that they also share at least one common effector: let-7-C miRNAs. Steroid hormone regulation of let-7-C miRNAs may therefore represent an ancestral pathway that plays widespread roles both developmentally and post-developmentally (Chawla, 2012).

Regulation of Drosophila circadian rhythms by miRNA let-7 is mediated by a regulatory cycle

MicroRNA-mediated post-transcriptional regulations are increasingly recognized as important components of the circadian rhythm. This study identified microRNA let-7, part of the Drosophila let-7-Complex, as a regulator of circadian rhythms mediated by a circadian regulatory cycle. Overexpression of let-7 in clock neurons lengthens circadian period and its deletion attenuates the morning activity peak as well as molecular oscillation. Let-7 regulates the circadian rhythm via repression of Clockwork Orange (Cwo). Conversely, upregulated cwo in cwo-expressing cells can rescue the phenotype of let-7-Complex overexpression. Moreover, circadian Prothoracicotropic hormone (PTTH) and Clock-regulated 20-OH ecdysteroid signalling contribute to the circadian expression of let-7 through the 20-OH Ecdysteroid receptor. Thus, this study has found a regulatory cycle involving PTTH, a direct target of Clock, and PTTH-driven miRNA let-7 (Chen, 2014).

Lin-28 regulates oogenesis and muscle formation in Drosophila melanogaster

Understanding the control of stem cell (SC) differentiation is important to comprehend developmental processes as well as to develop clinical applications. Lin28 is a conserved molecule that is involved in SC maintenance and differentiation by regulating let-7 miRNA maturation. However, little is known about the in vivo function of Lin28. This study reports critical roles for lin-28 during oogenesis. let-7 maturation was shown to be increased in lin-28 null mutant fly ovaries. lin-28 null mutant female flies display reduced fecundity, due to defects in egg chamber formation. More specifically, in mutant ovaries, the egg chambers were shown to fuse during early oogenesis resulting in abnormal late egg chambers. This phenotype is the combined result of impaired germline SC differentiation and follicle SC differentiation. A model is suggested in which these multiple oogenesis defects result from a misregulation of the ecdysone signaling network, through the fine-tuning of Abrupt and Fasciclin2 expression. These results give a better understanding of the evolutionarily conserved role of lin-28 on GSC maintenance and differentiation (Stratoulias, 2014).

The Cold-Shock Domain (CSD) protein Lin28 was initially identified in Caenorhabditis elegans (C. elegans) as a component of the heterochronic pathway that regulates the timing of cell fate specification (Ambros, 1984). Subsequent discovery of gene expression regulation through small non-coding RNAs clarified the role of Lin28 in this pathway. The lin-28 mRNA is a conserved target of the let-7 micro-RNA (miRNA) family both in C. elegans and vertebrates. On the other hand, Lin28 inhibits let-7 processing. At the molecular level, Lin28 protein interacts with the let-7 precursor (pre-let-7), resulting in inhibition of let-7 maturation. The let-7 inhibition occurs through the physical interaction of the pre-let-7 loop and Lin28 protein, preventing further processing of pre-let-7 towards the mature form of let-7. Together, these interactions create a feedback loop between Lin28 and let-7, leading to a strict regulation of let-7 maturation (Stratoulias, 2014 and references therein).

Lin28 raised further interest when it was used, along with Nanog, to replace the factors c-Myc and Klf4 in somatic cell reprogramming. These experiments, together with data from human embryonic stem cells, underscored the important role of lin-28 in pluripotency regulation and maintenance. Besides acting as a negative regulator of let-7 maturation, Lin28 has also been shown to have a direct effect on translation through the recruitment of the RNA Helicase A. This mode of function, independent of let-7 maturation, has been demonstrated in the case of Insulin-like Growth Factor 2 during mouse myogenesis. Lin28 binding on IGF-2 mRNA increases its translation efficiency and therefore facilitates skeletal myogenesis in mice (Stratoulias, 2014 and references therein).

The Lin28 protein is composed of four domains: a positively charged linker that binds two Cys-Cys-His-Cys (CCHC)-type zinc-binding motifs to the CSD. In mammalian genomes, two paralogs of lin-28 are found, Lin28A and Lin28B. While Lin28B represses let-7 processing in the nucleus to prevent the formation of the precursor form from the primary let-7, Lin28A also blocks cytoplasmic processing of let-7 (Piskounova, 2011). It has recently been shown in mouse that deletion of the Lin28 linker domain alters the protein’s three-dimensional structure and is sufficient to disrupt sequestration of the precursor form of let-7 (pre-let-7) (Stratoulias, 2014).

The miRNA let-7 family is conserved across diverse animals, functioning to control late temporal transitions during development. During the last decade, the involvement of let-7 in regulating cell differentiation has been analyzed in various contexts, including neural cell specification, stem cell maintenance and hematopoietic progenitor differentiation. While eight different let-7 miRNA genes are annotated in the human genome, only one is found in Drosophila melanogaster. Like in C. elegans, in Drosophila the loss of let-7 expression leads to the modification of temporal regulation of the metamorphosis process. During fly metamorphosis, the expression of let-7 complex (let-7C), a polycistronic locus encoding the let-7, miR-100 and miR-125 miRNAs, is under direct control by the steroid hormone ecdysone. Ecdysone is the central regulator of insect developmental transitions. Therefore, let-7 has been proposed to be part of a conserved, ecdysone regulated pathway that controls the timing of the larva to adult transition (Stratoulias, 2014).

In addition to affecting the metamorphosis clock, Sokol and colleagues have shown that the let-7 deletion also affects the neuromuscular remodeling that takes place during the larva to adult transition. During neuromuscular remodeling, and under normal conditions, the dorsal internal oblique muscles (DIOMs) disappear 12 hours after emergence of the adult fly from the pupa. However, the adult let-7 mutants retain the DIOMs through adulthood. Deletion of the let-7 gene is sufficient to induce this phenotype, while deletion of either miR-100 or miR-125 genes is not enough to recapitulate the DIOM phenotype. Furthermore, let-7 has been shown to govern the maturation of neuromuscular junction of adult abdominal muscles, through regulation of Abrupt expression (Stratoulias, 2014 and references therein).

While previous studies have demonstrated that the let-7 target Abrupt and ecdysone signaling are required for oogenesis in fruit fly ovaries, and that the let-7 miRNA family is abundantly expressed both in newborn mouse ovaries and in fly ovaries, no study has been conducted on the role of Lin-28/let-7 network in Drosophila ovaries. Therefore, a study was undertaken of the effects of lin-28 during Drosophila melanogaster development from the egg to the adult, and more particularly during oogenesis (Stratoulias, 2014).

A lin-28 mutant was generated, and the consequent increase of let-7 maturation was validated. lin-28 knockout resulted in reduced muscular performance and defects in DIOM morphogenesis. These results were in line with the let-7 knock out muscular phenotype described earlier. Moreover, this study identified multiple defects during oogenesis due to abnormal follicle and germline stem cell (FSCs and GSCs respectively) differentiation. A link is proposed between ovarian defects and ectopic expression of Fasciclin2 (Fas2), a known downstream target of the Ecdysone pathway, and a predicted let-7 target (Stratoulias, 2014).

Because of their role during stem cell differentiation, members of the let-7 miRNA family have been extensively studied. However, the role of lin-28 is still poorly documented. Deletion of let-7 in Drosophila impairs the musculature remodeling during the larva to adult metamorphosis. For instance the DIOMs, muscles which are required for eclosion and which are lost within 12 hours after eclosion, they are maintained during adulthood upon let-7 deletion. By generating the first lin-28 deletion in flies, this study has successfully confirmed the involvement of Lin-28/let-7 regulatory network in DIOM remodeling. This study has shown that deletion of lin-28 leads to over maturation of let-7, which negatively affects, and sometimes prevents DIOM formation. This drastic phenotype leads to a suboptimal muscular phenotype. However, due to a variable penetrance of the lin-28 deletion phenotype, a proportion of the flies could eclose and live as fertile animals (Stratoulias, 2014).

In addition, a link was discovered between Lin-28 function and oogenesis. The data indicates a role of let-7 during GSC differentiation and egg chamber formation. Because of the importance of these processes, let-7 maturation has to be strictly regulated by Lin-28 activity. It is suggested that a potential network involving Lin-28/let-7/Ecdysone signaling/Abrupt/Fas2 is needed during GSC differentiation and BC migration. The role of Abrupt in downregulating the steroid hormone Ecdysone has previously been demonstrated. Indeed, the loss of Taiman, a target of the transcription factor Abrupt and co-activator of Ecdysone receptor, leads to an increase of undifferentiated GSCs in the germarium due to disruption of Ecdysone signaling. Therefore, by regulating the expression pattern of Abrupt, Lin28/let-7 may adjust the domain of Ecdysone activity, providing a control over the GSCs differentiation and egg chamber maturation during the oogenesis. Indeed, it has been shown that the Ecdysone titre rises during oogenesis at stage 9. While the precise Ecdysone expression pattern is not known, it is suggested that the uniform EcR expression pattern in follicle cells in lin-28 mutants may break the Ecdysone signaling asymmetry needed during proper oogenesis (Stratoulias, 2014).

Furthermore, a previous study demonstrated the activation of let-7 expression via Ecdysone activity. This study showed that lin-28 deletion, resulted in the alleviation of Lin28's inhibitory role on let-7 maturation. This led to loss of Abrupt, which in turn inhibited Ecdysone activity and maintained Fas2 expression, resulting in BC migration impairment. To test whether the increase of Ecdysone signaling amplifies let-7 expression through a positive feedback loop, a system was generated in which there is no control of either let-7 expression nor of Ecdysone activity. This situation leads to an early cyst fusion, a loss of proper GSC differentiation and a mitotic defect, as was observed in the homozygous lin-28^dF30 ovaries. The accumulation of these defects may be enough to trigger apoptosis at mid-oogenesis, a well-known checkpoint previously described (Stratoulias, 2014).

Interestingly, the variable penetrance of the phenotype allows proper oogenesis and appearance of subfertile adult flies. This suggests a robust molecular network where feedback loops can rescue the system if one component disturbs the balance (Stratoulias, 2014).

By combining these results with previously published studies, a conserved link is suggested between hormonal signaling and germline stem cell differentiation, involving the let-7 miRNA family. This suggestion is reinforced in the last couple of years by the discovery of dormant ovarian follicles and mitotically active germ cells in adult mammalian ovaries, which are responsive to gonadotropin hormone. Moreover, it has been demonstrated that Lin-28 is involved in germline stem cell regulation in human ovary and in the ovarian surface epithelium of severe ovarian infertility patients axonal projection is critical for assembly of a functional sensory circuit (Stratoulias, 2014).

RNA maturation

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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).

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

Purification of RISC complex of Drosophila

RNA interference (RNAi) regulates gene expression by the cleavage of messenger RNA, by mRNA degradation and by preventing protein synthesis. These effects are mediated by a ribonucleoprotein complex known as RISC 1(RNA-induced silencing complex 1). Four Drosophila components (short interfering RNAs, Argonaute 2, VIG and FXR3) of a RISC enzyme have been identified that degrade specific mRNAs in response to a double-stranded-RNA trigger. Tudor-SN (tudor staphylococcal nuclease) -- a protein containing five staphylococcal/micrococcal nuclease domains and a tudor domain -- is a component of the RISC enzyme in Caenorhabditis elegans, Drosophila and mammals. Although Tudor-SN contains non-canonical active-site sequences, purified Tudor-SN exhibits nuclease activity similar to that of other staphylococcal nucleases. Notably, both purified Tudor-SN and RISC are inhibited by a specific competitive inhibitor of micrococcal nuclease. Tudor-SN is the first RISC subunit to be identified that contains a recognizable nuclease domain, and could therefore contribute to the RNA degradation observed in RNAi (Caudy, 2003).

Exposure of cells to double-stranded RNA (dsRNA) can elicit various types of sequence-specific gene silencing. A signature of these silencing events is the involvement of small RNAs of approximately 22-25 nucleotides (nt) that guide the selection of silencing targets. These short interfering RNAs (siRNAs) or microRNAs (miRNAs) are generated by the processing of silencing triggers by an RNaseIII family nuclease, Dicer. Small RNAs join multicomponent ribonucleoprotein (RNP) complexes, known generically as RISCs, which enforce silencing (Caudy, 2003 and references therein).

Both to address the nature of the RNAi effector machinery in detail, and to examine the relationship between the different effector mechanisms of RNAi, a RISC complex has been biochemically purified from Drosophila that degrades its mRNA target, and its protein and RNA components have been identified. In multiple, independent purifications of RISC, along with previously characterized proteins, a potentially novel component corresponding to a Drosophila candidate gene, CG7008, has been identified. This evolutionarily conserved 103 kDa protein contains five repeats of a staphylococcal/micrococcal nuclease domain. Four of these repeats are intact, whereas the fifth repeat is fused at its amino terminus to a tudor domain, which has been implicated in the binding of modified amino acids. On the basis of this characteristic domain structure, the protein was named Tudor-SN, for tudor staphylococcal nuclease. Through each purification step, Tudor-SN co-fractionates with known RISC components (Caudy, 2003).

Orthologs of Tudor-SN are found in plants (Arabidopsis9), C. elegans, mammals and Schizosaccharomyces pombe, but not in Saccharomyces cerevisiae. To investigate whether a role for Tudor-SN orthologs in RNAi is evolutionarily conserved, biochemical fractionation of extracts were carried out from C. elegans and mammalian cells (Caudy, 2003).

Cytosolic extracts were prepared from synchronized cultures of wild-type C. elegans. As in Drosophila, a large fraction of the miRNA population can be extracted from the ribosomes. Size fractionation of extracts derived from adult animals has revealed that miRNAs eluted from the column in two peaks, representing 500 kDa and 250 kDa complexes, similar to what has been observed previously in extracts from Drosophila S2 cells. Three different miRNAs -- lin-4, let-7 and mir-52 -- behaved identically in this assay. By contrast, size fractionation of C. elegans egg extract, and examination of complexes containing mir-40 and mir-52, suggests the presence of only the 500 kDa complex. Thus, it seems that miRNAs in C. elegans can inhabit multiple, distinct RNP complexes, and that the partitioning of miRNAs between these complexes may depend on both the identity of the miRNA and the developmental stage of the organism (Caudy, 2003).

RISC complexes in C. elegans have not previously been characterized. Therefore, whether Drosophila RISC components co-fractionate with miRNAs in C. elegans extracts was probed. Antibodies were raised to F56D12.5 (VIG-1), the worm homolog of Drosophila VIG, and F10G7.2 (TSN-1), the worm ortholog of Tudor-SN. Both VIG-1 and TSN-1 were enriched in the fractions that contained 250 kDa miRNA. By contrast, VIG-1 or TSN-1 did not appear in fractions containing 500 kDa miRNA complex (Caudy, 2003).

To test whether putative RISC components are present in the same complex, antibodies were used to immunoprecipitate individual components. In Drosophila, antibodies to either FXR or VIG co-immunoprecipitated Argonaute 2 (Ago-2), as was predicted by findings using epitope-tagged versions of these proteins. Similar amounts of Ago-2 were also co-immunoprecipitated using antibodies directed against Tudor-SN. Furthermore, antibodies directed against FXR and VIG co-immunoprecipitate Tudor-SN, and VIG and Tudor-SN antisera conversely recover FXR, indicating that all four proteins are present in a single complex. In C. elegans, VIG-1-specific antibodies can co-immunoprecipitate TSN-1, indicating a similar association of the C. elegans orthologs of Drosophila RISC proteins (Caudy, 2003).

In naive mammalian cells, it was difficult to detect interactions between orthologs of Drosophila RISC components. However, the formation of a complex containing these proteins was induced if an RNAi response was first triggered by transfection with siRNAs. Specifically, interactions were shown between an Argonaute family protein, AGO2 (human GERp95/EIF2C2/AGO2), the fragile X mental retardation protein (FMRP) and the mammalian Tudor-SN homolog, p100. Notably, complex formation occurred without changes in the expression levels of individual RISC components, indicating that association of pre-existing proteins is nucleated when a siRNA becomes available in the cell (Caudy, 2003).

RISC is an RNP complex that can contain either siRNAs or miRNAs. Consistent with their roles as components of RISC, both miRNAs and siRNAs can be co-immunoprecipitated from Drosophila cells using antisera that recognize FXR, VIG and Tudor-SN. Similarly, in C. elegans, TSN-1 and VIG-1 immunoprecipitates contain let-7 RNA. In addition, the mammalian let-7 miRNA was found in immunoprecipitates of p100, and in parallel the previously demonstrated association between miRNAs and AGO2 was confirmed. miRNAs were also detected in association with members of the fragile X family in mammalian cells, including FMRP, FXR1 and FXR2. Considered together, these results point to a common architecture for RISC in animals as a complex that contains a small RNA (miRNA or siRNA) and protein components that include an Argonaute family member, VIG, Tudor-SN and, at least in Drosophila and mammals, a fragile X family member (Caudy, 2003).

The mammalian homolog of Tudor-SN, known as p100, has been implicated as a co-activator for an Epstein-Barr virus transcription factor, EBNA-2. An exclusively nuclear localization of Tudor-SN would be inconsistent with a role in RNAi, since many studies of RNAi in C. elegans, Drosophila, Neurospora and mammals have shown that post-transcriptional gene silencing by RNAi occurs largely in the cytoplasm. In both Drosophila and mammalian cells, Tudor-SN/p100 is present predominantly in cytoplasmic fractions. Examination of Tudor-SN immunoreactivity also shows a predominantly cytoplasmic localization. In C. elegans, TSN-1 is found in significant amounts both in the nucleus and in the cytosol (Caudy, 2003).

RISC is a nuclease that catalyses endonucleolytic cleavage of substrates, as directed by the associated siRNA. In many cases, siRNAs also trigger mRNA degradation, and the results of biochemical purification are consistent with an association between RISC and a nuclease that catalyses complete destruction of targeted mRNAs. To assess the possibility that Tudor-SN might contribute to catalysis by RISC, its intrinsic nuclease activity was examined. All five staphylococcal nuclease domains of Tudor-SN contain mutations that alter the canonical active site, as derived from comparisons of bacterial family members and from structural data. A large panel of mutations has been made in staphyloccocal nuclease, some of which are similar to those that alter Tudor-SN domains away from the active-site consensus. Generally, these mutations lower the reaction rate but do not abolish catalysis (Caudy, 2003).

Recombinant Drosophila Tudor-SN was expressed and purified from Escherichia coli. Nuclease activity precisely co-fractionates with the recombinant protein, and monospecific carboxy-terminal Tudor-SN anti-peptide antibodies selectively deplete activity from purified Tudor-SN preparations. In these depletion experiments, nuclease activity was recovered on antibody-sepharose complexes. Micrococcal nucleases show broad substrate specificity, cutting both RNA and DNA. Similarly, Tudor-SN can cleave both RNA and DNA substrates (Caudy, 2003).

3',5'-Deoxythymidine bisphosphate (pdTp), known to be a specific competitive inhibitor of staphylococcal nucleases, inhibits Tudor-SN at 100 µM concentrations, whereas dTp (3'-deoxythymidine monophosphate) does not inhibit Tudor-SN activity. Importantly, RISC activity is also inhibited by pdTp. These data are consistent with the possibility that Tudor-SN contributes at least some of the nuclease activity observed in RNAi effector complexes, but are also consistent with other interpretations (Caudy, 2003).

To examine the role of Tudor-SN in dsRNA-mediated silencing in vivo, use was made of a reporter system in C. elegans. A lacZ-lin-41 fusion transgene expresses LacZ in the seam cells under the translational control of let-7. In wild-type animals, LacZ staining in the seam cells can be observed from the L1 stage through to L4. The staining is absent in adult animals as a consequence of let-7-mediated repression. As expected, suppression of alg-1 and dcr-1 by RNAi resulted in persistence of LacZ staining in adults. Next, RNAi was used to analyse the involvement of VIG-1 and TSN-1 in let-7 function. RNAi against either VIG-1 or TSN-1 results in persistent expression of the LacZ reporter in the seam cells of adult animals. This shows that VIG-1 and TSN-1 are required for proper function of the let-7 miRNA in vivo. By contrast, after RNAi against VIG-1, TSN-1 and DCR-1, no effect on RNAi efficiency was seed. The effects observed after RNAi against VIG-1, TSN-1 and DCR-1 probably reflect a partial reduction of function, since other phenotypes associated with let-7 loss of function (vulva and alae defects) are not observed (Caudy, 2003).

These data strongly indicate that Tudor-SN is a bona fide RISC component. This is reflected by the co-purification of Tudor-SN and RISC in Drosophila, C. elegans and mammalian cells. However, it remains open to question whether Tudor-SN is a catalytic engine of RNAi. Despite the aforementioned data, there are potential inconsistencies. (1) Purified, recombinant Tudor-SN is non-sequence-specific, in contrast to RISC, which shows a high degree of selectivity for its mRNA targets. (2)Tudor-SN will cleave both RNA and DNA, whereas no DNase activity is detected in RISC. (3) Several investigators have detected specific cleavage of mRNAs within the siRNA-mRNA hybrid, and this is difficult to rationalize with the known activities of Tudor-SN and related enzymes. It is certainly consistent with the biochemical data to suppose that RISC contains multiple nucleases, only one of which (the putative Slicer) can catalyse site-specific mRNA cleavage. In this scenario, Tudor-SN might act to degrade the remainder of the mRNA. In accord with this idea, targeting of an mRNA by a single siRNA often results in complete degradation of the mRNA. Alternatively, it is possible that Tudor-SN does not have a catalytic role in the RISC complex. Indeed, pdTp is a competitive inhibitor that engages the potential nucleic-acid-binding domains of Tudor-SN. Thus, inhibition of RISC by pdTp may reflect a block in the ability of Tudor-SN to engage RNAs, possibly including the mRNA target, in the context of the RISC complex. Answers to these questions will come only from understanding RISC in sufficient detail to allow reconstitution of its native activity from purified components such that the individual contributions of each to the varied roles of the RNAi effector machinery can be studied in detail (Caudy, 2003).

Temporal regulation of metamorphic processes in Drosophila by the let-7 and miR-125 heterochronic microRNAs

The let-7 and lin-4 microRNAs belong to a class of temporally expressed, noncoding regulatory RNAs that function as heterochronic switch genes in the nematode C. elegans. Heterochronic genes control the relative timing of events during development and are considered a major force in the rapid evolution of new morphologies. let-7 is highly conserved and in Drosophila is temporally coregulated with the lin-4 homolog, miR-125. Little is known, however, about their requirement outside the nematode or whether they universally control the timing of developmental processes. A Drosophila mutant that lacks let-7 and miR-125 activities has been generated; these mutants have a pleiotropic phenotype arising during metamorphosis. Loss of let-7 and miR-125 results in temporal delays in two distinct metamorphic processes: the terminal cell-cycle exit in the wing and maturation of neuromuscular junctions (NMJs) at adult abdominal muscles. The abrupt (ab) gene, encoding a nuclear protein, was identifed as a bona fide let-7 target, and evidence is provided that let-7 governs the maturation rate of abdominal NMJs during metamorphosis by regulating ab expression. It is concluded that Drosophila Iet-7 and miR-125 mutants exhibit temporal misregulation of specific metamorphic processes. As in C. elegans, Drosophila let-7 is both necessary and sufficient for the appropriate timing of a specific cell-cycle exit, indicating that its function as a heterochronic microRNA is conserved. The ab gene is a target of let-7, and its repression in muscle is essential for the timing of NMJ maturation during metamorphosis. These results suggest that let-7 and miR-125 serve as conserved regulators of events necessary for the transition from juvenile to adult life stages (Caygill, 2008).

The results indicate that in Drosophila, loss of the let-7 and miR-125 microRNAs leads to numerous defects that alter the morphology and behavior of adult flies. The expression of these microRNAs is restricted to the metamorphic stage of development, and both loss and premature expression of let-7 and miR-125 are detrimental to animal survival, indicating that temporal restriction of their expression is essential. Many defects in the mutants affect sensory and motor behaviors; the results indicate that neuromuscular connectivity in abdominal muscles is severely hampered and that there are direct consequences on the rate of adult eclosion. Both the NMJ and eclosion defects result from deregulated expression of a single let-7 target in abdominal muscles. Control of NMJ growth represents a new regulatory role for microRNAs in general, and given the extensive neurological problems of let-7, miR-125 mutants, it is possible that synapse development is broadly regulated by these particular micoRNAs (Caygill, 2008).

The data clearly implicate regulation of ab by let-7 as a critical factor in the rate of NMJ development during Drosophila metamorphosis. The aberrant persistence of Ab in muscle nuclei in the mutant significantly slows NMJ development, perhaps by interfering with synapse-strengthening signals exchanged between muscle and motoneurons. Because Ab expression is widespread during Drosophila development, the need for its downregulation late in development is in striking contrast to the requirement for Ab at earlier stages: in muscle for neuromuscular targeting and for epidermal attachment of specific muscle groups and in a class of embryonic and larval sensory neurons to limit dendritic branching. Based on its dose sensitivity, Ab has been proposed to function in a concentration-dependent manner. By examining the expression of Ab protein directly, it was found that Ab is downregulated to nearly undetectable levels in most abdominal-muscle nuclei by the pharate adult stage. Its decline in the myoblasts may begin as early as 26 hr APF, a time when let-7 expression is rising. However, the experiments indicate that deregulation of Ab cannot account for all of the mutant defects, indicating that other targets of let-7 and/or miR-125 must function as effectors for these processes. To date, Ab is the only target of Drosophila let-7 to be verified in vivo, and no miR-125 targets have been authenticated (Caygill, 2008).

In addition to its role in NMJ maturation, the results implicate let-7 in a wing defect that causes cells to continue to divide after wild-type wing cells have exited the cell cycle. This phenotype is strikingly similar to the reiterated divisions of hypodermal blast cells in C. elegans let-7 mutants. In both cases, the extra divisions do not continue indefinitely, nor do they occur in all tissues. Little is known about the mechanism of cell-cycle regulation by let-7 in either organism. In C. elegans, persistent expression of lin-41, encoding a RBCC (ring B-box coiled-coil) protein, in let-7 mutants accounts for much of the mutant phenotype. Drosophila homologs of lin-41 include dappled (shown to be a let-7 target in cell-culture assays: O'Farrell, 2008) and brat (brain tumor), a translational inhibitor. brat encodes a potent tumor suppressor whose absence causes metastatic brain tumors and that is predicted by one computational program to contain let-7 binding sites. Interestingly, overexpression of brat suppresses wing growth. Thus, a plausible hypothesis for the wing defect in let-7, miR-125 mutants is that loss of let-7 simultaneously deregulates a cell-cycle regulator(s) and brat and that the former drives additional cell divisions in the wing while the latter suppresses growth (Caygill, 2008).

The expression of many genes required during larval stages is downregulated during metamorphosis, in part because their presence during pupal development hinders the ordered progression of events such as the primary and secondary responses to ecdysone, the hormone that controls insect metamorphosis. Thus, a plausible role for let-7 and miR-125 is to rid cells of unnecessary larval mRNAs quickly at the transition to pupal development; such a role might be similar to the clearing of maternal mRNAs at the maternal-zygotic transition as recently demonstrated in zebrafish and in Drosophila. Heterochrony facilitates rapid evolution of new morphologies through changes in the timing of developmental processes. The pleiotropic phenotype associated with let-7, mir-125 mutants suggests that each microRNA contributes to multiple processes during metamorphosis, and the identification of additional targets of both is therefore an important future quest that should clarify their contribution to developmental timing and morphological evolution (Caygill, 2008).

In conclusion this study has generated a mutant of Drosophila Iet-7 and miR-125 that exhibits temporal misregulation of specific metamorphic processes. Drosophila let-7 is both necessary and sufficient for the appropriate timing of a specific cell-cycle exit and thus functions as a conserved heterochronic microRNA. The abrupt gene is a target of let-7, and its repression in muscle is essential for the timing of NMJ maturation during metamorphosis. let-7 and miR-125 could serve as conserved regulators of processes necessary for the transition from juvenile to adult life stages (Caygill, 2008).

Regulation of the Drosophila lin-41 homologue dappled by let-7 reveals conservation of a regulatory mechanism within the LIN-41 subclade

Drosophila Dappled (DPLD) is a member of the RBCC/TRIM superfamily, a protein family involved in numerous diverse processes such as developmental timing and asymmetric cell divisions. DPLD belongs to the LIN-41 subclade, several members of which are micro RNA (miRNA) regulated. The LIN-41 subclade members was examined and their relation to other RBCC/TRIMs and dpld paralogs was examined, and a new Drosophila muscle specific RBCC/TRIM, Another B-Box Affiliate (ABBA) was identifed. In silico predictions of candidate miRNA regulators of dpld identified let-7 as the strongest candidate. Overexpression of dpld led to abnormal eye development, indicating that strict regulation of dpld mRNA levels is crucial for normal eye development. This phenotype was sensitive to let-7 dosage, suggesting let-7 regulation of dpld in the eye disc. A cell-based assay verified let-7 miRNA down-regulation of dpld expression by means of its 3'-untranslated region. Thus, dpld seems also to be miRNA regulated, suggesting that miRNAs represent an ancient mechanism of LIN-41 regulation (O'Farrell, 2008).

The RING, B-box and coiled-coil/Tripartite Motif (RBCC/TRIM) proteins form a large protein superfamily with members spanning invertebrate to mammals. They have been shown to participate in numerous distinct processes, including roles in development, both normal and tumorous, asymmetric cell divisions and viral response, to name but a few. This protein superfamily is characterized by the presence of multiple protein-protein interaction domains, from which it has derived its name, that is, RING, B-box, and coiled-coil domains. In addition to these domains, members of this family often have distinct C-terminal domain(s), such as a series of NHL domains (first identified in NCL-1, HT2A and LIN-41 protein) (O'Farrell, 2008 and references therein).

The dappled (dpld) gene, encodes a RBCC/TRIM protein highly homologous to C elegans lin-41. Dappled protein (DPLD), however, lacks the N-terminal most RING domain found in many of the other members of this superfamily. Despite this, both DPLD and its paralog Brain tumour (BRAT) are considered to be RBCC/TRIM superfamily members due to the high sequence conservation of the remaining protein domains and the conservation of their order/positioning within the protein. dpld was first identified in a tumorigenesis screen. The name itself relates to the tumorous phenotype, causing large melanotic spots within the animal, giving a dappled (an equestrian term) or spotted appearance. dpld mutations resulting from the screen were classified as likely causing a defect in cell growth or proliferation control. Little else is known about either the function or regulation of dpld (O'Farrell, 2008).

BRAT, on the other hand, has been the subject of intense study in recent times and as the name suggests, gives rise to brain tumours when mutated. Tumours arising in brat animals are highly proliferative, invasive, transplantable and lethal to the animal. Of interest, tumorigenic alleles of brat disrupt the NHL repeat region, implicating it as having a direct role in tumor suppression. Furthermore, the BRAT NHL domain has been shown to mediate protein-protein interactions of various kinds, including direct binding to and promoting asymmetric distribution of the cell fate determinant Prospero. Thus, both dpld and brat can cause tumors when mutated, suggesting at least some functional overlap (O'Farrell, 2008 and references therein).

The functionally characterized DPLD-like proteins to date, in addition to BRAT, include Drosophila MEI-P26, known to be involved in meiotic cell cycle control, and DPLD's orthologous protein from C. elegans, LIN-41. lin-41 is a heterochronic gene, regulating the timing of specific developmental events, demonstrated to govern the number of cell divisions during C. elegans larval hypodermal development. Mutants lack a terminal cell division and instead precociously differentiate to a terminal cell fate. The molecular mechanism by which this occurs is unknown. However, certain aspects of lin-41 regulation have been elucidated. Specifically, C. elegans lin-41 mRNA was one of the first transcripts experimentally demonstrated to be under micro RNA (miRNA) translational regulation, imposed in this case by the let-7 miRNA (O'Farrell, 2008 and references therein).

miRNAs are short oligomers of RNA, 19-24 nucleotides long, encoded in the genome that are capable of recognizing and annealing to complementary sites within mRNA sequences. These are primarily located in the 3-untranslated region (3-UTR) of the target mRNAs. Formation of the double-stranded miRNA/mRNA duplex subsequently causes either a translational repression of the mRNA, or a targeted degradation of the RNA duplex. Regardless, the overall effect is a decrease in the level of translated protein from the targeted mRNA transcript. Recent work has shown that the avian and mammalian lin-41 orthologs are also likely candidates for this mode of regulation by means of conserved miRNA let-7 and miR-125 target sites, indicating possible conservation of this mode of regulation within the subclade (O'Farrell, 2008 and references therein).

Based on these observations, this study set out to ascertain the phylogenetic positioning of DPLD within the LIN-41 subclade and RBCC/TRIM superfamily and to consider the possibility of its translational regulation by miRNA. Indeed, it could be demonstrated that dpld is targeted and repressed by let-7 in Drosophila, an miRNA that also specifically targets other members of the LIN-41 subclade. This finding suggests wide conservation of this regulatory mechanism between lin-41 family members. Furthermore, through phylogenetic reconstructions, a novel Drosophila RBCC/TRIM family member was identified orthologous to NHL-1 and provide a brief description (O'Farrell, 2008).

To date, there are a total of nine annotated B-box proteins in the Drosophila melanogaster predicted proteome. Four of these additionally have NHL repeats in the C-terminal end. Two of the NHL containing proteins, Meiotic protein P26, MEI-P26, and CG15105 (which this study has named Another B-Box Affiliate, ABBA) are typical members of the RBCC/TRIM protein superfamily having RING, B-box, and coiled-coil domains. The others, Dappled, DPLD, and BRAT, while lacking the RING domain are also considered RBCC/TRIM family members due to the high sequence conservation of the remaining protein domains and the conservation of their positioning within the protein. Moreover, DPLD and LIN-41 belong to the same InParanoid metazoan ortholog pairs cluster. To provide an up-to-date understanding of the evolutionary relationships of the RBCC/TRIM proteins containing NHL domains in D. melanogaster, the RBCC/TRIM and B-box protein sequences were analyzed from several fully sequenced genomes (O'Farrell, 2008).

Previously, all Drosophila RBCC/TRIM NHL proteins have been likened to the mammalian RBCC/TRIM proteins TRIM2 and 3. However, recent release of vertebrate sequence data brings new mammalian and vertebrate proteins to the LIN-41 subclade, showing greater sequence similarity between proteins within this subclade than to any other vertebrate RBCC/TRIM proteins. DPLD associates with the LIN-41 subclade with a very high bootstrap value of 999/1000 for this node, indicating a very high degree of reliability (O'Farrell, 2008).

The majority of the conservation between fly, vertebrate, and worm LIN-41 proteins resides in the B-box domains and the C-terminal NHL-propeller region. Alignment of seven sequences, spanning from Homo sapiens to D. melanogaster, for these five NHL repeats reveals a high level of conservation, with 57% overall similarity and 41% identity. There is partial, weak conservation of a potential sixth C-terminal NHL domain in DPLD, which is considered a half-domain. Poor conservation of the sixth NHL domain seems common to those RBCC/TRIM proteins represented with the sixth NHL domain of C. elegans LIN-41, and D. melanogaster BRAT and MEI-P26 proteins having to be annotated manually. The three-dimensional structure of the NHL domain of BRAT has been determined and the sixth domain does form a portion the β-propeller structure. As such, the lack of conservation in this portion of the propeller may represent functional divergence and, for example, alter protein interaction capabilities. Accordingly, despite the poor conservation in the DPLD C-terminal-most NHL domain, it likely represents a genuine NHL domain (O'Farrell, 2008).

There are several interesting observations associated with this cladogram. First, the LIN-41 subclade of the RBCC/TRIM family contains members spanning from Drosophila/Caenorhabditis to mammals and as such highlights the potential for RBCC/TRIM protein and regulatory studies using the model organisms within this subclade. However, and a second point of interest, all the vertebrate members of the LIN-41 subclade possess N-terminal RING domains, based on both protein predictions and experimental evidence as does C. elegans LIN-41. This finding is in contrast to the dipteran insect members of the subclade, which lack the RING domain, illustrating a potential limit to functional comparisons. From their relative positions in the cladogram, the lack of a RING domain in the insect proteins DPLD and BRAT is interpreted to represent two independent events, whereby the RING domains of each protein were lost. Despite the presence of a RING domain in LIN-41 vertebrate proteins, these are more closely related to the RING-less insect members than to any vertebrate protein relatives (O'Farrell, 2008).

Manually searching up to 300 kb of ungapped sequence upstream to the dpld ATG, searching for cysteine and histidine residues appropriately spaced to fulfill the RING consensus sequence did not reveal any canonical RING domain to which DPLD could be associated. This observation, together with the presence of numerous dpld cDNAs (over 60), which do not encode RING domains, leads to the conclusion that DPLD is RING-less. As mentioned, no vertebrate members of the MEI-P26 and BRAT subclades have been described nor has any evidence been found suggesting that vertebrate members exist, indicating this branch to be protostome-specific; however, with constantly increasing genome sequence coverage, this may change (O'Farrell, 2008).

A notable addition to the cladogram is the protein CG15105, ABBA, which is most similar to NHL-1 in C. elegans, a known ubiquitin ligase. ABBA is a RBCC/TRIM family member possessing the N-terminal RING, suggesting a possible ubiquitinating ability of this protein. The data shows that ABBA is more closely related to the TRIM2 and 3 subclades of the RBCC/TRIM superfamily than DPLD or other Drosophila B-box proteins are. ABBA has an overall identity of 19% with 26% similarity to human TRIM2, the majority of the conserved amino acids lying in the RING and NHL domains (38% identical and 57% similar in a 272 amino acid stretch of the NHL repeat region) (O'Farrell, 2008).

The expression patterns of all four Drosophila RBCC/TRIMs (DPLD, BRAT, MEI-26, and ABBA) have been partially characterized during embryogenesis, and the expression pattern of brat has been extensively documented in developing adult tissues. The four RBCC/TRIM paralogs have very different zygotic transcription patterns, the only exception being an overlap between brat and dpld, which are coexpressed in the developing embryonic central nervous system. To gain a better understanding of the potential functions of those less-characterized Drosophila family members, abba and dpld, in situ hybridization (ISH) was performed on a variety of tissues at various developmental stages. This revealed that, in contrast to the other three RBCC/TRIMs which all are expressed maternally, there was no maternal contribution of abba. Rather, expression of abba was detectable only from mid-embryogenesis onward, specifically in developing muscles. This expression pattern was maintained in the differentiated larval muscles while absent in other tissues. The C. elegans ortholog of ABBA, NHL-1 is also predominantly expressed in muscle tissue (O'Farrell, 2008).

ISH experiments in Drosophila embryos revealed dpld mRNA to be present ubiquitously in the early embryo, thus maternally contributed. Later during embryogenesis, dpld mRNA was expressed in a spatially restricted but dynamic fashion during both CNS and peripheral nervous system (PNS) development, gradually assuming a more defined and stable expression in neurons of the CNS and PNS. Double labeling with antibodies directed against the neuron (anti-ELAV) and sheath (anti-PROS) cells of the PNS in conjunction with ISH, demonstrated that the neurons of the PNS (both chordotonal and multidendritic neurons) expressed dpld at the end of PNS development. Further examination of dpld mRNA distribution in the imaginal discs of late third instar larvae, focusing on the larger wing and eye-antennal discs, revealed dpld mRNA to be expressed in specific cells along the future anterior wing margin of the wing. These cells are likely precursors of the innervated mechano- and/or chemosensory bristles that line the wing margin, in analogy with dpld expression in other neuronal cell types. Some background staining is visible in the surrounding wing pouch; this is, however, unspecific signal, which unlike that of the wing margin staining was not reproducible. In addition, dpld was expressed in a particular pattern during eye development in the eye-antennal imaginal disc (O'Farrell, 2008).

Drosophila neuronal photoreceptor cells of the adult eye are known to be selected from a monolayer of undifferentiated, proliferating cells during larval third instar. During the process of eye cell specification and differentiation, a physical indent known as the morphogenetic furrow sweeps across the disc. Within this furrow, the first photoreceptor cells, the R8, are born at regular intervals. These then progressively recruit additional photoreceptor and accessory cells necessary to construct a complete ommatidium. dpld was found to be broadly expressed both immediately anterior and posterior to the morphogenetic furrow. Interestingly, a more discrete expression was observed at some distance posterior to the furrow in distinct regularly ordered cells, this in contrast to the broad band(s) of expression around the furrow. In an effort to identify the nature of this cell type, double-labeling experiments using ISH and anti-ELAV antibodies were performed, demonstrating dpld to be expressed in neuronal photoreceptor cells. dpld is reiteratively expressed in the neuron both before and after its specification, suggesting that DPLD may fulfill different roles in proliferating versus differentiated cells. This finding is similar to abba, which is expressed both during and after muscle cell differentiation (O'Farrell, 2008).

From the eye disc, the developing photoreceptor neurons send axons to synapse in the optic lobes of the larval CNS. Two distinct concentric arches of dpld expression were clearly visible in both lateral and dorsal views of the optic lobe, whereas another less distinct arch encompassing the inner two was also observed. The outermost arch was narrower and labeled individual, regularly spaced cells. From a dorsal view the expression pattern had the distinctive heart-shaped pattern particular to the proliferative zones of the optic lobe. Vertebrate lin-41 genes are also expressed during embryogenesis in, among other places, developing nervous tissue. One interesting conclusion from the ISH experiments is that the zone of dpld expression in the larval optic lobes is concurrent with dpld expression in neurons of the eye, suggesting a possible function for DPLD in both the photoreceptor neurons and their target brain region (O'Farrell, 2008).

Multiple computational approaches have indicated dpld as a possible miRNA target on the basis of one or more let-7 (in addition to other miRNA) regulatory sites present in the 3' region of dpld mRNA transcripts. Furthermore, recent studies demonstrated miRNA regulation of dpld homologues (lin-41) in other species. However, miRNA regulation of dpld in Drosophila has yet to be demonstrated. Toward this end, a combined in silico approach was used in which all possible miRNA sites in the dpld 3'-UTR were examined using miRNA prediction software, whereas independently the 3' region of a large number of insect dpld genes were compared and analyzed using standard multiple alignment techniques. Subsequently, target sites were sought within the highly conserved region of the 3'-UTR, in an effort to pinpoint potential miRNA target site(s). In this manner, the 3' region of dpld orthologs in several species were examined and several candidate miRNA sites were identified. The best candidate miRNA was let-7, which had three well-conserved sites (one of which was perfectly conserved within Drosophilidae) with favorable G folding values, followed by miR-31a and miR-31b. The miR-31a site M31a 1 was not perfectly conserved between all 11 species. In conclusion, the in silico approach enabled prediction of several candidate miRNA regulators of dpld mRNA (O'Farrell, 2008).

Next, the relevance of these potential dpld regulators was assessed in the fly. To do this, dpld was expressed in transgenic animals using the UAS/GAL4 system. dpld normally is expressed in a spatially and temporally restricted pattern in the developing eye field. Ectopic expression of dpld by the GMR-GAL4 driver, which drives expression of GAL4 in all cells behind the morphogenetic furrow caused a severe eye phenotype as revealed both by anti-ELAV staining of third instar larval eye discs and scanning electron images of the surface of the eye. The pattern of ELAV expression was altered in response to dpld ectopic expression, ommatidial units appeared enlarged as did the individual neurons and in places seemed to be shared between or overlap other ommatidial units. These ommatidial units, which normally arrange themselves into a highly organized crystal lattice-like array, were additionally disturbed on the surface of the eye in GMR>dpld flies, with many of the ommatidial units appearing fused together. At higher magnifications, the lens material, which is secreted by the underlying cone cells, appeared to have spread and to be shared between ommatidial units. This finding, together with the irregular spacing of the interommatidial bristles throughout the eye, accounts for most of the externally visible defects. In addition, a clear reduction in pigment was observed under the light microscope, indicating a lack of pigment cells or defects therein leading to lowered pigment production. Cone and pigment cells, are specified from cells born in the second mitotic wave of the eye disk, a time point and position where ISH experiments have shown dpld to normally be expressed in a spatially restricted manner. Hence, ectopic expression of dpld post furrow interfered with the growth/cell size of many cell types that constitute the adult eye, including neurons, and possibly the subsequent recruitment of cone and pigment cells. Studies directed toward elucidating the underlying mechanism by which dpld acts in the eye are currently under way. The phenotype resulting from ectopically expressed dpld could be modulated by either varying the GAL4 efficiency (by culturing GMR>dpld flies at 29°C or altering gene dosage). It is therefore concluded that the eye phenotype resulting from ectopic expression of dpld was sensitive to levels of DPLD, and an enhancer/suppressor screen for genetic modifiers was consequently performed (O'Farrell, 2008).

Typically, in enhancer/suppressor screens, altering levels of various factors involved in regulation of the level of active of protein, including in this context miRNA translational regulation, would affect the severity of the observable phenotype, in this case the rough eye resulting from GMR>dpld. Almost the entire Drosophila genome has chromosomal deletion coverage, publicly available as individual fly stocks collectively termed the Drosophila deletion kit. Included in this deletion coverage are most miRNA transcriptional units, facilitating a rapid screen through regions of the genome for genetic interactions. In this manner one can appraise the impact of lowering the gene dosage of candidate interactors in a dpld sensitized background. Importantly, specific mutations in the various miRNA are not available in flies, in fact, due to the small size of each miRNA locus, miRNA represent a very difficult target for traditional mutagenesis. Based on the results from the in silico approach predicting potential miRNA regulators of dpld, deletions and duplications including miRNA transcriptional units were selected for further analysis (O'Farrell, 2008).

Of interest, a duplication that included the let-7 locus, mildly suppressed the GMR>dpld phenotype, whereas a deletion uncovering let-7 enhanced the phenotype, consistent with a role for let-7 in dpld regulation in vivo. This was further examined using a range of independent deletions in the let-7 area, which either do or do not remove the let-7 locus. By means of the use of overlapping deletions, the chromosomal region genetically interacting with dpld was narrowed downto 23 loci. The enhancement of the GMR>dpld phenotype in a let-7 background most likely reflects an increase in levels of DPLD and/or an expansion of DPLD activity within the eye field (O'Farrell, 2008).

The use of independent deletions, uncovering miR-31a, miR-31b, and miR-210, produced milder but reproducible enhancements of the GMR>dpld eye phenotype. ISH was performed using antisense locked nucleic acid analogue (LNA) probes directed against miR-31a and let-7 (it having been shown to be present in pupal imaginal discs and CNS by means of Northern techniques only. It was found that let-7 was expressed in the two larger discs of wild-type third instar larvae, the wing and eye-antennal discs. Within the wing disc, let-7 expression was detected in the wing pouch, whereas in the eye, let-7 was found to be expressed in a specific pattern at a distance post-furrow in the portion of the disc where the assembling ommatidial units are located. Thus far the specific cell type(s) expressing let-7 have not been identified. miR-31a was expressed in the optic lobe region of the CNS. As neither the miR-31a or miR-210 potential targeting of the dpld 3'-UTR is conserved to other species, these findings were not investigated further. Similarly, the lack of potential conserved miR-125 target sites within the dpld 3'-UTR (being thought to regulate other lin-41 homologues) likely reflects the lack of conservation of this regulatory mechanism between vertebrate lin-41 and Drosophila dpld (O'Farrell, 2008).

An advantage of the enhancer/suppressor approach described is the availability of deletions and the relative ease and speed of screening. A limit to the system, however, is the large size of the genomic deletions. Although the approach used in this study facilitated rapid screening, from which non-interacting deletions containing potential miRNA regulators of dpld could be discounted, subsequent detailed verification of the putative miRNA is required. The strongest interactor, let-7, which additionally represents a potentially conserved mechanism of miRNA regulation of lin-41 transcripts, was chosen for further molecular study (O'Farrell, 2008).

To directly address the potential regulatory role of let-7 on dpld mRNA, a luciferase reporter assay was implemented in Drosophila S2 cell lines. Importantly, these cell lines have been previously shown not to express let-7 under normal circumstances, although they can be induced to upon exposure to ecdysone, the insect moulting hormone. Multiple versions of the dpld 3-UTR were created stemming from two publicly available cDNAs that differ in their 3'-UTR length, LD39167, which has a 3'-UTR 1527 nucleotides in length (luciferase constructs tagged with this 3'-UTR are referred to as dpld-L) and LD02463, which has a 3'-UTR 242 nucleotides in length (luciferase constructs tagged with this 3'-UTR are referred to as dpld-S). The entire sequence of the dpld-S 3'-UTR is in common to both UTRs. Within this region lies the first predicted let-7 target site (LCS1). The longer 3'-UTR variant, dpld-L, contains an additional two potential let-7 target sites, which lie in close proximity to each other. Mutated versions of both 3'-UTRs were generated in which specifically the let-7 target sites were removed, referred to as dpld-LD and dpld-S. The sensitivity of both the wild-type UTRs (dpld-L and dpld-S) and mutated versions (dpld-LD and dpld-S) was tested to determine whether or not these UTRs were, first, sensitive to let-7, and, second, whether the let-7 target sites were responsible for conferring this sensitivity and finally to compare the different versions of the 3'-UTRs to assess whether the naturally occurring length variation held any significance in terms of let-7 regulation. Both the wild-type and mutated dpld-L constructs promoted equal levels of luciferase reporter gene activity in the absence of let-7. Upon coexpression of a let-7 expression plasmid with the dpld-L 3'-UTR construct strong suppression of reporter gene activity was observed. In contrast, let-7 did not suppress the expression from the mutant dpld-LD construct. Furthermore, coexpression of either 3'-UTR reporter construct with the miRNA miR-92b (not predicted to target the dpld 3'-UTR) had no suppressing effect, indicating the let-7 miRNA effect to be specific. From this two conclusions are drawn: the dpld 3'-UTR is sensitive to let-7 expression and indeed the predicted, conserved let-7 target sites are responsible for this effect. Similarly, both the wild-type and mutated dpld-S constructs promoted roughly equal levels of luciferase reporter gene activity in the absence of let-7; however, in the presence of let-7, the mutated dpld-S, lacking the single predicted let-7 target site, was expressed at levels approximately threefold that of the intact version. It is concluded that the short version was also sensitive to let-7 and that again the predicted target site (LCS1) conferred this sensitivity. Moreover, it is concluded that the difference in length of the UTRs did not represent a plausible mechanism by which dpld could overcome let-7 repression. Furthermore, an additional construct was tested, dpld-L, minus only the let-7 site in common to both UTRs. This construct behaved in a similar fashion to dpld-LD in the presence of let-7, although expression levels were slightly lower, indicating a minor contribution of these two let-7 target sites to let-7 repression of the dpld-L construct. This finding suggested that the let-7 target site (LCS1) in common to both UTR variants, and the most widely conserved LCS within Drosophilidae conferred the majority of the dpld 3'-UTRs sensitivity to let-7 (O'Farrell, 2008).

DEVELOPMENTAL BIOLOGY

let-7 RNA is first detected in late third instar (L3) larvae (Pasquinelli, 2000) around the time a pulse of the steroid hormone 20-hydroxyecdysone (ecdysone) triggers puparium formation (PF) and the onset of metamorphosis. To explore the potential influence of ecdysone on let-7 RNA expression, the profile of let-7 expression was determined during a period from late third larval instar, through prepupal development (a period marked by the sequential expression of ecdysone-inducible genes), to the adult stage. let-7 RNA is first detected at low levels around 4 h before PF. This stage coincides with a short period of high ecdysone titer that triggers PF. let-7 RNA remains at low levels during prepupal development, and then rapidly accumulates to high levels throughout pupal development, reaching a maximum of expression during the second day of pupal life. This rise in let-7 RNA parallels a prolonged pulse of high-level ecdysone secretion. This correlation between the profile of let-7 expression and the time course of changes in ecdysone titer suggests that ecdysone could induce let-7 transcription (Sempere, 2002).

The tissue-specificity of let-7 expression was determined in prepupae and adult animals in order to correlate expression domains with function. Tanning prepupae (0-4 h after PF) were dissected to obtain a variety of tissues: brains, imaginal discs, fat bodies, salivary glands, and Malphigian tubules. Levels of let-7 RNA in these tissues were quantified by Northern analysis. let-7 RNA was detected in all tissues examined, though at relatively higher levels in fat bodies and imaginal discs. The widespread expression of let-7 RNA suggests that let-7 could mediate diverse metamorphic processes, such as the terminal differentiation of imaginal discs and apoptosis of salivary glands and fat bodies. Levels of let-7 RNA in adult ovaries and carcasses (tissues remaining after ovary dissection) were quantified by Northern analysis. let-7 is expressed in both somatic and gonadal tissues in the adult (Sempere, 2002).

EFFECTS OF MUTATION

The Drosophila let-7-Complex (let-7-C) is a polycistronic locus encoding three ancient microRNAs: let-7, miR-100, and fly lin-4 (miR-125). The let-7-C locus is principally expressed in the pupal and adult neuromusculature. let-7-C knockout flies appear normal externally but display defects in adult behaviors (e.g., flight, motility, and fertility) as well as clear juvenile features in their neuromusculature. The function of let-7-C to ensure the appropriate remodeling of the abdominal neuromusculature during the larval-to-adult transition is carried out predominantly by let-7 alone. This heterochronic role of let-7 is likely just one of the ways in which let-7-C promotes adult behavior (Sokol, 2008).

Mutations in heterochronic genes in Caenorhabditis elegans cause cells in particular lineages to express their stage-specific fates earlier or later than normal. Detailed analysis of these genes has revealed a regulatory pathway of heterochronic genes that specifies the timing of cellular development in diverse cell types and thereby ensures a coordinated schedule of developmental events throughout the worm. The existence of the heterochronic gene pathway in worms and the conservation of some of its components through animal evolution suggest that functionally analogous pathways could also coordinate developmental timing in higher organisms. Two of these highly conserved components of the heterochronic pathway, let-7 and lin-4, are microRNAs (miRNAs), a class of small RNAs that post-transcriptionally modulate the expression of target transcripts. The sequences and developmentally regulated expression profiles of let-7 and lin-4 are conserved among diverse bilaterians. For example, Drosophila let-7 and miR-125 (fly lin-4) are robustly up-regulated during metamorphosis, as is another highly conserved miRNA, miR-100. All three of these ancient miRNAs are encoded in a 1-kb region of the Drosophila genome, and their clustered organization has been conserved and duplicated in vertebrates. These findings suggest that miR-100, let-7, and miR-125 coordinately control gene expression to regulate developmental timing in animals. To test this hypothesis, the roles of miR-100, let-7, and miR-125 were analyzed in Drosophila; these miRNAs are required for normal adult behavior, suggesting roles in neural development and/or function. let-7 in particular is required for remodeling of the fly neuromusculature during the larval-to-adult transition, confirming that a general developmental timing function of let-7 has been evolutionarily conserved from worms to flies (Sokol, 2008).

The clustered organization of Drosophila miR-100, let-7, and miR-125 suggests that these miRNAs are co-transcribed as a single polycistronic transcript. To test this hypothesis, cDNAs generated from genomic regions between miR-100 and let-7 and between let-7 and miR-125 were isolated using 5' and 3' rapid amplification of cDNA ends (RACE). This analysis identified two overlapping cDNA fragments that corresponded to a 2435-nucleotide (nt) primary transcript that encoded the ~70-nt hairpin sequences of miR-100, let-7, and miR-125, and was comprised of three exons that spanned 17,400 kb of genomic DNA. It is concluded that miR-100, let-7, and miR-125 are cotranscribed from a single locus, which is referred to as the let-7-Complex (let-7-C) since let-7 was the first of these miRNAs identified in Drosophila. It is inferred that the miR-100, let-7, and miR-125 clusters in the genomes of other animals also represent single polycistronic loci. It should be noted that cotranscribed let-7-C miRNAs may not always be coexpressed, given that post-transcriptional processing of mature miRNAs from primary transcripts can be subject to developmental regulation (Sokol, 2008).

To investigate whether let-7-C miRNAs collectively regulate developmental timing in Drosophila, two independent let-7-C knockout strains, let-7-C^KO1 and let-7-C^GKI, were generated. Both strains lack expression of the mature processed forms of miR-100, let-7, and miR-125. To test whether the activity of each of the let-7-C miRNAs is required for let-7-C function, the phenotypes of three different let-7-C derivative strains were analyzed in which the expression of miR-100, let-7, or miR-125 had been eliminated individually. These singly mutant strains are referred to as miR-100^δ, let-7^δ, and miR-125^δ, respectively. miR-100^δ mutants functioned normally in all behavioral assays, indicating that miR-100 was not solely responsible for any of the identified let-7-C functions. None of the single mutant strains displayed strong male fertility or climbing defects, suggesting that for normal male fertility and climbing behavior, the combinatorial action of any two let-7-C microRNAs could suffice. In contrast, let-7^δ and miR-125^δ mutants displayed severely reduced spontaneous locomotion as well as partial defects in flight. The normal climbing and nearly normal flight of let-7^δ and miR-125^δ mutants suggested that their severely impaired spontaneous locomotory activity was not simply the consequence of physically or metabolically impaired mobility, but rather likely reflected a behavioral deficit of neurological origin. Finally, let-7^δ mutants alone displayed moderately severe defects in female fertility and oviposition, indicating that let-7 is required for an essential function to promote female reproduction (Sokol, 2008).

To identify the specific place where let-7-C miRNAs may function to promote adult behavior, the spatiotemporal expression pattern of the let-7-C locus was examined. The let-7-C^GKI strain, in which the yeast transcriptional activator Gal4 had been inserted into the let-7-C locus, was used to drive expression of Gal4-dependent transgenes encoding membrane-bound or nuclear forms of GFP. A UAS-let-7-C transgene placed under the control of the let-7-C::Gal4 insertion restored miR-100, let-7, and miR-125 expression as well as climbing activity to let-7-C^KO1/GKI mutants. Three characteristics of the let-7-C::Gal4 expression pattern are outlined below. Initially, let-7-C::Gal4 was expressed in neurons throughout the adult brain and ventral nerve cord, and this adult CNS expression was the culmination of a dramatic expansion in the spatial expression pattern of let-7-C::Gal4 that occurred in the CNS during the first half of metamorphosis. Second, let-7-C::Gal4 was expressed in neurons that innervated structures throughout the adult, including sensory organs in the head, flight muscles in the thorax, and the alimentary tract, the male and female reproductive tracts, and the male and female genitalia in the abdomen. It is noted that let-7-C::Gal4 was very densely expressed in the posterior tip of the adult abdominal ganglion, as well as in motoneurons that projected posteriorly and innervated two distinct sets of abdominal muscles, the dorsal internal oblique muscles (DIOM) and the dorsal muscles (DM). The DIOMs are remnants of the larval body wall that persist through metamorphosis (presumably to function in the process of eclosion) and in the wild type are fated to die within 12 h of eclosion. In contrast, the DMs are the adult body-wall muscles and are derived from larval myoblasts that undergo myogenesis during metamorphosis. Third, let-7-C::Gal4 was not only expressed in motoneurons but in muscle cells as well, including the DIOMs and DMs. Taken together, the expression of let-7-C::Gal4 in pupal and adult neurons and muscles is consistent with the hypothesis that the behavioral phenotypes of let-7-C mutant adults are the consequence of defects in the metamorphosis of the neuromusculature (Sokol, 2008).

To test whether let-7-C miRNAs play a role in specifying the configuration of the adult neuromusculature, the abdominal muscle system of let-7-C^KO1/GKI mutants was examined, since let-7-C is expressed in abdominal motoneurons and muscles. Two very clear and highly penetrant defects were found. (1) the DIOMs that ordinarily decay during post-eclosion maturation of wild-type flies failed to disappear in older let-7-C^KO1/GKI mutants. (2) The DMs of let-7-C^KO1/GKI mutant adults were clearly smaller than those in age-matched wild-type controls. Restoration of let-7-C expression rescued both the DIOM and DM phenotype. To test whether let-7-C^KO1/GKI mutant muscle phenotypes were the consequence of defects apparent prior to the onset of metamorphosis, the musculature and myoblasts of let-7-C^KO1/GKI larvae were examined, and it was found that both appeared normal. It is therefore concluded that the abdominal muscle system of let-7-C^KO1/GKI mutant adults failed to complete its larval-to-adult remodeling, displaying both persistent pupal as well as immature adult characteristics. This is interpreted as a heterochronic phenotype, since let-7-C^KO1/GKI mutant adults exhibited both juvenile features (e.g., muscle system morphology) as well as mature adult traits (e.g., external appearance) at the same time (Sokol, 2008).

To test whether let-7-C affects the remodeling of other internal tissues, the morphogenesis of the CNS during metamorphosis in let-7-C^KO1/GKI mutants was examined and found that at a gross level, CNS development appeared to have proceeded normally. To examine the results of nervous system remodeling in finer detail, focus was placed on the morphology of motoneurons that innervate the DIOMs or the DMs. DIOMs are innervated by DIOM motoneurons, which also degenerate after eclosion. The DIOMs and their DIOM motoneurons, however, are triggered to die at different times and therefore may be controlled by independent signals. Interestingly, it was found that the neuromuscular junctions (NMJs) connecting DIOMs and their innervating motoneurons failed to decay in let-7-C^KO1/GKI mutant adults, indicating that the DIOMs and DIOM motoneurons persisted together. These data suggest that let-7-C functions to coordinate the fates of DIOMs and DIOM motoneurons. Similarly, the reduced size of let-7-C^KO1/GKI mutant DMs was reflected in clear defects in let-7-C^KO1/GKI mutant DM NMJs, which were either completely absent, shorter in length than wild-type NMJs, or devoid of boutons, appearing as long, thin processes along the length of the DM. It is concluded that the heterochronic abdominal muscle defect was reflected in a corollary nervous system defect, supporting the hypothesis that disruption of neuromusculature remodeling could underlie at least some of the let-7-C^KO1/GKI mutant behavioral phenotypes (Sokol, 2008).

The striking similarity between let-7-C^KO1/GKI mutant phenotypes and the phenotypes associated with manual denervation of abdominal muscles prior to metamorphosis was noted. In both cases, adult DM muscles fail to grow to wild-type width, contain fewer nuclei, and display aberrant NMJs. However, the let-7-C mutation and denervation differ in at least one respect: their effect on the male-specific muscle of Lawrence (MOL). MOLs are present in let-7-C^KO1/GKI adult males but absent in manually denervated adult males. The overall similarity between the effects of genetic depletion of let-7-C and muscle denervation during metamorphosis supports the hypothesis that let-7-C is required to regulate an interaction between muscles and motoneurons during neuromusculature remodeling (Sokol, 2008).

To test whether the activities of miR-100, let-7, or miR-125 are required individually for neuromusculature remodeling, the abdominal muscle pattern as well as DM NMJs were examined in miR-100^δ, let-7^δ, and miR-125^δ single mutants. Two-day-old miR-100^δ and miR-125^δ males retained none of the six DIOMs, while let-7^δ males retained 61% ± 28.5% of DIOMs. Although the frequency of complete DIOM retention is lower in let-7^δ mutants compared to let-7-C^KO1/GKI mutants, it is noted that 83% ± 25% of let-7^δ mutant DIOMs had arrested at some stage in the process of degeneration. With respect to both the DM and DM NMJ phenotype, it was similarly found that miR-100^δ and miR-125^δ mutants appeared normal, whereas let-7^δ mutants phenocopied let-7-C^KO1/GKI mutants. miR-100^δ and miR-125^δ DMs were 18.3 ± 0.8 µm and 16.8 ± 0.8 µm in width, respectively, while let-7^δ DMs were 12 ± 1.8 µm in length. Similarly, miR-100^δ and miR-125^δ NMJs were 45.9 ± 10.2 µm and 55.1 ± 13.3 µm in length, respectively, while let-7^δ NMJs were 12.5 ± 5 µm in length. For the sake of consistency, all the morphological data quantified in this study were collected from adult males. However, let-7-C^KO1/GKI and let-7^δ mutant females exhibited DM and DIOM phenotypes identical to their male siblings, suggesting that the reduced egg-laying displayed by let-7^δ mutant females might be a consequence of defects in their abdominal neuromusculature. From these data, it is concluded that the activity of let-7 alone was predominantly responsible for let-7-C-dependent remodeling of the abdominal neuromusculature, and therefore that a heterochronic let-7 role in regulating developmental transitions had been evolutionarily conserved from worms to flies (Sokol, 2008).

The functional dissection of Drosophila let-7-C presented in this study indicates that let-7-C is required for adult behavior and that defects in neuromusculature remodeling correlate with some aspects of this requirement. The perdurance of juvenile features in adult Drosophila let-7 mutants is analogous to the reiteration of larval cell fates in adult Caenorhabditis elegans let-7 mutants (Reinhart et al. 2000), confirming the suggestion by Pasquinelli et al. in 2000 that let-7 might control developmental transitions in diverse bilateria. Future work in flies should extend this analysis to identify the relevant mRNA targets that Drosophila let-7 regulates in its heterochronic role and to examine how this heterochronic function is integrated into the more general requirements of the let-7-C locus in promoting adult behavior. For the most part, the set of targets predicted for Drosophila let-7 are distinct from those predicted for C. elegans let-7. Unpublished observations indicate that one of Drosophila let-7's targets is the transcription factor abrupt, although it was also found that ectopic expression of abrupt in a let-7-C::Gal4-driven pattern is not sufficient to recapitulate the let-7^δ phenotype. The conservation of the genomic clustering as well as neuronal expression of let-7, mir-125, and mir-100 from flies to vertebrates suggests that let-7-C loci could function in neuromuscular and/or neuronal remodeling in mammals. Future work on let-7-C should reveal how its diverse effects on temporal cell fates, developmental timing, and neuronal remodeling are related (Sokol, 2008).

A genome-wide survey of sexually dimorphic expression of Drosophila miRNAs identifies the steroid hormone-induced miRNA let-7 as a regulator of sexual identity

miRNAs bear an increasing number of functions throughout development and in the aging adult. This study addresses their role in establishing sexually dimorphic traits and sexual identity in male and female Drosophila. A survey of miRNA populations in each sex identifies sets of miRNAs differentially expressed in male and female tissues across various stages of development. The pervasive sex-biased expression of miRNAs generally increases with the complexity and sexual dimorphism of tissues, gonads revealing the most striking biases. The male-specific regulation of the X chromosome was found to be relevant to miRNA expression on two levels. First, in the male gonad, testis-biased miRNAs tend to reside on the X chromosome. Second, in the soma, X- linked miRNAs do not systematically rely on dosage compensation. The importance of a sex-biased expression of miRNAs in establishing sexually dimorphic traits was addressed. The study of the conserved let-7-C miRNA cluster controlled by the sex-biased hormone ecdysone places let-7 as a primary modulator of the sex determination hierarchy. Flies with modified let-7 levels present doublesex-related phenotypes and express sex determination genes normally restricted to the opposite sex. In testes and ovaries, alterations of the ecdysone induced let-7 result in aberrant gonadal somatic cell behavior and non cell-autonomous defects in early germline differentiation. Gonadal defects as well as aberrant expression of sex determination genes persist in aging adults under hormonal control. Together, these findings place ecdysone and let-7 as modulators of a somatic systemic signal that helps establish and sustain sexual identity in males and females and differentiation in gonads. This work establishes the foundation for a role of miRNAs in sexual dimorphism and demonstrates that similar to vertebrate hormonal control of cellular sexual identity exists in Drosophila (Fagegaltier, 2014).

EVOLUTIONARY HOMOLOGS

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

MicroRNAs (miRNAs) are a large family of small regulatory RNAs that are poorly understood. The let-7 miRNA regulates the timing of the developmental switch from larval to adult cell fates during Caenorhabditis elegans development. Expression of let-7 RNA is temporally regulated, with robust expression in the fourth larval and adult stages. Like let-7 RNA, a transcriptional fusion of the let-7 promoter to gfp is temporally regulated, indicating that let-7 is transcriptionally controlled. Temporal upregulation of let-7 transcription requires an enhancer element, the temporal regulatory element (TRE), situated about 1200 base pairs upstream of the start of the mature let-7 RNA. The TRE is both necessary and sufficient for this temporal upregulation. A TRE binding factor (TREB) is able to bind to the TRE, and a 22-base pair inverted repeat within the TRE is necessary and sufficient for this binding. The nuclear hormone receptor DAF-12 and the RNA binding protein LIN-28 are both required for the correct timing of let-7 RNA and let-7::gfp expression. It is speculated that these heterochronic genes regulate let-7 expression through its TRE (Johnson, 2003).

One model for the action of this cis-acting TRE is one of positive regulation, where in the early larval stages (L1-L3) the TRE is free and unbound by transcriptional activators and does not induce let-7 expression. Upon entering the L3/L4 transition, TREB binds to the IR in the TRE and activates the transcription of let-7. TRE therefore acts as an enhancer element. Since let-7 expression appears coupled to the L3 molt, it is predicted that TREB activity is likely to be regulated by a hormone that signals the molt. TREB could itself be a nuclear hormone receptor or another factor that is regulated by hormonal signaling. An alternative model would be that the TREB is always bound to the TRE and becomes activated by binding of an early-L4-produced hormone or other ligand. Future work will distinguish between these two models and reveal the identity of TREB. Additionally, it is suspected that TREB activity is likely to be controlled by lin-4, lin-14, daf-12, and lin-28, the heterochronic genes upstream of let-7 (Johnson, 2003).

The identity of the TREB is open to speculation, but various nuclear hormone receptors in C. elegans play roles in molting and may correspond to TREB or be activators of TREB. There are approximately 270 nuclear receptor genes found in the C. elegans genome, at least 10 of which have been shown to be expressed in seam cells. All of these genes are potential TREB candidates, but only 1, daf-12, plays a role in the heterochronic pathway. daf-12 indeed regulates the timing of let-7 expression: mutations in daf-12 that result in retarded heterochronic phenotypes cause retarded expression of let-7 and let-7::gfp. A DAF-12::GFP fusion reveals high expression in the hypodermal seam cells, and both the DAF-12::GFP fusion protein and daf-12 mRNA are expressed throughout development, placing DAF-12 in the right place at the right time. One possibility is that DAF-12 regulation of let-7 could be direct with DAF-12 filling the role of the TREB and binding to the TRE to activate the transcription of let-7. However, TREB binding activity is not affected in a daf-12(rh61) mutant, suggesting that DAF-12 may act indirectly in promoting let-7 expression (Johnson, 2003).

This study has addressed some fundamental questions about let-7 regulation in C. elegans, but the answers found might have broader implications. let-7 is a broadly conserved stRNA, and is also a member of the large class of recently discovered miRNAs. miRNAs are nonprotein coding genes that encode mature RNA products of about 20-24 nt in length. Each of these genes is hypothesized to be transcribed as a longer precursor molecule that can fold back on itself to form a hairpin loop. Three newly discovered miRNAs, mir-48, mir-69, and mir-84, have similar temporal expression patterns to let-7, and mir-48 and mir-84 share sequence identity to let-7. The homologies between both the expression patterns and the sequences of let-7, mir-48, and mir-84 suggest a possible common role for these genes and, more strongly, the possibility of common transcriptional regulation. In support of this, possible common promoter elements upstream of these genes have been identified (Johnson, 2003).

The let-7 microRNA is phylogenetically conserved and temporally expressed in many animals. C. elegans let-7 controls terminal differentiation in a stem cell-like lineage in the hypodermis, while human let-7 has been implicated in lung cancer. To elucidate let-7's role in temporal control of nematode development, sequence analysis and reverse genetics were used to identify candidate let-7 target genes. The nuclear hormone receptor daf-12 is a let-7 target in seam cells, while the forkhead transcription factor pha-4 is a target in the intestine. Additional likely targets are the zinc finger protein die-1 and the putative chromatin remodeling factor lss-4. Together with the previous identification of the hunchback ortholog hbl-1 as a let-7 target in the ventral nerve cord, these findings show that let-7 acts in at least three tissues to regulate different transcription factors, raising the possibility of let-7 as a master temporal regulator (Grosshans, 2005).

Post-transcriptional regulation of the let-7 microRNA during neural cell specification

The let-7 miRNA regulates developmental timing in C. elegans and is an important paradigm for investigations of miRNA functions in mammalian development. This study investigated the role of miRNA precursor processing in the temporal control and lineage specificity of the let-7 miRNA. In situ hybridization (ISH) in E9.5 mouse embryos revealed early induction of let-7 in the developing central nervous system. The expression pattern of three let-7 family members closely resembled that of the brain-enriched miRNAs mir-124, mir-125 and mir-128. Comparison of primary, precursor, and mature let-7 RNA levels during both embryonic brain development and neural differentiation of embryonic stem cells and embryocarcinoma (EC) cells suggest post-transcriptional regulation of let-7 accumulation. Reflecting these results, let-7 sensor constructs were strongly down-regulated during neural differentiation of EC cells and displayed lineage specificity in primary cells. Neural differentiation of EC cells was accompanied by an increase in let-7 precursor processing activity in vitro. Furthermore, undifferentiated and differentiated cells contained distinct precursor RNA binding complexes. A neuron-enhanced binding complex was shown by antibody challenge to contain the miRNA pathway proteins Argonaute1 and FMRP. Developmental regulation of the processing pathway correlates with differential localization of the proteins Argonaute, FMRP, MOV10, and TNRC6B in self-renewing stem cells and neurons (Wulczyn, 2007).

The C. elegans hunchback-like gene lin-57/hbl-1 controls developmental time and is regulated by let-7

Temporal control of development is an important aspect of pattern formation that awaits complete molecular analysis. lin-57 has been identified as a member of the C. elegans heterochronic gene pathway, which ensures that postembryonic developmental events are appropriately timed. Loss of lin-57 function causes the hypodermis to terminally differentiate and acquire adult character prematurely. lin-57 has been identified as hbl-1, revealing a role for the worm hunchback homolog in control of developmental time. Significantly, fly hunchback (hb) temporally specifies cell fates in the nervous system. The hbl-1/lin-57 3'UTR is required for postembryonic downregulation in the hypodermis and nervous system and contains multiple putative binding sites for temporally regulated microRNAs (miRNAs), including let-7. Indeed, hbl-1/lin-57 is regulated by let-7, at least in the nervous system. Examination of the hb 3'UTR reveals potential binding sites for known fly miRNAs. Thus, evolutionary conservation of hunchback genes may include temporal control of cell fate specification and microRNA-mediated regulation (Abrahante, 2003).

Postembryonic temporal downregulation of hbl-1 in the worm nervous system and hypodermis is programmed, at least in part, through its 3'UTR, which contains multiple putative let-7 binding sites that are evolutionarily conserved. In the nervous system, an hbl-1::gfp::hbl-1 reporter construct is temporally deregulated in a let-7 mutant background; enhanced expression is observed in the ventral nerve cord and anterior nerve ring of adults. Together, these results imply that the hbl-1 3'UTR is a direct target of the let-7 miRNA (Abrahante, 2003).

The extent of hbl-1::gfp::hbl-1 misexpression in let-7 mutants is less than might be expected if let-7 acts alone to downregulate neuronal expression and suggests that additional factors, perhaps other microRNAs, act together with let-7. Indeed, a large and diverse family of miRNAs has been discovered in C. elegans. Among the worm miRNAs reported, three (mir-84, mir-48, and mir-241) share sequence identity with let-7 RNA and are expressed with the same temporal specificity as let-7. The sequence conservation among these miRNAs, particularly between mir-84 and let-7 (81% identical), suggests that they may have target sites in common. Thus, complete temporal deregulation of the hbl-1 reporter may require simultaneous inactivation of multiple miRNAs (Abrahante, 2003).

The role of let-7 in control of hbl-1 in the hypodermis is less clear. The simplest way to interpret let-7 suppression by hbl-1, together with let-7 binding sites in the hbl-1 3'UTR, is that hbl-1 is a direct target of the let-7 miRNA. However, hypodermal hbl-1::gfp expression begins to subside in the L2 and disappears in the early L3, prior to let-7 accumulation in the mid to late L3 stage. Assuming that the hbl-1::gfp construct (which contains a 6.4 kb 5' flanking sequence through the first three introns) contains all relevant enhancer regions, this implies that 3'UTR-mediated downregulation of hbl-1 in hyp7 is controlled by other factors, perhaps including earlier-acting miRNAs (Abrahante, 2003).

let-7 could add to the repression of hbl-1 mRNA from the mid L3 stage onward, ensuring its silence at late developmental stages. However, consistent hbl-1::gfp::hbl-1 misexpression was not detected in the hypodermis of let-7 mutants, suggesting only a minor role for let-7 or redundant action by let-7-related genes. Alternatively, a low threshold level of the HBL-1 presumed transcription factor (not detectable by gfp assay) may be required for hypodermal function. Thus, small changes in HBL-1 level could lead to major developmental consequences through deregulation of target genes (Abrahante, 2003).

Temporal regulation of hbl-1 differs from that of lin-41, the other known let-7 target. lin-41::gfp is expressed in both neurons and hypodermis but is temporally downregulated only in the hypodermis. The discordant patterns of regulation suggest inherent differences between the hbl-1 and lin-41 3'UTRs and the assembled factors that orchestrate their function (Abrahante, 2003).

Reduction of hbl-1 activity by mutation or RNAi does not fully suppress let-7 null mutations. Explanations for this partial epistasis include incomplete loss of hbl-1 function, misexpression of let-7 targets, or redundancy at the hbl-1 step in the pathway. This work suggests that the let-7 target, lin-41, is at least part of the answer. Simultaneous removal of hbl-1 and lin-41 activities produces stronger suppression of the let-7 phenotype than does single depletion of either gene. In let-7(+) animals, depletion of hbl-1 and lin-41 activities produces a fully penetrant L3 molt phenotype and can cause terminal differentiation at the L2 molt, one stage earlier than in either single mutant. Together, these results indicate that let-7 acts through both hbl-1 and lin-41 and that these genes function with partial redundancy to inhibit premature activation of the adult hypodermal program at the L2 and L3 molts in wild-type animals (Abrahante, 2003).

These findings extend the intriguing parallels between the early and late timers of the heterochronic gene pathway, which together mediate stage-specific temporal identities. Each timer is initiated by a microRNA that has two known targets; in the early timer, lin-4 downregulates lin-14 and lin-28, and, in the late timer, let-7 acts through hbl-1 and lin-41. In each case, one target encodes a transcription factor (LIN-14 and HBL-1), and the other encodes a protein with hallmarks of a translational regulator (LIN-28 and LIN-41). Since loss-of-function for each pair of targets causes enhanced precocious phenotypes, it appears that both transcriptional and translational controls are necessarily integrated into both timers to ensure proper timing of cell fate specification (Abrahante, 2003).

Previous studies have generally supported a linear pathway of heterochronic genes, with lin-4 acting as the most upstream and global regulator. These analyses suggest that the pathway is branched. Concomitant loss of hbl-1 and lin-41 activities suppresses the let-7 mutant phenotype more completely than that of lin-4. Loss of hbl-1 and lin-41 activities only weakly restores alae synthesis at the L4 molt in lin-4 mutants, whereas it leads to essentially complete execution of the adult seam cell program at the L3 molt in a let-7 mutant background. These observations indicate that either lin-4 or the genes it regulates have additional targets that time the adult hypodermal program independently of hbl-1 and lin-41. Thus, multiple temporal inputs converge upon the transcription factor LIN-29, indicating that a branched pathway functions to ensure proper timing of seam cell terminal differentiation. Elaboration of these proposed branches will require searches for additional components of the heterochronic gene pathway (Abrahante, 2003).

hbl-1, the C. elegans hunchback ortholog, also controls temporal patterning. Furthermore, hbl-1 is a probable target of microRNA regulation through its 3'UTR. hbl-1 loss-of-function causes the precocious expression of adult seam cell fates. This phenotype is similar to loss-of-function of lin-41, a known target of the let-7 microRNA. Like lin-41 mutations, hbl-1 loss-of-function partially suppresses a let-7 mutation. The hbl-1 3'UTR is both necessary and sufficient to downregulate a reporter gene during development, and the let-7 and lin-4 microRNAs are both required for HBL-1/GFP downregulation. Multiple elements in the hbl-1 3'UTR show complementarity to regulatory microRNAs, suggesting that microRNAs directly control hbl-1. MicroRNAs may likewise function to regulate Drosophila hunchback during temporal patterning of the nervous system (Lin, 2003).

HBL-1/GFP is expressed strongly in hypodermal cells, including the embryonic seam cell precursors, and in neurons like those of the ventral nerve cord (VNC) during postembryonic stages. HBL-1/GFP expression was reexamined, focusing on hypodermal and VNC expression at postembryonic C. elegans developmental stages. Strain BW1932 contains an integrated array with the hbl-1 promoter, the first 133 amino acids of HBL-1 fused to GFP, and the hbl-1 3'UTR. During the L1 stage, HBL-1/GFP expression is observed in the hypodermal syncitial cells (e.g., hyp7), in the ventral hypodermal cells (P cells), and weakly in the lateral hypodermal seam cells (H, V, and T cells). By the L2 stage, HBL-1/GFP was no longer expressed in the seam cells but was still observed in P cell descendants and weakly in the non-seam cell hypodermis. By the L3 stage, HBL-1/GFP was virtually absent in the hypodermis and Pn.p cell descendants, but was still highly expressed in the ventral nerve cord (generated from Pn.a cells) and other unidentified neurons. Early L4 animals express high HBL-1/GFP levels in the VNC, while late L4 and adult animals express HBL-1/GFP very weakly in the VNC. In some adult VNCs, expression is undetectable. As judged by this HBL-1/GFP fusion, HBL-1 expression is downregulated during the course of postembryonic development, with highest expression in L1 animals and lowest expression in adults (Lin, 2003).

let-7 RNA is expressed predominantly in the L4 and adult stages. HBL-1/GFP expression in the VNC is downregulated during the L4 and adult stages by a 3'UTR-dependent mechanism. The similar timing of these two events suggest that let-7 might be involved in downregulation of hbl-1 in the VNC. Indeed, it was found that while 45% of let-7(n2853) adults expressed intense HBL-1/GFP in the VNC, only 4% of wild-type animals did the same. lin-4 RNA is also present in the L4 stage. Intense HBL-1/GFP expression is seen in the VNC of 100% of lin-4(e912) adult animals. Thus, both wild-type let-7 and lin-4 RNAs are required for proper hbl-1 downregulation in the VNC (Lin, 2003).

The heterochronic gene lin-46 functions in the let-7 pathway

The succession of developmental events in the C. elegans larva is governed by the heterochronic genes. When mutated, these genes cause either precocious or retarded developmental phenotypes, in which stage-specific patterns of cell division and differentiation are either skipped or reiterated, respectively. A new heterochronic gene, lin-46, has been identified from mutations that suppress the precocious phenotypes caused by mutations in the heterochronic genes lin-14 and lin-28. lin-46 mutants on their own display retarded phenotypes in which cell division patterns are reiterated and differentiation is prevented in certain cell lineages. The analysis indicates that lin-46 acts at a step immediately downstream of lin-28, affecting both the regulation of the heterochronic gene pathway and execution of stage-specific developmental events at two stages: the third larval stage and adult. lin-46 is required prior to the third stage for normal adult cell fates, suggesting that it acts once to control fates at both stages, and that it affects adult fates through the let-7 branch of the heterochronic pathway. Interestingly, lin-46 encodes a protein homologous to MoeA of bacteria and the C-terminal domain of mammalian gephyrin, a multifunctional scaffolding protein. These findings suggest that the LIN-46 protein acts as a scaffold for a multiprotein assembly that controls developmental timing, and expand the known roles of gephyrin-related proteins to development (Pepper, 2004).

lin-46 encodes a 391 amino acid protein with homology along its entire length to MoeA of bacteria and the C-terminal domains (referred to as the E-domain) of the mammalian protein gephyrin; other related proteins are Cinnamon of Drosophila and CNX1 of Arabidopsis. Gephyrin is a submembraneous scaffolding protein that aids in clustering glycine and GABA receptors at postsynaptic neurons. MoeA is involved in the last step of the biosynthesis of molybdenum co-factor, a metal coordinating molecule in many molybdo-enzymes, although the exact function of MoeA in this process is not known. Gephyrin, as well as Cinnamon and CNX1, are essentially fusions of homologs of the bacterial proteins MoeA and MogA. Indeed, all three of these proteins from higher eukaryotes are believed to participate in molybdenum cofactor biosynthesis, as the bacterial proteins do. C. elegans is unusual among multicellular eukaryotes in having its MoeA and MogA homologs encoded separately, and, furthermore, having two MoeA paralogs, one of which is encoded by lin-46 (Pepper, 2004).

It is possible that lin-28 and lin-46 directly affect the microRNA let-7, which then regulates other genes that control the larval to adult switch. This is consistent with the failure of lin-46 mutant to suppress the precocious phenotypes of the later-acting genes lin-41 and lin-57. Further analysis will determine whether lin-46 and lin-28 indeed affect the expression or activity of let-7 (Pepper, 2004).

The let-7 microRNA family members mir-48, mir-84, and mir-241 function together to regulate developmental timing in Caenorhabditis elegans

The microRNA let-7 is a critical regulator of developmental timing events at the larval-to-adult transition in C. elegans. Recently, microRNAs with sequence similarity to let-7 have been identified. Doubly mutant animals lacking the let-7 family microRNA genes mir-48 and mir-84 exhibit retarded molting behavior and retarded adult gene expression in the hypodermis. Triply mutant animals lacking mir-48, mir-84, and mir-241 exhibit repetition of L2-stage events in addition to retarded adult-stage events. mir-48, mir-84, and mir-241 function together to control the L2-to-L3 transition, likely by base pairing to complementary sites in the 3′ UTR of the hunchback homolog hbl-1 and downregulating hbl-1 activity. Genetic analysis indicates that mir-48, mir-84, and mir-241 specify the timing of the L2-to-L3 transition in parallel to the heterochronic genes lin-28 and lin-46. These results indicate that let-7 family microRNAs function in combination to affect both early and late developmental timing decisions (Abbott, 2005).

The C. elegans genome encodes at least 19 microRNA gene families containing from 2 to 8 members with significant sequence conservation within the ~22 nt microRNA sequence. Sequence conservation among family members is strongest near the 5' end of the microRNA in the region known as the 'seed',which has been proposed to reflect a potential for family members to direct the repression of shared target genes. Because mir-48, mir-84, and mir-241 display complete sequence conservation in the seed region at the 5′ end, it is possible that they repress a common set of targets and hence may be functionally equivalent. The current findings suggest that let-7, mir-48, mir-84, and mir-241 may all act to repress a shared target, hbl-1. let-60 RAS also has been proposed to be a target of mir-84 based on overexpression experiments. Elevated levels of let-60 RAS expression lead to a multivulva phenotype; however, no multivulva phenotype was observed in mir-84 single mutants nor in mir-48 mir-241; mir-84 triple mutants (Abbott, 2005).

The results leave open the possibility that mir-48, mir-84, and mir-241 are not functionally equivalent in all respects. Sequence differences in the 3′ end of the let-7 family microRNAs may direct the repression of some distinct sets of targets, the repression of which could function coordinately to regulate developmental timing. Target sites that lack strong complementarity at the microRNA 5′ end can direct repression if there is extensive compensatory pairing at the 3′ end, thus allowing for distinct activities of microRNA family members. Indeed, let-7 complementary sites in the lin-41 mRNA have extensive complementarity to the let-7 3′ region, along with imperfect pairing to the let-7 5′ seed region. The specificity imparted by compensatory 3′ pairing may function to enable repression of lin-41 by let-7 and not allow for the repression of lin-41 by mir-48, mir-84, or mir-241. Similarly, extensive 3′ pairing to one of the other three let-7 family members might compensate for a lack of strong 5′ pairing and therefore could restrict the repression of specific targets to individual let-7 family members (Abbott, 2005).

The findings suggest that the four let-7 family microRNAs may all act to repress hbl-1. Reduction of hbl-1 activity can suppress the heterochronic defects observed in both mir-48 mir-241; mir-84 and let-7 mutant animals, indicating that hbl-1 functions downstream of the let-7 family microRNAs. Moreover, the failure to appropriately downregulate hbl-1 can be detected in the hypodermis of mir-48 mir-241; mir-84 mutants and in neuronal cells of let-7 mutants. The hbl-1 3′ UTR contains eight let-7 complementary sites. Because these potential binding sites differ in sequence, each may be able to bind the individual let-7 family microRNAs with differing efficacies. The relative contribution of individual let-7 family microRNAs to the repression of hbl-1 activity remains to be tested (Abbott, 2005).

Previous studies showed a role for hbl-1 activity in controlling the L4-to-adult transition. The current findings indicate that hbl-1 also controls the L2-to-L3 transition in the hypodermis. This early role for hbl-1 is consistent with the observation that reduction of hbl-1 activity by RNAi results in a decreased number of seam cells in L2-stage animals. A reduced number of seam cells likely reflects a partial omission of the L2-stage proliferative program. This precocious phenotype is relatively weak in comparison to that of lin-28(lf) mutants, in which all seam cells generated from the V lineage fail to execute the L2-stage program. This weak phenotype may be a consequence of residual hbl-1 activity of the partial loss-of-function allele, ve18. It is possible that complete loss of hbl-1 activity would result in a stronger precocious L2-omission phenotype similar to that seen in lin-28(lf) mutants (Abbott, 2005).

An important regulator of the L2-to-L3 transition is lin-28, yet multiple lines of evidence suggest that the control of the L2-to-L3 transition by mir-48, mir-84, and mir-241 does not occur through regulation of lin-28 activity. (1) A lin-28::gfp::lin-28 reporter transgene that recapitulates the wild-type temporal regulation of LIN-28 protein and that rescues the phenotype of lin-28(lf) worms is not derepressed in mir-48 mir-241; mir-84 triple mutants. (2) It was found that the level of endogenous LIN-28 protein was not significantly elevated in mir-48 mir-241; mir-84 triple mutants, whereas, in lin-4 retarded mutants, LIN-28 protein is abnormally abundant at later larval stages. (3) Two alleles of lin-58 that contain mutations upstream of mir-48, and hence lead to the misexpression of mir-48, enhance the precocious phenotype of a lin-28 null mutant, indicating that mir-48 does not act exclusively through lin-28. (4) It was found that the L2 reiteration phenotype of mir-48 mir-241; mir-84 triple mutants could occur independently of lin-28 activity. These data together indicate that mir-48, mir-84, and mir-241 control the L2-to-L3 transition primarily through downstream effectors other than lin-28, even though the lin-28 3′ UTR contains a let-7 complementary site. It is possible that the let-7 family microRNAs may contribute to the repression of lin-28 expression, but to a degree undetectable by the assays used (Abbott, 2005).

Genetic epistasis analysis indicates that mir-48, mir-84, and mir-241 function in parallel with the lin-28 and lin-46 pathway to downregulate hbl-1 activity and hence control the L2-to-L3 transition. One model to account for this convergence of pathways on hbl-1 would be that LIN-46, in its putative role as a scaffolding protein, could control assembly of a protein complex that directly interacts with HBL-1 protein to inhibit its activity in parallel with the repression of hbl-1 mRNA translation exerted by mir-48, mir-84, and mir-241. Alternatively, LIN-46 could interact with RNA binding protein(s) and directly potentiate the activity of the mir-48, mir-84, and mir-241 microRNAs (Abbott, 2005).

These data suggest that mir-48, mir-84, and mir-241 control developmental timing in two physically associated but distinct cell types in the hypodermis: the postmitotic main body hypodermal syncytial cell, hyp7, and the proliferative seam cells. Two lines of evidence point to a role in hyp7 for mir-48, mir-84, and mir-241 to repress hbl-1 and control hyp7 temporal behavior. (1) mir-48; mir-84 mutant worms displayed heterochronic defects in hyp7; the expression of the adult-specific transgene col-19::gfp was retarded in hyp7 but was regulated normally in the seam cells. Thus, the supernumerary molt observed in mir-48; mir-84 double mutants may be a consequence of a heterochronic defect in hyp7. (2) The data indicate that mir-48, mir-84, and mir-241 act in hyp7 to repress hbl-1 activity. In mir-48 mir-241; mir-84 worms, hbl-1::gfp::hbl-1 was misregulated in hyp7. Thus, the 3′ UTR-dependent downregulation of hbl-1::gfp::hbl-1 in hyp7 that occurs in wild-type animals can be accounted for largely by the regulation of hbl-1 by mir-48, mir-84, and mir-241 (Abbott, 2005).

mir-48, mir-84, and mir-241 may also function in the hypodermal seam cells to control developmental timing. Reduction of hbl-1 activity genetically or by hbl-1 RNAi affected stage-specific behavior of seam cells, resulting in suppression of the retarded seam cell and alae phenotypes of mir-48 mir-241; mir-84 worms. This could be a consequence of the repression of hbl-1 by mir-48, mir-84, and mir-241 in the seam cells. Interestingly, hbl-1::gfp::hbl-1 cannot be detected in the seam cells after the L1 stage, suggesting that, at the time of the L2-to-L3 transition, the amount of hbl-1 expression in seam cells is relatively low. Thus, mir-48, mir-84, and mir-241 may function cell autonomously in the seam cells at the L3 stage to downregulate hbl-1, albeit beginning from a level already below the threshold of detection by the assays. Alternatively, since repression of hbl-1::gfp::hbl-1 is readily observed at the L2-to-L3 transition in hyp7 (the main body hypodermal syncytial cell), it is conceivable that the stage-specific behavior of seam cells may be controlled non-cell autonomously by a hbl-1-regulated signal from hyp7. Non-cell autonomous signaling from hyp7 to neighboring cells has been proposed in the pathway to specify the fates of vulval precursor cells (VPCs). Mosaic analyses suggest that the sites of action of the multivulva (Muv) gene locus lin-15 and of the synthetic Muv genes lin-37 and lin-35 are in hyp7. One model is that hyp7 generates a signal to neighboring VPCs to inhibit vulval cell fate specification. Similarly, a signal from hyp7 to the lateral hypodermal seam cells may regulate the temporal behavior of seam cells and thereby help coordinate developmental timing throughout the hypodermis (Abbott, 2005).

In summary, the results presented in this study demonstrate a role for the let-7 family microRNA genes mir-48, mir-84, and mir-241 in the heterochronic pathway to control the L2-to-L3 cell fate transitions in the hypodermis. Proper progression through the L1 and L2 larval stages requires downregulation of lin-14 and lin-28, primarily through the action of the microRNA lin-4. These findings extend the involvement of microRNAs in the regulation of C. elegans developmental timing to include a requirement for the downregulation of hbl-1 by the combined action of the three let-7 family microRNAs, mir-48, mir-84, and mir-241, in the hypodermis. The L2-to-L3 transition is controlled by complex genetic mechanisms involving two microRNA-regulated pathways that converge on hbl-1: the lin-4, lin-28, lin-46 pathway and the mir-48, mir-84, mir-241 pathway. These parallel inputs to hbl-1 may serve to couple hbl-1 downregulation to distinct upstream temporal signals. Further, the functional redundancy among mir-48, mir-84, and mir-241 could reflect alternative mechanisms for triggering the L2-to-L3 transition throughout the hypodermis. mir-48, mir-84, and mir-241 seem to have more minor roles, compared to let-7, at the L4-to-adult transition in the hypodermis, indicating that different microRNA family members can be deployed for distinct roles, perhaps through differences in temporal or spatial expression patterns and/or differences in target specificity. These findings suggest analogous forms of genetic redundancy and regulatory complexity may be expected in pathways involving other families of related microRNAs (Abbott, 2005).

Regulatory mutations of mir-48, a C. elegans let-7 family microRNA, cause developmental timing defects

The C. elegans heterochronic genes program stage-specific temporal identities in multiple tissues during larval development. These genes include the first two miRNA-encoding genes discovered, lin-4 and let-7. lin-58 alleles, identified as lin-4 suppressors, define another miRNA that controls developmental time. These alleles are unique in that they contain point mutations in a gene regulatory element of mir-48, a let-7 family member. mir-48 is expressed prematurely in lin-58 mutants, whereas expression of mir-241, another let-7 family member residing immediately upstream of mir-48, appears to be unaffected. A mir-48 transgene bearing a lin-58 point mutation causes strong precocious phenotypes in the hypodermis and vulva when expressed from multicopy arrays. mir-48::gfp fusions reveal expression in these tissues, and inclusion of a lin-58 mutation causes precocious and enhanced gfp expression. These results suggest that lin-58 alleles disrupt a repressor binding site that restricts the time of miR-48 action in wild-type animals (Li, 2005).

The lin-58 mutations described here appear to reveal a negative regulatory element that prevents mir-48 accumulation until the proper time during the mid-to-late L1 stage. Intriguingly, the 11 bp inverted repeat disrupted by lin-58 mutations spans a 7-8 bp match to a computationally identified consensus motif (5′-CTCCGCCC-3′; underlined residues are mutated in lin-58 alleles) found 5′ to most worm miRNA genes that are independently expressed. A perfect match to this sequence also resides another ~500 bp upstream. The functional significance of the motif, and whether it relates directly to the repressive element defined by lin-58 lesions, is as yet unclear. Replacement of the entire 11 bp inverted repeat in mir-48::gfp with the AT sequence has the same effect upon GFP expression as does insertion of the ve33 point mutation; it results in enhanced and precocious expression, indicating that the GC repeat is not required for mir-48 transcriptional activation. In addition, the lin-58(ve33) and lin-58(ve12) point mutations cause precocious accumulation of miR-48, but do not appear to interfere with the processing of pre-miR-48, suggesting that the site is not required for recruitment of RNA processing machinery. Thus, these data are consistent with a model in which the lin-58 lesions disrupt a repressor binding site that acts to restrict the timing of microRNA action (Li, 2005).

The mir-84 and let-7 paralogous microRNA genes of Caenorhabditis elegans direct the cessation of molting via the conserved nuclear hormone receptors NHR-23 and NHR-25

The let-7 microRNA (miRNA) gene of Caenorhabditis elegans controls the timing of developmental events. let-7 is conserved throughout bilaterian phylogeny and has multiple paralogs. The paralog mir-84 acts synergistically with let-7 to promote terminal differentiation of the hypodermis and the cessation of molting in C. elegans. Loss of mir-84 exacerbates phenotypes caused by mutations in let-7, whereas increased expression of mir-84 suppresses a let-7 null allele. Adults with reduced levels of mir-84 and let-7 express genes characteristic of larval molting as they initiate a supernumerary molt. mir-84 and let-7 promote exit from the molting cycle by regulating targets in the heterochronic pathway and also nhr-23 and nhr-25, genes encoding conserved nuclear hormone receptors essential for larval molting. The synergistic action of miRNA paralogs in development may be a general feature of the diversified miRNA gene family (Hayes, 2006).

The C. elegans genes nhr-23 and nhr-25 encode orphan nuclear hormone receptors orthologous, respectively, to DHR3 and ßFTZ-F1, which are related to mammalian ROR/RZR/RevErb and SF-1, respectively. Both receptors are essential for completion of the larval molts, suggesting that particular functions of nhr-23/DHR3 and nhr-25/ ßFTZ-F1 might be conserved and, further, that regulation by steroid hormones might be a common feature of molting in C. elegans and Drosophila. However, a steroid hormone regulating molting of C. elegans has not yet been identified and the genome lacks orthologs of ECR or USP (Hayes, 2006).

A genetic model is presented for the function of mir-84 and let-7 in epithelial differentiation, as related to the molting cycle. The let-7 miRNA targets lin-41 mRNA and also hbl-1 mRNA, in combination with paralogous miRNAs. During early larval development, LIN-41 and HBL-1 together repress production of the zinc-finger transcription factor LIN-29. Expression of let-7 and related miRNAs late in larval development represses lin-41 and hbl-1, thereby activating LIN-29. LIN-29 promotes expression of col-19 and possibly other collagen genes characteristic of an adult cuticle and also represses expression of col-17 and possibly other collagen genes characteristic of larval cuticle. LIN-29 is likely to regulate additional genes that control the molting cycle that have not yet been identified (Hayes, 2006).

Inactivation of either one of the nuclear hormone receptor genes nhr-23 or nhr-25 is sufficient to prevent the aberrant supernumerary molt caused by reduced levels of mir-84 and let-7. NHR-23 and NHR-25 thus serve as key downstream effectors of the miRNAs in regulation of the molting cycle. One model is that LIN-29, or a transcription factor regulated by LIN-29, represses nhr-23 and nhr-25 following the fourth molt. Accordingly, GFP expression from an nhr-23 reporter gene increases fourfold in the hypodermis of let-7 mir-84 adults. The relationship between nhr-23 and nhr-25 in C. elegans remains to be determined; however, DHR3 stimulates transcription of ßFTZ-F1 in flies (Hayes, 2006).

The identification of sites in the 3' UTR of nhr-25 that are complementary to let-7 family members and are also conserved in other nematodes suggests that the let-7 family targets the nhr-25 message to negatively regulate production of NHR-25 in adults. Consistent with this model, increasing the abundance of mir-84 partly suppresses the supernumerary molt caused by a probable null mutation in the lin-29 gene. Also, in preliminary experiments RNA species attributable to cleavage of the nhr-25 message upon binding of let-7-like miRNAs were detected in extracts from wildtype adults. Steroid hormones and co-factors probably also regulate activity of NHR-23 and NHR-25 during the life cycle (Hayes, 2006).

Regulation by miRNAs thus converges on transcription factors upstream in the genetic networks regulating molting. NHR-23 coordinates several aspects of larval molting by promoting expression of genes required for patterning the new cuticle and ecdysis, including, respectively, the collagen gene dpy-7 and the collagenase gene nas-37. Inactivation of either nhr-23 or nhr-25 abrogates the reiterated expression of gfp reporters for mlt-10 and nas-37 caused by mutation of let-7 and mir-84. NHR-25 might promote expression of the corresponding genes during larval development, even though RNAi of nhr-25 is not sufficient to abrogate expression of the gfp reporters in wild-type larvae. Interestingly, inactivation of nhr-23 or nhr-25 causes an earlier blockade in the molting program in let-7 mir-84 adults than in wild-type larvae, such that the mutant adults do not enter lethargus or attempt to ecdyse. Parallel pathways might drive early steps of molting during larval development (Hayes, 2006).

Intriguingly, adults with reduced levels of mir-84 and let-7 are unable to shed their cuticle to complete the supernumerary molt. One possibility is that particular genes required for ecdysis are not induced. Whereas the hypodermis and seam cells retain some larval character in let-7 mir-84 adults, other cells, perhaps particular neurons or specialized epithelia, might be fully differentiated and therefore unable to coordinate with the molting program. Consistent with this idea, let-7 mir-84 adults spend an atypically long time in lethargus, suggesting a failure to exit the behavioral program. Alternatively, particular structural features of the fifth cuticle might be physically incompatible with shedding the exoskeleton (Hayes, 2006).

Considering an aberrant ecdysis as the terminal phenotype of let-7 mir-84 mutants, it is intriguing to speculate that the let-7 family and possibly other miRNAs regulate aspects of the larval molting cycle. Indeed, increased expression of either mir-84 or let-7 causes some larvae to arrest development, trapped inside partly shed cuticle, indicating that levels of let-7-like miRNAs can impact molting of larvae (Hayes, 2006).

Mechanisms that set the pace of the molting cycle are not well understood, although physiologic cues such as nutritional status and environmental cues such as temperature impact the duration of larval stages. Interestingly, let-7 and let-7 mir-84 mutants initiate the supernumerary molt in synchrony, rather than in a stochastic fashion, relative to the time of hatching. Thus, a timing mechanism for molting persists in these particular miRNA mutants (Hayes, 2006).

The let-7 gene is perfectly conserved throughout bilaterian phylogeny, and vertebrate genomes specify many miRNAs homologous to let-7. Vertebrate let-7 and protein-coding genes orthologous to targets of let-7 identified in C. elegans play crucial roles in development. Moreover, reduced expression of human let-7 correlates with shortened survival in lung cancer patients, and let-7 might regulate the RAS oncogene. The possibility of functional conservation among homologs of let-7 in humans and worms intimates the importance of understanding how let-7 and its paralogs function in C. elegans. This work shows how analysis of double mutants can reveal how the many miRNAs that form paralogous families work together to regulate their targets (Hayes, 2006).

A genome-wide map of conserved microRNA targets in C. elegans

Metazoan miRNAs regulate protein-coding genes by binding the 3' UTR of cognate mRNAs. Identifying targets for the 115 known C. elegans miRNAs is essential for understanding their function. By using a new version of PicTar and sequence alignments of three nematodes, it is predicted that miRNAs regulate at least 10% of C. elegans genes through conserved interactions. A new experimental pipeline was developed to assay 3' UTR-mediated posttranscriptional gene regulation via an endogenous reporter expression system amenable to high-throughput cloning, demonstrating the utility of this system using one of the most intensely studied miRNAs, let-7. Expression analyses uncover several new potential let-7 targets and suggest a new let-7 activity in head muscle and neurons. To explore genome-wide trends in miRNA function, functional categories of predicted target genes were analyzed; one-third of C. elegans miRNAs target gene sets are enriched for specific functional annotations. miRNA target predictions were integrated with other functional genomic data from C. elegans. At least 10% of C. elegans genes are predicted miRNA targets, and a number of nematode miRNAs seem to regulate biological processes by targeting functionally related genes. An in vivo system was developed for testing miRNA target predictions in likely endogenous expression domains. The thousands of genome-wide miRNA target predictions for nematodes, humans, and flies are available from the PicTar website and are linked to an accessible graphical network-browsing tool allowing exploration of miRNA target predictions in the context of various functional genomic data resources (Lall, 2006).

To molecularly test PicTar predictions of let-7 targets, 12 novel putative targets were selected without input from the phenotypic suppression test. T14B1.1 (a novel gene, PicTar rank 2) and unc-129 (a TGF-β homolog, rank 19) were tested as targets of let-7. The T14B1.1 3′ UTR contains several predicted conserved let-7 sites. T14B1.1 reporter constructs are expressed in multiple tissues including head neurons and the hypodermis, consistent with in situ hybridization data. Strikingly, a reporter gene carrying the T14B1.1 3′ UTR is expressed in main body hypodermal tissue during the L2 and early L3 larval stages, but hypodermal expression decreases dramatically during the L4 stage, consistent with the appearance of high levels of mature let-7. Decreased expression depends on the T14B1.1 3′ UTR, since the decrease in expression is alleviated when the unc-54 3′ UTR is substituted. These observations are consistent with the hypothesis that T14B1.1 is repressed by let-7. T14B1.1 is a novel gene that has no known RNAi phenotype and cannot suppress the let-7 vulval bursting phenotype; thus, these results also illustrate that a combined computational and experimental pipeline can identify targets that may not have been found by conventional experimental means, such as a genetic suppressor screen (Lall, 2006).

unc-129, a TGF-β homolog shown to be involved in axon guidance and which contains two predicted let-7 sites in its 3′ UTR, was tested. By using upstream sequence from the unc-129 locus, expression was observed in head muscle and ventral motor neurons, but not in body wall muscle. Expression in head cells decreases in late larval stages, concomitant with a rise in let-7 levels. This decrease is mildly alleviated when the unc-129 3′ UTR is replaced with the unc-54 3′ UTR (Lall, 2006).

Upregulation of the let-7 microRNA with precocious development in lin-12/Notch hypermorphic Caenorhabditis elegans mutants

The lin-12/Notch signaling pathway is conserved from worms to humans and is a master regulator of metazoan development. lin-12/Notch gain-of-function (gf) animals display precocious alae at the L4 larval stage with a significant increase in let-7 expression levels. Furthermore, lin-12(gf) animals display a precocious and higher level of let-7 gfp transgene expression in seam cells at L3 stage. Interestingly, lin-12(gf) mutant rescued the lethal phenotype of let-7 mutants similar to other known heterochronic mutants. It is proposed that lin-12/Notch signaling pathway functions in late developmental timing, upstream of or in parallel to the let-7 heterochronic pathway. Importantly, the human microRNA let-7a was also upregulated in various human cell lines in response to Notch1 activation, suggesting an evolutionarily conserved cross-talk between let-7 and the canonical lin-12/Notch signaling pathway (Solomon, 2008).

Ascl1a regulates Müller glia dedifferentiation and retinal regeneration through a Lin-28-dependent, let-7 microRNA signalling pathway

Unlike mammals, teleost fish mount a robust regenerative response to retinal injury that culminates in restoration of visual function. This regenerative response relies on dedifferentiation of Müller glia into a cycling population of progenitor cells. However, the mechanism underlying this dedifferentiation is unknown. This paper reports that genes encoding pluripotency factors are induced following retinal injury. Interestingly, the proneural transcription factor, Ascl1a, and the pluripotency factor, Lin-28, are induced in Müller glia within 6 h following retinal injury and are necessary for Müller glia dedifferentiation. Ascl1a is necessary for lin-28 expression, and Lin-28 suppresses let-7 microRNA (miRNA) expression. Furthermore, let-7 represses expression of regeneration-associated genes such as, ascl1a, hspd1, lin-28, oct4, pax6b and c-myc. hspd1, oct4 and c-myc(a) exhibit basal expression in the uninjured retina and let-7 may inhibit this expression to prevent premature Müller glia dedifferentiation. The opposing actions of Lin-28 and let-7 miRNAs on Müller glia differentiation and dedifferentiation are similar to that of embryonic stem cells and suggest novel targets for stimulating Müller glia dedifferentiation and retinal regeneration in mammals (Ramachandran, 2010).

Characterization of let-7 in mammalian cells

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

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).

HMGA2, a high-mobility group protein, is oncogenic in a variety of tumors, including benign mesenchymal tumors and lung cancers. Knockdown of Dicer in HeLa cells revealed that the HMGA2 gene is transcriptionally active, but its mRNA is destabilized in the cytoplasm through the microRNA (miRNA) pathway. HMGA2 is derepressed upon inhibition of let-7 in cells with high levels of the miRNA. Ectopic expression of let-7 reduces HMGA2 and cell proliferation in a lung cancer cell. The effect of let-7 on HMGA2 is dependent on multiple target sites in the 3' untranslated region (UTR), and the growth-suppressive effect of let-7 on lung cancer cells is rescued by overexpression of the HMGA2 ORF without a 3'UTR. These results provide a novel example of suppression of an oncogene by a tumor-suppressive miRNA and suggest that some tumors activate the oncogene through chromosomal translocations that eliminate the oncogene’s 3'UTR with the let-7 target sites (Lee, 2007).

HuR recruits let-7/RISC to repress c-Myc expression

RNA-binding proteins (RBPs) and microRNAs (miRNAs) are potent post-transcriptional regulators of gene expression. This study shows that the RBP HuR reduces c-Myc expression by associating with the c-Myc 3' untranslated region (UTR) next to a miRNA let-7-binding site. Lowering HuR or let-7 levels relieves the translational repression of c-Myc. Unexpectedly, HuR and let-7 repressed c-Myc through an interdependent mechanism; let-7 requires HuR to reduce c-Myc expression and HuR required let-7 to inhibit c-Myc expression. These findings suggest a regulatory paradigm wherein HuR inhibits c-Myc expression by recruiting let-7-loaded RISC (RNA miRNA-induced silencing complex) to the c-Myc 3'UTR (Kim, 2009).

An epigenetic switch involving NF-kappaB, Lin28, Let-7 MicroRNA, and IL6 links inflammation to cell transformation

Inflammation is linked clinically and epidemiologically to cancer, and NF-kappaB appears to play a causative role, but the mechanisms are poorly understood. An experimental model of oncogenesis is described involving a derivative of MCF10A, a spontaneously immortalized cell line derived from normal mammary epithelial cells, that contains ER-Src, a fusion of the Src kinase oncoprotein (v-Src) and the ligand binding domain of the estrogen receptor. Treatment of these cells with estrogen receptor antagonist tamoxifen (TAM) for 36 hr results in phenotypic transformation, formation of multiple foci, the ability to form colonies in soft agar, increased motility and invasive ability, and tumor formation upon injection in nude mice. This model permits the opportunity to kinetically follow the pathway of cellular transformation in a manner similar to that used to study viral infection and other temporally ordered processes. Transient activation of Src oncoprotein can mediate an epigenetic switch from immortalized breast cells to a stably transformed line that forms self-renewing mammospheres that contain cancer stem cells. Src activation triggers an inflammatory response mediated by NF-kappaB that directly activates Lin28 transcription and rapidly reduces let-7 microRNA levels. Let-7 directly inhibits IL6 expression, resulting in higher levels of IL6 than achieved by NF-kappaB activation. IL6-mediated activation of the STAT3 transcription factor is necessary for transformation, and IL6 activates NF-kappaB, thereby completing a positive feedback loop. This regulatory circuit operates in other cancer cells lines, and its transcriptional signature is found in human cancer tissues. Thus, inflammation activates a positive feedback loop that maintains the epigenetic transformed state for many generations in the absence of the inducing signal (Iliopoulos, 2009).

The TRIM-NHL protein TRIM32 activates microRNAs and prevents self-renewal in mouse neural progenitors

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).

Lin28 enhances tissue repair by reprogramming cellular metabolism

Regeneration capacity declines with age, but why juvenile organisms show enhanced tissue repair remains unexplained. Lin28a, a highly conserved RNA-binding protein expressed during embryogenesis, plays roles in development, pluripotency, and metabolism. To determine whether Lin28a might influence tissue repair in adults, the reactivation of Lin28a expression was engineered in several models of tissue injury. Lin28a reactivation improved hair regrowth by promoting anagen in hair follicles and accelerated regrowth of cartilage, bone, and mesenchyme after ear and digit injuries. Lin28a inhibits let-7 microRNA biogenesis; however, let-7 repression was necessary but insufficient to enhance repair. Lin28a bound to and enhanced the translation of mRNAs for several metabolic enzymes, thereby increasing glycolysis and oxidative phosphorylation (OxPhos). Lin28a-mediated enhancement of tissue repair was negated by OxPhos inhibition, whereas a pharmacologically induced increase in OxPhos enhanced repair. Thus, Lin28a enhances tissue repair in some adult tissues by reprogramming cellular bioenergetics (Shyh-Chang, 2013).

Mechanism of Dis3l2 substrate recognition in the Lin28-let-7 pathway

The pluripotency factor Lin28 inhibits the biogenesis of the let-7 family of mammalian microRNAs. Lin28 is highly expressed in embryonic stem cells and has a fundamental role in regulation of development, glucose metabolism and tissue regeneration. Overexpression of Lin28 is correlated with the onset of numerous cancers, whereas let-7, a tumour suppressor, silences several human oncogenes. Lin28 binds to precursor let-7 (pre-let-7) hairpins, triggering the 3' oligo-uridylation activity of TUT4 and TUT7. The oligoU tail added to pre-let-7 serves as a decay signal, as it is rapidly degraded by the exonuclease Dis3l2, a homologue of the catalytic subunit of the RNA exosome. The molecular basis of Lin28-mediated recruitment of TUT4 and TUT7 to pre-let-7 and its subsequent degradation by Dis3l2 is largely unknown. To examine the mechanism of Dis3l2 substrate recognition this study determined the structure of mouse Dis3l2 in complex with an oligoU RNA to mimic the uridylated tail of pre-let-7. Three RNA-binding domains form an open funnel on one face of the catalytic domain that allows RNA to navigate a path to the active site different from that of its exosome counterpart. The resulting path reveals an extensive network of uracil-specific interactions spanning the first 12 nucleotides of an oligoU-tailed RNA. This study identified three U-specificity zones that explain how Dis3l2 recognizes, binds and processes uridylated pre-let-7 in the final step of the Lin28-let-7 pathway (Faehnle, 2014).

LIN28 zinc knuckle domain is required and sufficient to induce let-7 oligouridylation

LIN28 (see Drosophila Lin28) is an RNA binding protein that plays crucial roles in pluripotency, glucose metabolism, tissue regeneration, and tumorigenesis. LIN28 binds to the let-7 (see Drosophila let-7) primary and precursor microRNAs through bipartite recognition and induces degradation of let-7 precursors (pre-let-7) by promoting oligouridylation by terminal uridylyltransferases (TUTases; see Drosophila Tailor). This study report that the zinc knuckle domain (ZKD) of mouse LIN28 recruits TUT4 to initiate the oligouridylation of let-7 precursors. Crystal structure of human LIN28 in complex with a fragment of pre-let-7f-1 determined to 2.0 Å resolution shows that the interaction between ZKD and RNA is constrained to a small cavity with a high druggability score. The specific interaction between ZKD and pre-let-7 was shown to be necessary and sufficient to induce oligouridylation by recruiting the N-terminal fragment of TUT4 (NTUT4) and the formation of a stable ZKD:NTUT4:pre-let-7 ternary complex is crucial for the acquired processivity of TUT4 (Wang, 2017).

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Search PubMed for articles about Drosophila let-7

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