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).
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).
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).
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 loqsf00791 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).
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).
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).
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).
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'UTRmut) 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'UTRWT 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'UTRWT expression in the presence of let-7, relative to control miRNA. A gfp-Imp-3'UTRmut 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 (ImpT21) 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).
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