RNAi is a gene-silencing phenomenon triggered by double-stranded (ds) RNA and involves the generation of 21 to 26 nt RNA segments that guide mRNA destruction. In Caenorhabditis elegans, lin-4 and let-7 encode small temporal RNAs (stRNAs) of 22 nt that regulate stage-specific development. Inactivation of genes related to RNAi pathway genes, a homolog of Drosophila Dicer (dcr-1), and two homologs of rde-1 (alg-1 and alg-2), cause heterochronic phenotypes similar to lin-4 and let-7 mutations. dcr-1, alg-1, and alg-2 are necessary for the maturation and activity of the lin-4 and let-7 stRNAs. These findings suggest that a common processing machinery generates guide RNAs that mediate both RNAi and endogenous gene regulation (Grishok, 2001).
Genetic studies in C. elegans have identified several genes essential for RNA interference. Probable null mutations in rde-1 (for RNAi defective) cause a complete lack of RNAi but no other discernible phenotypes. rde-1 encodes a 1020 amino acid protein that is a member of a large family of proteins found in a wide range of eukaryotes. Members of the RDE-1 family have two conserved domains of unknown biochemical function. The 300 amino acid PIWI domain located in the C-terminal region of these homologs shows the highest degree of sequence conservation. The 110 amino acid PAZ domain is located N-terminal to the PIWI domain and is also found in the Dicer family of proteins. RDE-1 homologs in the fungus, Neurospora, and the plant, Arabidopsis, have also been implicated in PTGS (post-transcriptional gene silencing) mechanisms suggesting that RDE-1 family members not only share conserved structures but also have conserved functions in gene silencing in three kingdoms of eukaryotic organisms (Grishok, 2001 and references therein).
Mutations in rde-1 homologs have also been shown to have developmental consequences. For example, in Drosophila, the ago1 gene is required for embryogenesis (Kataoka, 2001), the piwi gene is required for the maintenance of the germline stem cell population, and aubergine is required for the proper expression of the germline determinant Oskar (Wilson, 1996). Additionally, aubergine (also known as Sting) has been implicated in the PTGS-like suppression of the repetitive Stellate locus in the Drosophila germline (Schmidt, 1999). In Arabidopsis two very similar genes, argonaute (ago1) and pinhead/zwille, are required for stem cell patterning of the plant meristem. argonaute is also necessary for PTGS in Arabidopsis. The C. elegans genome contains 23 homologs of rde-1 including orthologs of both piwi and ago1. Previous studies have shown that the C. elegans piwi and ago1 orthologs have germline and possibly additional developmental functions. The pleiotropic nature of the defects associated with loss-of-function mutations in members of this family could reflect discrete regulatory functions in numerous developmental events or alternatively might reflect a more general misregulation of silencing mechanisms that are necessary to insure proper stem cell maintenance and differentiation (Grishok, 2001 and references therein).
The combination of vulval and adult cuticle maturation defects caused by RNAi of alg-1/alg-2 and dcr-1 is reminiscent of phenotypes resulting from mutations in the genes lin-4 and let-7. The lin-4 and let-7 genes promote transitions from earlier to later cell fates and, thus, mutations in these genes cause reiteration of cell divisions typical of earlier larval stages, a hallmark of genes that regulate developmental timing (such genes have been termed 'heterochronic genes'). For example, loss-of-function mutations in let-7 result in a failure of larval seam cells in the hypodermis to progress to the adult-specific program of terminal differentiation indicated by the production of the adult-specific alae -- instead, the cells repeat the late larval type of divisions. These reiterated divisions contribute to an unstable vulval structure and failure to form a cuticle with adult alae (Grishok, 2001).
The similarity of phenotypes described above to those of the heterochronic genes lin-4 and let-7 raised the possibility that alg-1, alg-2, and dcr-1 might act upstream of the lin-4 or let-7 stRNAs or might be necessary for their regulatory activities. lin-4 and let-7 are expressed as longer, approximately 70 nt RNAs that are predicted to fold into structures containing regions of double-stranded RNA. Because Drosophila Dicer cleaves introduced dsRNAs into fragments of approximately 22 nt (Bernstein, 2001), it was hypothesized that the heterochronic phenotypes caused by dcr-1 (RNAi) may be due to a defect in the processing of the larger, potentially dsRNA, forms of lin-4 and let-7 into the 22 nt stRNAs. To test this idea progeny were collected from mothers subjected to dcr-1(RNAi) and Northern blot analyses were performed to monitor the size and abundance of the lin-4 and let-7 RNAs. Because alg-1/alg-2 (RNAi) causes a similar heterochronic phenotype but acts at an unknown step in the pathway, lin-4 and let-7 processing were also monitored in alg-1/alg-2 (RNAi) animals (Grishok, 2001).
Both dcr-1 and alg-1/alg-2(RNAi) animals exhibited a marked accumulation of the lin-4 long form at both L3-L4 and adult stages. The same RNA preparations from the dcr-1 or alg-1/alg-2 (RNAi) animals were probed for the expression of let-7. It was found that, as with lin-4, let-7 processing depends on dcr-1 activity but, in contrast, does not appear to depend on alg-1/alg-2 activity. lin-4 and let-7 stRNA processing were monitored in dcr-1(ok247) homozygotes and in animals specifically depleted for either alg-1 or alg-2. In this experiment RNAs prepared from each population were simultaneously probed for expression of lin-4 and let-7 RNA. As with dcr-1(RNAi), the ok247 homozygotes exhibit a significant accumulation of both lin-4 and let-7 long forms. A gene-specific dsRNA targeting alg-1 induces accumulation of the pre-lin-4 RNA but not pre-let-7, and similarly, alg-2(ok304) animals exhibits a slight accumulation of pre-lin-4 and little or no accumulation of pre-let-7 (Grishok, 2001).
The quantity of the short forms of the lin-4 and let-7 stRNAs consistently appeared to be reduced in RNA populations prepared from alg-1/alg-2(RNAi), dcr-1(RNAi), and dcr-1(ok247) animals, while control RNA populations prepared from animals undergoing RNAi of the cuticle collagen gene rol-6 exhibited normal levels of lin-4 and let-7 stRNAs. This apparent reduction in let-7 stRNA level was observed even in alg-1/alg-2(RNAi) populations where no significant accumulation of pre-let-7 was observed. These findings suggest that alg-1/alg-2 activities may be more important for the stability or function of let-7 stRNA than for its processing from the larger form. Alternatively, alg-1/alg-2 might also be involved in let-7 processing but the let-7 long form may be less stable, so that unprocessed let-7 does not accumulate in the absence of alg-1/alg-2 activity (Grishok, 2001).
Thus, the efficient processing of the lin-4 and let-7 stRNAs from larger precursors depends on the activity of DCR-1, a C. elegans homolog of the Drosophila multifunctional RNase III related protein, Dicer, that has been shown in Drosophila cell extracts to process dsRNA into siRNAs that can mediate RNAi (Bernstein, 2001). Further, alg-1 and alg-2, two homologs of the RNAi pathway gene rde-1, are required for efficient stRNA expression, and along with dcr-1 function to promote lin-4 and let-7 activities in temporal development. Thus, the expression of the tiny RNAs that mediate RNAi and developmental gene regulation appear to share a requirement for DCR-1 activity, while RDE-1 and its homologs provide parallel functions in these pathways. These findings are consistent with a model in which members of the RDE-1 and DCR-1 families act not only in gene silencing but also with naturally expressed dsRNAs to execute cellular and developmental gene regulatory events (Grishok, 2001).
Although there are compelling similarities between RNAi and developmental regulation by lin-4 and let-7 there are also several important differences. In RNAi, the dsRNAs utilized, typically contain long stretches of perfect base pairing. The stRNA precursors, however, are predicted to contain at most 6, for lin-4, and 13, for let-7, uninterrupted Watson-Crick base pairs. Whereas cleavage of the perfectly base-paired RNAs that initiate RNAi yields both sense and antisense, or potentially double-stranded siRNAs, only one strand of the lin-4 and let-7 stRNAs is detected. Thus, after generation of the mature stRNA, the remaining sequences must undergo rapid degradation (Grishok, 2001).
The RNAi and stRNA pathways also appear to induce distinct outcomes: RNA destruction versus translation inhibition. In RNAi the target mRNA is rapidly degraded. Although the RNase responsible for target RNA destruction is not yet known, it is thought that the antisense strand of the siRNA acts as a guide in mRNA destruction, by base-pairing with the target mRNA. The stRNAs also specifically downregulate the expression of their target genes. Although details of the mechanism by which stRNAs cause decreased expression are unknown, the regulation of lin-14 by lin-4 occurs at the translational level. Upon expression of lin-4 RNA, the levels of LIN-14 protein rapidly decline, but lin-14 mRNA levels remain constant and appear to remain associated with polyribosomes. Because let-7-mediated regulation of LIN-41 protein expression may only occur in a subset of cells, it is, as yet, unclear if the mRNA levels or polyribosome loading of this target is affected by the expression of let-7 RNA (Grishok, 2001).
A role for RDE-1 family members in both small RNA production and targeting could explain why the inhibition of alg-1/alg-2 induces such a dramatic effect on lin-4 and let-7 function while at best reducing but not eliminating the processed stRNA. Similarly, recent studies of small RNA accumulation during RNAi suggest that rde-1 is not essential for small RNA production after exposure to dsRNA and yet rde-1(+) activity is absolutely required for interference. Conceivably, dsRNA processing might still occur in the absence of RDE-1 or its homologs but the resulting siRNAs or stRNAs may not be assembled into the appropriate downstream complexes and therefore fail to function. Nevertheless, the finding that alg-1/alg-2(RNAi) dramatically affects the accumulation of the lin-4 precursor supports a role for these factors either upstream of, or at the same step as DCR-1 (Grishok, 2001).
The combination of a maternally provided dcr-1 activity and zygotic sterility make it difficult to unambiguously answer the question of whether this protein is absolutely essential for RNAi and stRNA pathways. Nevertheless, the reiteration of L2 fates revealed by the seam cell lineage analysis of dcr-1(RNAi) animals, and the suppression of those phenotypes by mutations in lin-14 or lin-41 are unique phenotypic and genetic signatures that strongly support the model where lin-4 and let-7 processing is dependent on dcr-1(+) activity. Perhaps the embryonic and larval lethal phenotypes associated with dcr-1 inhibition and the developmental phenotypes associated with the Arabidopsis homolog, caf 1, reflect a role for members of this gene family in the processing of other as yet unidentified small regulatory RNAs. Thus, tiny RNAs may function in a broader range of gene regulatory and developmental events than the temporal transitions mediated by the founding members of the class, the lin-4 and let-7 stRNAs (Grishok, 2001).
Double-stranded RNAs can suppress expression of homologous genes through an evolutionarily conserved process named RNA interference (RNAi) or post-transcriptional gene silencing (PTGS). One mechanism underlying silencing is degradation of target mRNAs by an RNP complex, which contains ~22 nt of siRNAs as guides to substrate selection. A bidentate nuclease called Dicer has been implicated as the protein responsible for siRNA production. This study characterizes the C. elegans ortholog of Dicer (K12H4.8; dcr-1) in vivo and in vitro. dcr-1 mutants show a defect in RNAi. Furthermore, a combination of phenotypic abnormalities and RNA analysis suggests a role for dcr-1 in a regulatory pathway comprised of small temporal RNA (let-7) and its target (e.g., lin-41) (Ketting, 2001).
The let-7 gene product is a small, noncoding RNA that regulates the timing of developmental events in C. elegans (therefore named small temporal RNA or stRNA. Of interest, the let-7 RNA is 21 nt in length, and it has been hypothesized that the let-7 RNA is produced by post-transcriptional processing of a longer precursor that is predicted to form an extended hairpin structure, which may be a substrate for DCR-1. Regulation by let-7 occurs at the translational level and presumably is mediated by complementary base-pairing between let-7 and the 3'-untranslated regions of target genes (Ketting, 2001 and references therein).
One of the in vivo targets of let-7 is lin-41 (Drosophila homolog: dappled), and the increased expression of this protein in let-7 mutants leads to the burst vulva phenotype. Interestingly, dcr-1 homozygous mutants also display a burst vulva phenotype, up to 80% (17/21), which can be rescued by introducing the wild-type dcr-1 gene. Tests were performed to see if this phenotype can be partially suppressed by down-regulating LIN-41 protein through RNAi; and indeed it can: after RNAi of lin-41, only 25% burst vulva (5/20) are found. This suggests that the burst vulva phenotype in dcr-1 mutant animals is at least partially caused by an up-regulation of LIN-41, and the epistatic effect is an indication that dcr-1 and lin-41 indeed act in the same pathway. Conversely, hypomorphic alleles of lin-41 have an Egl phenotype (an egg-laying defect), whereas null alleles of lin-41 are sterile owing to the absence of oocytes. Accordingly, different levels of ectopic expression of DCR-1 might, via down-regulation of lin-41, induce an Egl phenotype or sterility. This is indeed what is found. Although the phenotypes described above are not specific enough to directly imply dcr-1 as an actor in the let-7/lin-41 pathway, the phenotypic relationship between animals with altered DCR-1 levels and animals with alterations in the let-7/lin-41 pathway, are suggestive (Ketting, 2001).
To test this more directly, two approaches were undertaken. (1) Using Drosophila embryo extracts and immunoprecipitates as a source of Dicer, tests were performed to see whether Dicer could process Drosophila let-7 precursor RNA into its mature form in vitro. Indeed, the ~75-nt hairpin was processed into an ~21-nt mature RNA with a disproportionately high efficiency as compared to perfect duplexes of similar size. (2) It was asked whether the dcr-1 mutation had an effect on the levels of let-7 RNA in vivo. Levels of mature let-7 RNA are reduced in dcr-1 mutant animals, and that this reduction is accompanied by an accumulation of the longer let-7 RNA precursor. Together these results show that dcr-1 is directly involved in the conversion of the double-stranded let-7 precursor RNA into the active, 21-nt species (Ketting, 2001).
The mechanisms by which RNAi and stRNAs regulate the expression of target genes are quite distinct. In the former case, mRNAs are destroyed, whereas in the latter, expression is inhibited at the translational level. This raises the possibility that 22-nt RNAs produced by Dicer might act in multiple, distinct regulatory pathways that are not otherwise mechanistically related. Alternatively, the effector machinery may be shared by both processes, with an altered outcome of target recognition. The let-7 RNA is not perfectly homologous to its target substrates, and such a mismatch may inhibit the ability of RISC to cleave its substrates, effectively switching the mode of regulation from degradation to translational repression. It should be noted that let-7 is, most likely, not the only substrate for Dicer that is required for normal development. There may be many other endogenously encoded dsRNAs that are processed by Dicer to produce stRNA molecules, for example, lin-4. For this gene it has been shown that the mismatch between lin-4 and its target is critically required for proper regulation (Ketting, 2001 and references therein).
MicroRNAs (miRNAs) are a large family of small regulatory RNAs that are poorly understood. The let-7 miRNA regulates the timing of the developmental switch from larval to adult cell fates during Caenorhabditis elegans development. Expression of let-7 RNA is temporally regulated, with robust expression in the fourth larval and adult stages. Like let-7 RNA, a transcriptional fusion of the let-7 promoter to gfp is temporally regulated, indicating that let-7 is transcriptionally controlled. Temporal upregulation of let-7 transcription requires an enhancer element, the temporal regulatory element (TRE), situated about 1200 base pairs upstream of the start of the mature let-7 RNA. The TRE is both necessary and sufficient for this temporal upregulation. A TRE binding factor (TREB) is able to bind to the TRE, and a 22-base pair inverted repeat within the TRE is necessary and sufficient for this binding. The nuclear hormone receptor DAF-12 and the RNA binding protein LIN-28 are both required for the correct timing of let-7 RNA and let-7::gfp expression. It is speculated that these heterochronic genes regulate let-7 expression through its TRE (Johnson, 2003).
One model for the action of this cis-acting TRE is one of positive regulation, where in the early larval stages (L1-L3) the TRE is free and unbound by transcriptional activators and does not induce let-7 expression. Upon entering the L3/L4 transition, TREB binds to the IR in the TRE and activates the transcription of let-7. TRE therefore acts as an enhancer element. Since let-7 expression appears coupled to the L3 molt, it is predicted that TREB activity is likely to be regulated by a hormone that signals the molt. TREB could itself be a nuclear hormone receptor or another factor that is regulated by hormonal signaling. An alternative model would be that the TREB is always bound to the TRE and becomes activated by binding of an early-L4-produced hormone or other ligand. Future work will distinguish between these two models and reveal the identity of TREB. Additionally, it is suspected that TREB activity is likely to be controlled by lin-4, lin-14, daf-12, and lin-28, the heterochronic genes upstream of let-7 (Johnson, 2003).
The identity of the TREB is open to speculation, but various nuclear hormone receptors in C. elegans play roles in molting and may correspond to TREB or be activators of TREB. There are approximately 270 nuclear receptor genes found in the C. elegans genome, at least 10 of which have been shown to be expressed in seam cells. All of these genes are potential TREB candidates, but only 1, daf-12, plays a role in the heterochronic pathway. daf-12 indeed regulates the timing of let-7 expression: mutations in daf-12 that result in retarded heterochronic phenotypes cause retarded expression of let-7 and let-7::gfp. A DAF-12::GFP fusion reveals high expression in the hypodermal seam cells, and both the DAF-12::GFP fusion protein and daf-12 mRNA are expressed throughout development, placing DAF-12 in the right place at the right time. One possibility is that DAF-12 regulation of let-7 could be direct with DAF-12 filling the role of the TREB and binding to the TRE to activate the transcription of let-7. However, TREB binding activity is not affected in a daf-12(rh61) mutant, suggesting that DAF-12 may act indirectly in promoting let-7 expression (Johnson, 2003).
This study has addressed some fundamental questions about let-7 regulation in C. elegans, but the answers found might have broader implications. let-7 is a broadly conserved stRNA, and is also a member of the large class of recently discovered miRNAs. miRNAs are nonprotein coding genes that encode mature RNA products of about 20-24 nt in length. Each of these genes is hypothesized to be transcribed as a longer precursor molecule that can fold back on itself to form a hairpin loop. Three newly discovered miRNAs, mir-48, mir-69, and mir-84, have similar temporal expression patterns to let-7, and mir-48 and mir-84 share sequence identity to let-7. The homologies between both the expression patterns and the sequences of let-7, mir-48, and mir-84 suggest a possible common role for these genes and, more strongly, the possibility of common transcriptional regulation. In support of this, possible common promoter elements upstream of these genes have been identified (Johnson, 2003).
The let-7 microRNA is phylogenetically conserved and temporally expressed in many animals. C. elegans let-7 controls terminal differentiation in a stem cell-like lineage in the hypodermis, while human let-7 has been implicated in lung cancer. To elucidate let-7's role in temporal control of nematode development, sequence analysis and reverse genetics were used to identify candidate let-7 target genes. The nuclear hormone receptor daf-12 is a let-7 target in seam cells, while the forkhead transcription factor pha-4 is a target in the intestine. Additional likely targets are the zinc finger protein die-1 and the putative chromatin remodeling factor lss-4. Together with the previous identification of the hunchback ortholog hbl-1 as a let-7 target in the ventral nerve cord, these findings show that let-7 acts in at least three tissues to regulate different transcription factors, raising the possibility of let-7 as a master temporal regulator (Grosshans, 2005).
The let-7 miRNA regulates developmental timing in C. elegans and is an important paradigm for investigations of miRNA functions in mammalian development. This study investigated the role of miRNA precursor processing in the temporal control and lineage specificity of the let-7 miRNA. In situ hybridization (ISH) in E9.5 mouse embryos revealed early induction of let-7 in the developing central nervous system. The expression pattern of three let-7 family members closely resembled that of the brain-enriched miRNAs mir-124, mir-125 and mir-128. Comparison of primary, precursor, and mature let-7 RNA levels during both embryonic brain development and neural differentiation of embryonic stem cells and embryocarcinoma (EC) cells suggest post-transcriptional regulation of let-7 accumulation. Reflecting these results, let-7 sensor constructs were strongly down-regulated during neural differentiation of EC cells and displayed lineage specificity in primary cells. Neural differentiation of EC cells was accompanied by an increase in let-7 precursor processing activity in vitro. Furthermore, undifferentiated and differentiated cells contained distinct precursor RNA binding complexes. A neuron-enhanced binding complex was shown by antibody challenge to contain the miRNA pathway proteins Argonaute1 and FMRP. Developmental regulation of the processing pathway correlates with differential localization of the proteins Argonaute, FMRP, MOV10, and TNRC6B in self-renewing stem cells and neurons (Wulczyn, 2007).
Temporal control of development is an important aspect of pattern formation that awaits complete molecular analysis. lin-57 has been identified as a member of the C. elegans heterochronic gene pathway, which ensures that postembryonic developmental events are appropriately timed. Loss of lin-57 function causes the hypodermis to terminally differentiate and acquire adult character prematurely. lin-57 has been identified as hbl-1, revealing a role for the worm hunchback homolog in control of developmental time. Significantly, fly hunchback (hb) temporally specifies cell fates in the nervous system. The hbl-1/lin-57 3'UTR is required for postembryonic downregulation in the hypodermis and nervous system and contains multiple putative binding sites for temporally regulated microRNAs (miRNAs), including let-7. Indeed, hbl-1/lin-57 is regulated by let-7, at least in the nervous system. Examination of the hb 3'UTR reveals potential binding sites for known fly miRNAs. Thus, evolutionary conservation of hunchback genes may include temporal control of cell fate specification and microRNA-mediated regulation (Abrahante, 2003).
Postembryonic temporal downregulation of hbl-1 in the worm nervous system and hypodermis is programmed, at least in part, through its 3'UTR, which contains multiple putative let-7 binding sites that are evolutionarily conserved. In the nervous system, an hbl-1::gfp::hbl-1 reporter construct is temporally deregulated in a let-7 mutant background; enhanced expression is observed in the ventral nerve cord and anterior nerve ring of adults. Together, these results imply that the hbl-1 3'UTR is a direct target of the let-7 miRNA (Abrahante, 2003).
The extent of hbl-1::gfp::hbl-1 misexpression in let-7 mutants is less than might be expected if let-7 acts alone to downregulate neuronal expression and suggests that additional factors, perhaps other microRNAs, act together with let-7. Indeed, a large and diverse family of miRNAs has been discovered in C. elegans. Among the worm miRNAs reported, three (mir-84, mir-48, and mir-241) share sequence identity with let-7 RNA and are expressed with the same temporal specificity as let-7. The sequence conservation among these miRNAs, particularly between mir-84 and let-7 (81% identical), suggests that they may have target sites in common. Thus, complete temporal deregulation of the hbl-1 reporter may require simultaneous inactivation of multiple miRNAs (Abrahante, 2003).
The role of let-7 in control of hbl-1 in the hypodermis is less clear. The simplest way to interpret let-7 suppression by hbl-1, together with let-7 binding sites in the hbl-1 3'UTR, is that hbl-1 is a direct target of the let-7 miRNA. However, hypodermal hbl-1::gfp expression begins to subside in the L2 and disappears in the early L3, prior to let-7 accumulation in the mid to late L3 stage. Assuming that the hbl-1::gfp construct (which contains a 6.4 kb 5' flanking sequence through the first three introns) contains all relevant enhancer regions, this implies that 3'UTR-mediated downregulation of hbl-1 in hyp7 is controlled by other factors, perhaps including earlier-acting miRNAs (Abrahante, 2003).
let-7 could add to the repression of hbl-1 mRNA from the mid L3 stage onward, ensuring its silence at late developmental stages. However, consistent hbl-1::gfp::hbl-1 misexpression was not detected in the hypodermis of let-7 mutants, suggesting only a minor role for let-7 or redundant action by let-7-related genes. Alternatively, a low threshold level of the HBL-1 presumed transcription factor (not detectable by gfp assay) may be required for hypodermal function. Thus, small changes in HBL-1 level could lead to major developmental consequences through deregulation of target genes (Abrahante, 2003).
Temporal regulation of hbl-1 differs from that of lin-41, the other known let-7 target. lin-41::gfp is expressed in both neurons and hypodermis but is temporally downregulated only in the hypodermis. The discordant patterns of regulation suggest inherent differences between the hbl-1 and lin-41 3'UTRs and the assembled factors that orchestrate their function (Abrahante, 2003).
Reduction of hbl-1 activity by mutation or RNAi does not fully suppress let-7 null mutations. Explanations for this partial epistasis include incomplete loss of hbl-1 function, misexpression of let-7 targets, or redundancy at the hbl-1 step in the pathway. This work suggests that the let-7 target, lin-41, is at least part of the answer. Simultaneous removal of hbl-1 and lin-41 activities produces stronger suppression of the let-7 phenotype than does single depletion of either gene. In let-7(+) animals, depletion of hbl-1 and lin-41 activities produces a fully penetrant L3 molt phenotype and can cause terminal differentiation at the L2 molt, one stage earlier than in either single mutant. Together, these results indicate that let-7 acts through both hbl-1 and lin-41 and that these genes function with partial redundancy to inhibit premature activation of the adult hypodermal program at the L2 and L3 molts in wild-type animals (Abrahante, 2003).
These findings extend the intriguing parallels between the early and late timers of the heterochronic gene pathway, which together mediate stage-specific temporal identities. Each timer is initiated by a microRNA that has two known targets; in the early timer, lin-4 downregulates lin-14 and lin-28, and, in the late timer, let-7 acts through hbl-1 and lin-41. In each case, one target encodes a transcription factor (LIN-14 and HBL-1), and the other encodes a protein with hallmarks of a translational regulator (LIN-28 and LIN-41). Since loss-of-function for each pair of targets causes enhanced precocious phenotypes, it appears that both transcriptional and translational controls are necessarily integrated into both timers to ensure proper timing of cell fate specification (Abrahante, 2003).
Previous studies have generally supported a linear pathway of heterochronic genes, with lin-4 acting as the most upstream and global regulator. These analyses suggest that the pathway is branched. Concomitant loss of hbl-1 and lin-41 activities suppresses the let-7 mutant phenotype more completely than that of lin-4. Loss of hbl-1 and lin-41 activities only weakly restores alae synthesis at the L4 molt in lin-4 mutants, whereas it leads to essentially complete execution of the adult seam cell program at the L3 molt in a let-7 mutant background. These observations indicate that either lin-4 or the genes it regulates have additional targets that time the adult hypodermal program independently of hbl-1 and lin-41. Thus, multiple temporal inputs converge upon the transcription factor LIN-29, indicating that a branched pathway functions to ensure proper timing of seam cell terminal differentiation. Elaboration of these proposed branches will require searches for additional components of the heterochronic gene pathway (Abrahante, 2003).
hbl-1, the C. elegans hunchback ortholog, also controls temporal patterning. Furthermore, hbl-1 is a probable target of microRNA regulation through its 3'UTR. hbl-1 loss-of-function causes the precocious expression of adult seam cell fates. This phenotype is similar to loss-of-function of lin-41, a known target of the let-7 microRNA. Like lin-41 mutations, hbl-1 loss-of-function partially suppresses a let-7 mutation. The hbl-1 3'UTR is both necessary and sufficient to downregulate a reporter gene during development, and the let-7 and lin-4 microRNAs are both required for HBL-1/GFP downregulation. Multiple elements in the hbl-1 3'UTR show complementarity to regulatory microRNAs, suggesting that microRNAs directly control hbl-1. MicroRNAs may likewise function to regulate Drosophila hunchback during temporal patterning of the nervous system (Lin, 2003).
HBL-1/GFP is expressed strongly in hypodermal cells, including the embryonic seam cell precursors, and in neurons like those of the ventral nerve cord (VNC) during postembryonic stages. HBL-1/GFP expression was reexamined, focusing on hypodermal and VNC expression at postembryonic C. elegans developmental stages. Strain BW1932 contains an integrated array with the hbl-1 promoter, the first 133 amino acids of HBL-1 fused to GFP, and the hbl-1 3'UTR. During the L1 stage, HBL-1/GFP expression is observed in the hypodermal syncitial cells (e.g., hyp7), in the ventral hypodermal cells (P cells), and weakly in the lateral hypodermal seam cells (H, V, and T cells). By the L2 stage, HBL-1/GFP was no longer expressed in the seam cells but was still observed in P cell descendants and weakly in the non-seam cell hypodermis. By the L3 stage, HBL-1/GFP was virtually absent in the hypodermis and Pn.p cell descendants, but was still highly expressed in the ventral nerve cord (generated from Pn.a cells) and other unidentified neurons. Early L4 animals express high HBL-1/GFP levels in the VNC, while late L4 and adult animals express HBL-1/GFP very weakly in the VNC. In some adult VNCs, expression is undetectable. As judged by this HBL-1/GFP fusion, HBL-1 expression is downregulated during the course of postembryonic development, with highest expression in L1 animals and lowest expression in adults (Lin, 2003).
let-7 RNA is expressed predominantly in the L4 and adult stages. HBL-1/GFP expression in the VNC is downregulated during the L4 and adult stages by a 3'UTR-dependent mechanism. The similar timing of these two events suggest that let-7 might be involved in downregulation of hbl-1 in the VNC. Indeed, it was found that while 45% of let-7(n2853) adults expressed intense HBL-1/GFP in the VNC, only 4% of wild-type animals did the same. lin-4 RNA is also present in the L4 stage. Intense HBL-1/GFP expression is seen in the VNC of 100% of lin-4(e912) adult animals. Thus, both wild-type let-7 and lin-4 RNAs are required for proper hbl-1 downregulation in the VNC (Lin, 2003).
The succession of developmental events in the C. elegans larva is governed by the heterochronic genes. When mutated, these genes cause either precocious or retarded developmental phenotypes, in which stage-specific patterns of cell division and differentiation are either skipped or reiterated, respectively. A new heterochronic gene, lin-46, has been identified from mutations that suppress the precocious phenotypes caused by mutations in the heterochronic genes lin-14 and lin-28. lin-46 mutants on their own display retarded phenotypes in which cell division patterns are reiterated and differentiation is prevented in certain cell lineages. The analysis indicates that lin-46 acts at a step immediately downstream of lin-28, affecting both the regulation of the heterochronic gene pathway and execution of stage-specific developmental events at two stages: the third larval stage and adult. lin-46 is required prior to the third stage for normal adult cell fates, suggesting that it acts once to control fates at both stages, and that it affects adult fates through the let-7 branch of the heterochronic pathway. Interestingly, lin-46 encodes a protein homologous to MoeA of bacteria and the C-terminal domain of mammalian gephyrin, a multifunctional scaffolding protein. These findings suggest that the LIN-46 protein acts as a scaffold for a multiprotein assembly that controls developmental timing, and expand the known roles of gephyrin-related proteins to development (Pepper, 2004).
lin-46 encodes a 391 amino acid protein with homology along its entire length to MoeA of bacteria and the C-terminal domains (referred to as the E-domain) of the mammalian protein gephyrin; other related proteins are Cinnamon of Drosophila and CNX1 of Arabidopsis. Gephyrin is a submembraneous scaffolding protein that aids in clustering glycine and GABA receptors at postsynaptic neurons. MoeA is involved in the last step of the biosynthesis of molybdenum co-factor, a metal coordinating molecule in many molybdo-enzymes, although the exact function of MoeA in this process is not known. Gephyrin, as well as Cinnamon and CNX1, are essentially fusions of homologs of the bacterial proteins MoeA and MogA. Indeed, all three of these proteins from higher eukaryotes are believed to participate in molybdenum cofactor biosynthesis, as the bacterial proteins do. C. elegans is unusual among multicellular eukaryotes in having its MoeA and MogA homologs encoded separately, and, furthermore, having two MoeA paralogs, one of which is encoded by lin-46 (Pepper, 2004).
It is possible that lin-28 and lin-46 directly affect the microRNA let-7, which then regulates other genes that control the larval to adult switch. This is consistent with the failure of lin-46 mutant to suppress the precocious phenotypes of the later-acting genes lin-41 and lin-57. Further analysis will determine whether lin-46 and lin-28 indeed affect the expression or activity of let-7 (Pepper, 2004).
The microRNA let-7 is a critical regulator of developmental timing events at the larval-to-adult transition in C. elegans. Recently, microRNAs with sequence similarity to let-7 have been identified. Doubly mutant animals lacking the let-7 family microRNA genes mir-48 and mir-84 exhibit retarded molting behavior and retarded adult gene expression in the hypodermis. Triply mutant animals lacking mir-48, mir-84, and mir-241 exhibit repetition of L2-stage events in addition to retarded adult-stage events. mir-48, mir-84, and mir-241 function together to control the L2-to-L3 transition, likely by base pairing to complementary sites in the 3′ UTR of the hunchback homolog hbl-1 and downregulating hbl-1 activity. Genetic analysis indicates that mir-48, mir-84, and mir-241 specify the timing of the L2-to-L3 transition in parallel to the heterochronic genes lin-28 and lin-46. These results indicate that let-7 family microRNAs function in combination to affect both early and late developmental timing decisions (Abbott, 2005).
The C. elegans genome encodes at least 19 microRNA gene families containing from 2 to 8 members with significant sequence conservation within the ~22 nt microRNA sequence. Sequence conservation among family members is strongest near the 5' end of the microRNA in the region known as the 'seed',which has been proposed to reflect a potential for family members to direct the repression of shared target genes. Because mir-48, mir-84, and mir-241 display complete sequence conservation in the seed region at the 5′ end, it is possible that they repress a common set of targets and hence may be functionally equivalent. The current findings suggest that let-7, mir-48, mir-84, and mir-241 may all act to repress a shared target, hbl-1. let-60 RAS also has been proposed to be a target of mir-84 based on overexpression experiments. Elevated levels of let-60 RAS expression lead to a multivulva phenotype; however, no multivulva phenotype was observed in mir-84 single mutants nor in mir-48 mir-241; mir-84 triple mutants (Abbott, 2005).
The results leave open the possibility that mir-48, mir-84, and mir-241 are not functionally equivalent in all respects. Sequence differences in the 3′ end of the let-7 family microRNAs may direct the repression of some distinct sets of targets, the repression of which could function coordinately to regulate developmental timing. Target sites that lack strong complementarity at the microRNA 5′ end can direct repression if there is extensive compensatory pairing at the 3′ end, thus allowing for distinct activities of microRNA family members. Indeed, let-7 complementary sites in the lin-41 mRNA have extensive complementarity to the let-7 3′ region, along with imperfect pairing to the let-7 5′ seed region. The specificity imparted by compensatory 3′ pairing may function to enable repression of lin-41 by let-7 and not allow for the repression of lin-41 by mir-48, mir-84, or mir-241. Similarly, extensive 3′ pairing to one of the other three let-7 family members might compensate for a lack of strong 5′ pairing and therefore could restrict the repression of specific targets to individual let-7 family members (Abbott, 2005).
The findings suggest that the four let-7 family microRNAs may all act to repress hbl-1. Reduction of hbl-1 activity can suppress the heterochronic defects observed in both mir-48 mir-241; mir-84 and let-7 mutant animals, indicating that hbl-1 functions downstream of the let-7 family microRNAs. Moreover, the failure to appropriately downregulate hbl-1 can be detected in the hypodermis of mir-48 mir-241; mir-84 mutants and in neuronal cells of let-7 mutants. The hbl-1 3′ UTR contains eight let-7 complementary sites. Because these potential binding sites differ in sequence, each may be able to bind the individual let-7 family microRNAs with differing efficacies. The relative contribution of individual let-7 family microRNAs to the repression of hbl-1 activity remains to be tested (Abbott, 2005).
Previous studies showed a role for hbl-1 activity in controlling the L4-to-adult transition. The current findings indicate that hbl-1 also controls the L2-to-L3 transition in the hypodermis. This early role for hbl-1 is consistent with the observation that reduction of hbl-1 activity by RNAi results in a decreased number of seam cells in L2-stage animals. A reduced number of seam cells likely reflects a partial omission of the L2-stage proliferative program. This precocious phenotype is relatively weak in comparison to that of lin-28(lf) mutants, in which all seam cells generated from the V lineage fail to execute the L2-stage program. This weak phenotype may be a consequence of residual hbl-1 activity of the partial loss-of-function allele, ve18. It is possible that complete loss of hbl-1 activity would result in a stronger precocious L2-omission phenotype similar to that seen in lin-28(lf) mutants (Abbott, 2005).
An important regulator of the L2-to-L3 transition is lin-28, yet multiple lines of evidence suggest that the control of the L2-to-L3 transition by mir-48, mir-84, and mir-241 does not occur through regulation of lin-28 activity. (1) A lin-28::gfp::lin-28 reporter transgene that recapitulates the wild-type temporal regulation of LIN-28 protein and that rescues the phenotype of lin-28(lf) worms is not derepressed in mir-48 mir-241; mir-84 triple mutants. (2) It was found that the level of endogenous LIN-28 protein was not significantly elevated in mir-48 mir-241; mir-84 triple mutants, whereas, in lin-4 retarded mutants, LIN-28 protein is abnormally abundant at later larval stages. (3) Two alleles of lin-58 that contain mutations upstream of mir-48, and hence lead to the misexpression of mir-48, enhance the precocious phenotype of a lin-28 null mutant, indicating that mir-48 does not act exclusively through lin-28. (4) It was found that the L2 reiteration phenotype of mir-48 mir-241; mir-84 triple mutants could occur independently of lin-28 activity. These data together indicate that mir-48, mir-84, and mir-241 control the L2-to-L3 transition primarily through downstream effectors other than lin-28, even though the lin-28 3′ UTR contains a let-7 complementary site. It is possible that the let-7 family microRNAs may contribute to the repression of lin-28 expression, but to a degree undetectable by the assays used (Abbott, 2005).
Genetic epistasis analysis indicates that mir-48, mir-84, and mir-241 function in parallel with the lin-28 and lin-46 pathway to downregulate hbl-1 activity and hence control the L2-to-L3 transition. One model to account for this convergence of pathways on hbl-1 would be that LIN-46, in its putative role as a scaffolding protein, could control assembly of a protein complex that directly interacts with HBL-1 protein to inhibit its activity in parallel with the repression of hbl-1 mRNA translation exerted by mir-48, mir-84, and mir-241. Alternatively, LIN-46 could interact with RNA binding protein(s) and directly potentiate the activity of the mir-48, mir-84, and mir-241 microRNAs (Abbott, 2005).
These data suggest that mir-48, mir-84, and mir-241 control developmental timing in two physically associated but distinct cell types in the hypodermis: the postmitotic main body hypodermal syncytial cell, hyp7, and the proliferative seam cells. Two lines of evidence point to a role in hyp7 for mir-48, mir-84, and mir-241 to repress hbl-1 and control hyp7 temporal behavior. (1) mir-48; mir-84 mutant worms displayed heterochronic defects in hyp7; the expression of the adult-specific transgene col-19::gfp was retarded in hyp7 but was regulated normally in the seam cells. Thus, the supernumerary molt observed in mir-48; mir-84 double mutants may be a consequence of a heterochronic defect in hyp7. (2) The data indicate that mir-48, mir-84, and mir-241 act in hyp7 to repress hbl-1 activity. In mir-48 mir-241; mir-84 worms, hbl-1::gfp::hbl-1 was misregulated in hyp7. Thus, the 3′ UTR-dependent downregulation of hbl-1::gfp::hbl-1 in hyp7 that occurs in wild-type animals can be accounted for largely by the regulation of hbl-1 by mir-48, mir-84, and mir-241 (Abbott, 2005).
mir-48, mir-84, and mir-241 may also function in the hypodermal seam cells to control developmental timing. Reduction of hbl-1 activity genetically or by hbl-1 RNAi affected stage-specific behavior of seam cells, resulting in suppression of the retarded seam cell and alae phenotypes of mir-48 mir-241; mir-84 worms. This could be a consequence of the repression of hbl-1 by mir-48, mir-84, and mir-241 in the seam cells. Interestingly, hbl-1::gfp::hbl-1 cannot be detected in the seam cells after the L1 stage, suggesting that, at the time of the L2-to-L3 transition, the amount of hbl-1 expression in seam cells is relatively low. Thus, mir-48, mir-84, and mir-241 may function cell autonomously in the seam cells at the L3 stage to downregulate hbl-1, albeit beginning from a level already below the threshold of detection by the assays. Alternatively, since repression of hbl-1::gfp::hbl-1 is readily observed at the L2-to-L3 transition in hyp7 (the main body hypodermal syncytial cell), it is conceivable that the stage-specific behavior of seam cells may be controlled non-cell autonomously by a hbl-1-regulated signal from hyp7. Non-cell autonomous signaling from hyp7 to neighboring cells has been proposed in the pathway to specify the fates of vulval precursor cells (VPCs). Mosaic analyses suggest that the sites of action of the multivulva (Muv) gene locus lin-15 and of the synthetic Muv genes lin-37 and lin-35 are in hyp7. One model is that hyp7 generates a signal to neighboring VPCs to inhibit vulval cell fate specification. Similarly, a signal from hyp7 to the lateral hypodermal seam cells may regulate the temporal behavior of seam cells and thereby help coordinate developmental timing throughout the hypodermis (Abbott, 2005).
In summary, the results presented in this study demonstrate a role for the let-7 family microRNA genes mir-48, mir-84, and mir-241 in the heterochronic pathway to control the L2-to-L3 cell fate transitions in the hypodermis. Proper progression through the L1 and L2 larval stages requires downregulation of lin-14 and lin-28, primarily through the action of the microRNA lin-4. These findings extend the involvement of microRNAs in the regulation of C. elegans developmental timing to include a requirement for the downregulation of hbl-1 by the combined action of the three let-7 family microRNAs, mir-48, mir-84, and mir-241, in the hypodermis. The L2-to-L3 transition is controlled by complex genetic mechanisms involving two microRNA-regulated pathways that converge on hbl-1: the lin-4, lin-28, lin-46 pathway and the mir-48, mir-84, mir-241 pathway. These parallel inputs to hbl-1 may serve to couple hbl-1 downregulation to distinct upstream temporal signals. Further, the functional redundancy among mir-48, mir-84, and mir-241 could reflect alternative mechanisms for triggering the L2-to-L3 transition throughout the hypodermis. mir-48, mir-84, and mir-241 seem to have more minor roles, compared to let-7, at the L4-to-adult transition in the hypodermis, indicating that different microRNA family members can be deployed for distinct roles, perhaps through differences in temporal or spatial expression patterns and/or differences in target specificity. These findings suggest analogous forms of genetic redundancy and regulatory complexity may be expected in pathways involving other families of related microRNAs (Abbott, 2005).
The C. elegans heterochronic genes program stage-specific temporal identities in multiple tissues during larval development. These genes include the first two miRNA-encoding genes discovered, lin-4 and let-7. lin-58 alleles, identified as lin-4 suppressors, define another miRNA that controls developmental time. These alleles are unique in that they contain point mutations in a gene regulatory element of mir-48, a let-7 family member. mir-48 is expressed prematurely in lin-58 mutants, whereas expression of mir-241, another let-7 family member residing immediately upstream of mir-48, appears to be unaffected. A mir-48 transgene bearing a lin-58 point mutation causes strong precocious phenotypes in the hypodermis and vulva when expressed from multicopy arrays. mir-48::gfp fusions reveal expression in these tissues, and inclusion of a lin-58 mutation causes precocious and enhanced gfp expression. These results suggest that lin-58 alleles disrupt a repressor binding site that restricts the time of miR-48 action in wild-type animals (Li, 2005).
The lin-58 mutations described here appear to reveal a negative regulatory element that prevents mir-48 accumulation until the proper time during the mid-to-late L1 stage. Intriguingly, the 11 bp inverted repeat disrupted by lin-58 mutations spans a 7-8 bp match to a computationally identified consensus motif (5′-CTCCGCCC-3′; underlined residues are mutated in lin-58 alleles) found 5′ to most worm miRNA genes that are independently expressed. A perfect match to this sequence also resides another ~500 bp upstream. The functional significance of the motif, and whether it relates directly to the repressive element defined by lin-58 lesions, is as yet unclear. Replacement of the entire 11 bp inverted repeat in mir-48::gfp with the AT sequence has the same effect upon GFP expression as does insertion of the ve33 point mutation; it results in enhanced and precocious expression, indicating that the GC repeat is not required for mir-48 transcriptional activation. In addition, the lin-58(ve33) and lin-58(ve12) point mutations cause precocious accumulation of miR-48, but do not appear to interfere with the processing of pre-miR-48, suggesting that the site is not required for recruitment of RNA processing machinery. Thus, these data are consistent with a model in which the lin-58 lesions disrupt a repressor binding site that acts to restrict the timing of microRNA action (Li, 2005).
The let-7 microRNA (miRNA) gene of Caenorhabditis elegans controls the timing of developmental events. let-7 is conserved throughout bilaterian phylogeny and has multiple paralogs. The paralog mir-84 acts synergistically with let-7 to promote terminal differentiation of the hypodermis and the cessation of molting in C. elegans. Loss of mir-84 exacerbates phenotypes caused by mutations in let-7, whereas increased expression of mir-84 suppresses a let-7 null allele. Adults with reduced levels of mir-84 and let-7 express genes characteristic of larval molting as they initiate a supernumerary molt. mir-84 and let-7 promote exit from the molting cycle by regulating targets in the heterochronic pathway and also nhr-23 and nhr-25, genes encoding conserved nuclear hormone receptors essential for larval molting. The synergistic action of miRNA paralogs in development may be a general feature of the diversified miRNA gene family (Hayes, 2006).
The C. elegans genes nhr-23 and nhr-25 encode orphan nuclear hormone receptors orthologous, respectively, to DHR3 and ßFTZ-F1, which are related to mammalian ROR/RZR/RevErb and SF-1, respectively. Both receptors are essential for completion of the larval molts, suggesting that particular functions of nhr-23/DHR3 and nhr-25/ ßFTZ-F1 might be conserved and, further, that regulation by steroid hormones might be a common feature of molting in C. elegans and Drosophila. However, a steroid hormone regulating molting of C. elegans has not yet been identified and the genome lacks orthologs of ECR or USP (Hayes, 2006).
A genetic model is presented for the function of mir-84 and let-7 in epithelial differentiation, as related to the molting cycle. The let-7 miRNA targets lin-41 mRNA and also hbl-1 mRNA, in combination with paralogous miRNAs. During early larval development, LIN-41 and HBL-1 together repress production of the zinc-finger transcription factor LIN-29. Expression of let-7 and related miRNAs late in larval development represses lin-41 and hbl-1, thereby activating LIN-29. LIN-29 promotes expression of col-19 and possibly other collagen genes characteristic of an adult cuticle and also represses expression of col-17 and possibly other collagen genes characteristic of larval cuticle. LIN-29 is likely to regulate additional genes that control the molting cycle that have not yet been identified (Hayes, 2006).
Inactivation of either one of the nuclear hormone receptor genes nhr-23 or nhr-25 is sufficient to prevent the aberrant supernumerary molt caused by reduced levels of mir-84 and let-7. NHR-23 and NHR-25 thus serve as key downstream effectors of the miRNAs in regulation of the molting cycle. One model is that LIN-29, or a transcription factor regulated by LIN-29, represses nhr-23 and nhr-25 following the fourth molt. Accordingly, GFP expression from an nhr-23 reporter gene increases fourfold in the hypodermis of let-7 mir-84 adults. The relationship between nhr-23 and nhr-25 in C. elegans remains to be determined; however, DHR3 stimulates transcription of ßFTZ-F1 in flies (Hayes, 2006).
The identification of sites in the 3' UTR of nhr-25 that are complementary to let-7 family members and are also conserved in other nematodes suggests that the let-7 family targets the nhr-25 message to negatively regulate production of NHR-25 in adults. Consistent with this model, increasing the abundance of mir-84 partly suppresses the supernumerary molt caused by a probable null mutation in the lin-29 gene. Also, in preliminary experiments RNA species attributable to cleavage of the nhr-25 message upon binding of let-7-like miRNAs were detected in extracts from wildtype adults. Steroid hormones and co-factors probably also regulate activity of NHR-23 and NHR-25 during the life cycle (Hayes, 2006).
Regulation by miRNAs thus converges on transcription factors upstream in the genetic networks regulating molting. NHR-23 coordinates several aspects of larval molting by promoting expression of genes required for patterning the new cuticle and ecdysis, including, respectively, the collagen gene dpy-7 and the collagenase gene nas-37. Inactivation of either nhr-23 or nhr-25 abrogates the reiterated expression of gfp reporters for mlt-10 and nas-37 caused by mutation of let-7 and mir-84. NHR-25 might promote expression of the corresponding genes during larval development, even though RNAi of nhr-25 is not sufficient to abrogate expression of the gfp reporters in wild-type larvae. Interestingly, inactivation of nhr-23 or nhr-25 causes an earlier blockade in the molting program in let-7 mir-84 adults than in wild-type larvae, such that the mutant adults do not enter lethargus or attempt to ecdyse. Parallel pathways might drive early steps of molting during larval development (Hayes, 2006).
Intriguingly, adults with reduced levels of mir-84 and let-7 are unable to shed their cuticle to complete the supernumerary molt. One possibility is that particular genes required for ecdysis are not induced. Whereas the hypodermis and seam cells retain some larval character in let-7 mir-84 adults, other cells, perhaps particular neurons or specialized epithelia, might be fully differentiated and therefore unable to coordinate with the molting program. Consistent with this idea, let-7 mir-84 adults spend an atypically long time in lethargus, suggesting a failure to exit the behavioral program. Alternatively, particular structural features of the fifth cuticle might be physically incompatible with shedding the exoskeleton (Hayes, 2006).
Considering an aberrant ecdysis as the terminal phenotype of let-7 mir-84 mutants, it is intriguing to speculate that the let-7 family and possibly other miRNAs regulate aspects of the larval molting cycle. Indeed, increased expression of either mir-84 or let-7 causes some larvae to arrest development, trapped inside partly shed cuticle, indicating that levels of let-7-like miRNAs can impact molting of larvae (Hayes, 2006).
Mechanisms that set the pace of the molting cycle are not well understood, although physiologic cues such as nutritional status and environmental cues such as temperature impact the duration of larval stages. Interestingly, let-7 and let-7 mir-84 mutants initiate the supernumerary molt in synchrony, rather than in a stochastic fashion, relative to the time of hatching. Thus, a timing mechanism for molting persists in these particular miRNA mutants (Hayes, 2006).
The let-7 gene is perfectly conserved throughout bilaterian phylogeny, and vertebrate genomes specify many miRNAs homologous to let-7. Vertebrate let-7 and protein-coding genes orthologous to targets of let-7 identified in C. elegans play crucial roles in development. Moreover, reduced expression of human let-7 correlates with shortened survival in lung cancer patients, and let-7 might regulate the RAS oncogene. The possibility of functional conservation among homologs of let-7 in humans and worms intimates the importance of understanding how let-7 and its paralogs function in C. elegans. This work shows how analysis of double mutants can reveal how the many miRNAs that form paralogous families work together to regulate their targets (Hayes, 2006).
Metazoan miRNAs regulate protein-coding genes by binding the 3' UTR of cognate mRNAs. Identifying targets for the 115 known C. elegans miRNAs is essential for understanding their function. By using a new version of PicTar and sequence alignments of three nematodes, it is predicted that miRNAs regulate at least 10% of C. elegans genes through conserved interactions. A new experimental pipeline was developed to assay 3' UTR-mediated posttranscriptional gene regulation via an endogenous reporter expression system amenable to high-throughput cloning, demonstrating the utility of this system using one of the most intensely studied miRNAs, let-7. Expression analyses uncover several new potential let-7 targets and suggest a new let-7 activity in head muscle and neurons. To explore genome-wide trends in miRNA function, functional categories of predicted target genes were analyzed; one-third of C. elegans miRNAs target gene sets are enriched for specific functional annotations. miRNA target predictions were integrated with other functional genomic data from C. elegans. At least 10% of C. elegans genes are predicted miRNA targets, and a number of nematode miRNAs seem to regulate biological processes by targeting functionally related genes. An in vivo system was developed for testing miRNA target predictions in likely endogenous expression domains. The thousands of genome-wide miRNA target predictions for nematodes, humans, and flies are available from the PicTar website and are linked to an accessible graphical network-browsing tool allowing exploration of miRNA target predictions in the context of various functional genomic data resources (Lall, 2006).
To molecularly test PicTar predictions of let-7 targets, 12 novel putative targets were selected without input from the phenotypic suppression test. T14B1.1 (a novel gene, PicTar rank 2) and unc-129 (a TGF-β homolog, rank 19) were tested as targets of let-7. The T14B1.1 3′ UTR contains several predicted conserved let-7 sites. T14B1.1 reporter constructs are expressed in multiple tissues including head neurons and the hypodermis, consistent with in situ hybridization data. Strikingly, a reporter gene carrying the T14B1.1 3′ UTR is expressed in main body hypodermal tissue during the L2 and early L3 larval stages, but hypodermal expression decreases dramatically during the L4 stage, consistent with the appearance of high levels of mature let-7. Decreased expression depends on the T14B1.1 3′ UTR, since the decrease in expression is alleviated when the unc-54 3′ UTR is substituted. These observations are consistent with the hypothesis that T14B1.1 is repressed by let-7. T14B1.1 is a novel gene that has no known RNAi phenotype and cannot suppress the let-7 vulval bursting phenotype; thus, these results also illustrate that a combined computational and experimental pipeline can identify targets that may not have been found by conventional experimental means, such as a genetic suppressor screen (Lall, 2006).
unc-129, a TGF-β homolog shown to be involved in axon guidance and which contains two predicted let-7 sites in its 3′ UTR, was tested. By using upstream sequence from the unc-129 locus, expression was observed in head muscle and ventral motor neurons, but not in body wall muscle. Expression in head cells decreases in late larval stages, concomitant with a rise in let-7 levels. This decrease is mildly alleviated when the unc-129 3′ UTR is replaced with the unc-54 3′ UTR (Lall, 2006).
The lin-12/Notch signaling pathway is conserved from worms to humans and is a master regulator of metazoan development. lin-12/Notch gain-of-function (gf) animals display precocious alae at the L4 larval stage with a significant increase in let-7 expression levels. Furthermore, lin-12(gf) animals display a precocious and higher level of let-7 gfp transgene expression in seam cells at L3 stage. Interestingly, lin-12(gf) mutant rescued the lethal phenotype of let-7 mutants similar to other known heterochronic mutants. It is proposed that lin-12/Notch signaling pathway functions in late developmental timing, upstream of or in parallel to the let-7 heterochronic pathway. Importantly, the human microRNA let-7a was also upregulated in various human cell lines in response to Notch1 activation, suggesting an evolutionarily conserved cross-talk between let-7 and the canonical lin-12/Notch signaling pathway (Solomon, 2008).
Unlike mammals, teleost fish mount a robust regenerative response to retinal injury that culminates in restoration of visual function. This regenerative response relies on dedifferentiation of Müller glia into a cycling population of progenitor cells. However, the mechanism underlying this dedifferentiation is unknown. This paper reports that genes encoding pluripotency factors are induced following retinal injury. Interestingly, the proneural transcription factor, Ascl1a, and the pluripotency factor, Lin-28, are induced in Müller glia within 6 h following retinal injury and are necessary for Müller glia dedifferentiation. Ascl1a is necessary for lin-28 expression, and Lin-28 suppresses let-7 microRNA (miRNA) expression. Furthermore, let-7 represses expression of regeneration-associated genes such as, ascl1a, hspd1, lin-28, oct4, pax6b and c-myc. hspd1, oct4 and c-myc(a) exhibit basal expression in the uninjured retina and let-7 may inhibit this expression to prevent premature Müller glia dedifferentiation. The opposing actions of Lin-28 and let-7 miRNAs on Müller glia differentiation and dedifferentiation are similar to that of embryonic stem cells and suggest novel targets for stimulating Müller glia dedifferentiation and retinal regeneration in mammals (Ramachandran, 2010).
The bidentate RNase III Dicer cleaves microRNA precursors to generate the 21-23 nt long mature RNAs. These precursors are 60-80 nt long; they fold into a characteristic stem-loop structure and they are generated by an unknown mechanism. To gain insights into the biogenesis of microRNAs, the precise 5' and 3' ends of the let-7 precursors in human cells have been characterized. They harbor a 5'-phosphate and a 3'-OH and remarkably, they contain a 1-4 nt 3' overhang. These features are characteristic of RNase III cleavage products. Since these precursors are present in both the nucleus and the cytoplasm of human cells, these results suggest that they are generated in the nucleus by the nuclear RNase III. Additionally, these precursors fit the minihelix export motif and are thus likely exported by this pathway (Basyuk, 2003)
MicroRNAs (miRNAs) are approximately 21-nucleotide-long RNA molecules regulating gene expression in multicellular eukaryotes. In metazoa, miRNAs act by imperfectly base-pairing with the 3' untranslated region of target messenger RNAs (mRNAs) and repressing protein accumulation by an unknown mechanism. Endogenous let-7 microribonucleoproteins (miRNPs) or the tethering of Argonaute (Ago) proteins to reporter mRNAs in human cells inhibit translation initiation. M(7)G-cap-independent translation is not subject to repression, suggesting that miRNPs interfere with recognition of the cap. Repressed mRNAs, Ago proteins, and miRNAs were all found to accumulate in processing bodies. It is proposed that localization of mRNAs to these structures is a consequence of translational repression (Pillai, 2005).
HMGA2, a high-mobility group protein, is oncogenic in a variety of tumors, including benign mesenchymal tumors and lung cancers. Knockdown of Dicer in HeLa cells revealed that the HMGA2 gene is transcriptionally active, but its mRNA is destabilized in the cytoplasm through the microRNA (miRNA) pathway. HMGA2 is derepressed upon inhibition of let-7 in cells with high levels of the miRNA. Ectopic expression of let-7 reduces HMGA2 and cell proliferation in a lung cancer cell. The effect of let-7 on HMGA2 is dependent on multiple target sites in the 3' untranslated region (UTR), and the growth-suppressive effect of let-7 on lung cancer cells is rescued by overexpression of the HMGA2 ORF without a 3'UTR. These results provide a novel example of suppression of an oncogene by a tumor-suppressive miRNA and suggest that some tumors activate the oncogene through chromosomal translocations that eliminate the oncogene’s 3'UTR with the let-7 target sites (Lee, 2007).
RNA-binding proteins (RBPs) and microRNAs (miRNAs) are potent post-transcriptional regulators of gene expression. This study shows that the RBP HuR reduces c-Myc expression by associating with the c-Myc 3' untranslated region (UTR) next to a miRNA let-7-binding site. Lowering HuR or let-7 levels relieves the translational repression of c-Myc. Unexpectedly, HuR and let-7 repressed c-Myc through an interdependent mechanism; let-7 requires HuR to reduce c-Myc expression and HuR required let-7 to inhibit c-Myc expression. These findings suggest a regulatory paradigm wherein HuR inhibits c-Myc expression by recruiting let-7-loaded RISC (RNA miRNA-induced silencing complex) to the c-Myc 3'UTR (Kim, 2009).
Inflammation is linked clinically and epidemiologically to cancer, and NF-kappaB appears to play a causative role, but the mechanisms are poorly understood. An experimental model of oncogenesis is described involving a derivative of MCF10A, a spontaneously immortalized cell line derived from normal mammary epithelial cells, that contains ER-Src, a fusion of the Src kinase oncoprotein (v-Src) and the ligand binding domain of the estrogen receptor. Treatment of these cells with estrogen receptor antagonist tamoxifen (TAM) for 36 hr results in phenotypic transformation, formation of multiple foci, the ability to form colonies in soft agar, increased motility and invasive ability, and tumor formation upon injection in nude mice. This model permits the opportunity to kinetically follow the pathway of cellular transformation in a manner similar to that used to study viral infection and other temporally ordered processes. Transient activation of Src oncoprotein can mediate an epigenetic switch from immortalized breast cells to a stably transformed line that forms self-renewing mammospheres that contain cancer stem cells. Src activation triggers an inflammatory response mediated by NF-kappaB that directly activates Lin28 transcription and rapidly reduces let-7 microRNA levels. Let-7 directly inhibits IL6 expression, resulting in higher levels of IL6 than achieved by NF-kappaB activation. IL6-mediated activation of the STAT3 transcription factor is necessary for transformation, and IL6 activates NF-kappaB, thereby completing a positive feedback loop. This regulatory circuit operates in other cancer cells lines, and its transcriptional signature is found in human cancer tissues. Thus, inflammation activates a positive feedback loop that maintains the epigenetic transformed state for many generations in the absence of the inducing signal (Iliopoulos, 2009).
Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.