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Repression of C. elegans microRNA targets at the initiation level of translation requires GW182 proteins

2009; Springer Nature; Volume: 28; Issue: 3 Linguagem: Inglês

10.1038/emboj.2008.275

1460-2075

Xavier C. Ding, Helge Großhans,

MicroRNA in disease regulation

Article8 January 2009free access Repression of C. elegans microRNA targets at the initiation level of translation requires GW182 proteins Xavier C Ding Xavier C Ding Friedrich Miescher Institute for Biomedical Research (FMI), Basel, Switzerland Search for more papers by this author Helge Großhans Corresponding Author Helge Großhans Friedrich Miescher Institute for Biomedical Research (FMI), Basel, Switzerland Search for more papers by this author Xavier C Ding Xavier C Ding Friedrich Miescher Institute for Biomedical Research (FMI), Basel, Switzerland Search for more papers by this author Helge Großhans Corresponding Author Helge Großhans Friedrich Miescher Institute for Biomedical Research (FMI), Basel, Switzerland Search for more papers by this author Author Information Xavier C Ding1 and Helge Großhans 1 1Friedrich Miescher Institute for Biomedical Research (FMI), Basel, Switzerland *Corresponding author. Friedrich Miescher Institute for Biomedical Research (FMI), Maulbeerstrasse 66, WRO-1066.1.38, 4002 Basel, Switzerland. Tel.: +41 61 6976675; Fax: +41 61 6973976; E-mail: [email protected] The EMBO Journal (2009)28:213-222https://doi.org/10.1038/emboj.2008.275 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info MicroRNAs (miRNAs) repress target genes through a poorly defined antisense mechanism. Cell-free and cell-based assays have supported the idea that miRNAs repress their target mRNAs by blocking initiation of translation, whereas studies in animal models argued against this possibility. We examined endogenous targets of the let-7 miRNA, an important regulator of stem cell fates. We report that let-7 represses translation initiation in Caenorhabditis elegans, demonstrating this mode of action for the first time in an organism. Unexpectedly, although the lin-4 miRNA was previously reported to repress its targets at a step downstream of translation initiation, we also observe repression of translation initiation for this miRNA. This repressive mechanism, which frequently but not always coincides with transcript degradation, requires the GW182 proteins AIN-1 and AIN-2, and acts on several mRNAs targeted by different miRNAs. Our analysis of an expanded set of endogenous miRNA targets therefore indicates widespread repression of translation initiation under physiological conditions and establishes C. elegans as a genetic system for dissection of the underlying mechanisms. Introduction MicroRNAs (miRNAs) are small, untranslated RNAs involved in numerous developmental pathways in plants and animals (reviewed in Bushati and Cohen, 2007). They regulate a large fraction of cellular mRNAs by binding to complementary sequences in their target mRNAs (‘cognate mRNAs’), but the mechanisms involved in subsequent repression of the mRNA are less clear (reviewed in Eulalio et al, 2008a; Filipowicz et al, 2008). In the best understood example, prevalent in plants, miRNAs function as small interfering (si)RNAs and induce mRNA cleavage through the RNA-induced silencing complex (RISC) when binding to perfectly complementary sites in their target mRNAs (Jones-Rhoades et al, 2006). In animals, this appears to be the exception, as most animal miRNAs are only partially complementary to their targets (Bushati and Cohen, 2007), thus precluding RISC-mediated cleavage. Early work on the Caenorhabditis elegans lin-4 miRNA established, instead, the paradigm that miRNAs functioned by translationally repressing their targets at a step downstream of translation initiation, without substantially affecting transcript levels (Olsen and Ambros, 1999; Seggerson et al, 2002). By contrast, recent studies aimed at recapitulating miRNA function in cell-free systems concluded that miRNAs inhibit target mRNA translation at the initiation step (Wang et al, 2006; Mathonnet et al, 2007; Thermann and Hentze, 2007; Wakiyama et al, 2007). Inhibition of translation initiation, as evidenced by the hallmark shift of target mRNAs from heavy to light polysomal or monosomal fractions of sucrose density gradients in response to the miRNA, has also been observed in a number of cell-based studies. However, such studies also identified additional and sometimes conflicting miRNA modes of action (Eulalio et al, 2008a; Filipowicz et al, 2008). These mechanisms include inhibition of target mRNA translation after initiation, target mRNA degradation in a non-endonucleolytic manner, which may or may not result from deadenylation, and co-translational protein degradation. Target mRNA degradation has also been observed for some miRNA targets in vivo, in C. elegans and zebrafish (Bagga et al, 2005; Giraldez et al, 2006). Only a single study has so far demonstrated regulation of an endogenous mRNA, CAT1, by its cognate miR-122 miRNA at the level of translation initiation (Bhattacharyya et al, 2006). The other studies that examined endogenous miRNA targets instead provided evidence against repression of translation initiation (Olsen and Ambros, 1999; Seggerson et al, 2002; Kong et al, 2008), and this includes the only two studies that have tested this mechanism in an animal model, under physiological conditions (Olsen and Ambros, 1999; Seggerson et al, 2002). It is currently unclear whether this divergence of results denotes specific mechanisms operating for individual miRNAs and/or targets. Alternatively, the transfected miRNA target reporters that were used in the bulk of studies showing repression of translation initiation by miRNAs might be particularly conducive to this mode of action, consistent with reports that transfection modalities (Lytle et al, 2007) and choice of the promoters that drive reporter gene expression (Kong et al, 2008) can affect the apparent mode of target repression. Consistent with the elusive nature of miRNA mechanism(s), few molecular players have been identified. Mature miRNAs occur in a complex with Argonaute (AGO) family proteins, and it has been suggested that direct binding of AGO to the mRNA cap may be responsible for miRNA target repression (Kiriakidou et al, 2007), but this has been controversial (Eulalio et al, 2008b). The translation initiation factor eIF6 has been identified as a component of a large AGO2-containing complex in human cells and eIF6 depletion was shown to impair miRNA target gene silencing in human cells and C. elegans (Chendrimada et al, 2007). However, it has been suggested that the involvement of eIF6 may be indirect (Filipowicz et al, 2008), and studies of Drosophila cells have indicated that eIF6 may not be generally required for miRNA function (Eulalio et al, 2007, 2008b). Consistent with the latter notion, depletion of C. elegans eIF6 appears to enhance rather than diminish let-7 miRNA activity by genetic criteria (Ding et al, 2008). AGO proteins also bind to members of the GW182 protein family in various organisms and this interaction contributes to miRNA function (reviewed in Ding and Han, 2007). Tethering of GW182 to an mRNA leads to degradation of this mRNA, and, conversely, GW182 depletion impairs miRNA activity (Liu et al, 2005; Behm-Ansmant et al, 2006; Eulalio et al, 2008b). In C. elegans, combined loss of the two GW182-like proteins AIN-1/-2 partially phenocopies loss of the AGO proteins ALG-1/-2 and causes upregulation of reporter genes under miRNA control (Ding et al, 2005; Zhang et al, 2007). The level and extent to which AIN-1/-2 contribute to miRNA function have remained unknown, although it has been suggested that they might localize repressed miRNA targets to P-bodies to enable their degradation (Ding et al, 2005). We have focused here on the C. elegans let-7 miRNA to examine the mechanism of action of miRNAs in vivo. let-7 was originally identified as a component of the C. elegans heterochronic pathway (Reinhart et al, 2000), which controls the temporal fate of cells during postembryonic development. Several let-7 target genes have been identified (Slack et al, 2000; Abrahante et al, 2003; Lin et al, 2003; Grosshans et al, 2005; Lall et al, 2006) and among these, lin-41 and daf-12 have been characterized most extensively and their let-7-binding sites partially mapped (Reinhart et al, 2000; Slack et al, 2000; Vella et al, 2004; Grosshans et al, 2005). This availability of in vivo validated targets combined with the fact that the sequence of let-7 is perfectly conserved in animals (Pasquinelli et al, 2000; Lagos-Quintana et al, 2002), and that it has been used to examine miRNA mechanisms of action in diverse experimental systems (Bagga et al, 2005; Pillai et al, 2005; Nottrott et al, 2006; Mathonnet et al, 2007; Wakiyama et al, 2007), makes let-7 particularly suitable for our analysis. In addition, understanding the mode of action of this specific miRNA is of particular interest because of its important developmental and pathological functions as a potent regulator of stem cell fates and a tumour suppressor (reviewed in Büssing et al, 2008). We report that let-7 causes repression of translation initiation as well as degradation of its endogenous lin-41 and daf-12 target mRNAs. Other miRNAs silence their targets by the same mechanisms, and this includes lin-4 miRNA, previously reported to repress translation at a level after initiation (Olsen and Ambros, 1999; Seggerson et al, 2002). Translational repression requires the GW182 proteins AIN-1/-2, as does mRNA degradation. Our findings indicate that downregulation of translation initiation is widely used under physiological conditions in C. elegans and establish the nematode as a system for genetic dissection of this process. Results Translational blockade of endogenous let-7 target genes We recently observed widespread genetic interaction between let-7 and the translational machinery in C. elegans (Ding et al, 2008). These findings prompted us to examine whether let-7 regulates its targets translationally in vivo. To this end, we fractionated whole animal lysates by sucrose density gradient ultracentrifugation to analyse the polyribosome association of endogenous let-7 targets in wild-type and let-7(n2853) mutant C. elegans at the L3 developmental stage, when let-7 activity is low, and at the late L4 stage, when let-7 activity is high (Reinhart et al, 2000) (Figure 1A; Supplementary Figure S1). As the two let-7 targets daf-12 and lin-41 (Slack et al, 2000; Grosshans et al, 2005) are expressed at very low levels in L4 stage larvae (Snow and Larsen, 2000; Bagga et al, 2005 and this study, below), we used reverse transcription–quantitative PCR (RT–qPCR) to quantify them. It is to be noted that all experiments were performed using random hexamer oligonucleotides to prime RT, to include even mRNA, the poly(A) tail of which might be short due to the action of the miRNA (Eulalio et al, 2008a; Filipowicz et al, 2008). Additional control experiments, described below, further confirmed that we are detecting full-length mRNAs rather than partially stable degradation fragments. Figure 1.let-7 decreases translation initiation on daf-12 and lin-41 mRNAs. (A) Typical polysome profile of wild-type and let-7(n2853) animals with or without EDTA treatment. (B) Distribution of daf-12 and act-1 mRNAs across polysome profiles from synchronized wild-type and let-7(n2853) animals at the L3 and late L4 stage, with or without EDTA treatment. (C) Polysomal fraction of daf-12, lin-41, ama-1 and act-1 mRNAs in L3 and late L4 wild-type and let-7(n2853) animals as a percentage of the total (*P<0.05, **P<0.01). mRNA levels were analysed by RT–qPCR. EDTA treatment was performed in duplicate, one representative experiment is shown. All other panels in this and subsequent figures show the averages of n⩾3 independent experiments. For this and all subsequent figures, ‘WT’ denotes the wild-type N2 strain, and error bars are s.e.m. Download figure Download PowerPoint We found that both lin-41 and daf-12 mRNAs were moderately, but consistently depleted from the highly translated polysomal fractions in wild-type relative to let-7 mutant animals at the late L4 stage (Figure 1B and C; Supplementary Figure S1), in agreement with decreased translation initiation (Eulalio et al, 2008a). By contrast, ama-1 and act-1 mRNAs, which are not targeted by let-7, displayed similar translational profiles in both strains (Figure 1B and C; Supplementary Figure S1). L3 stage animals express little or no let-7 (Reinhart et al, 2000); accordingly, we see no difference when comparing polysomal association of daf-12 and lin-41 mRNAs between let-7 mutant and wild-type animals at this stage (Figure 1B and C; Supplementary Figure S1). Moreover, in wild-type animals, polysome association of daf-12 and lin-41 is decreased at L4 compared with L3 stage, consistent with the establishment of an inhibitory mechanism affecting translation initiation as let-7 expression starts. A more moderate decrease of polysome association is also seen when performing this comparison for let-7 mutant animals, suggesting that the n2853 allele may provide residual let-7 activity or that alternative mechanisms, possibly the let-7 sister miRNAs mir-48, mir-84 and mir-241 (Abbott et al, 2005; Li et al, 2005), may contribute. To exclude the possibility that the RNA that we analysed in our sucrose gradients was not representative for the total pool of cellular RNA, we performed the following control experiments. We used TRIzol to extract total RNA directly from ground worms, from the cleared lysate used for sucrose gradient centrifugation and from the pellet left behind upon lysate clearing. We found, first, that ∼90% of the RNA is in the lysate supernatant and will thus be loaded on the sucrose gradient. Second, composition of RNA in the supernatant and pellet is comparable, neither let-7 target nor control mRNAs are preferentially enriched in, or depleted from, the supernatant relative to RNA retained in the pellet (Supplementary Figure S2). Finally, although increasing sucrose concentration in total lysates decreased the yield of extracted RNA, RNA composition was largely unaffected (Supplementary Figure S2). To ensure comparable recovery from each fraction and greatest possible reproducibility, we equalized sucrose concentration in all fractions to 30% (w/v) prior to RNA extractions in all our experiments. This set of control experiments confirms that any results that we obtained in our analysis can be considered representative of the total pool of cellular RNA. To determine further whether the fast-sedimenting mRNA was indeed associated with polyribosomes, we treated lysates with EDTA and observed that all four mRNAs were shifted to the top of gradients. Distributions became indistinguishable for late L4 wild-type and let-7(n2853) animals (Figure 1; Supplementary Figure S1). As EDTA also disrupts non-ribosomal ribonucleoprotein complexes, we further used puromycin to disassemble specifically polysomes by inducing premature termination of the elongating peptide chains. Puromycin treatment of extracts collapsed polysomes and shifted the mRNAs deeper into the gradient (Supplementary Figure S3), possibly reflecting aggregation of the mRNA and not further pursued by us. The resulting sedimentation patterns were indistinguishable for wild-type and let-7 animals and occurred for let-7 target as well as control mRNAs. The coincident loss of polysomes and shift of mRNAs demonstrates that our assay examines mRNAs associated with translation-competent ribosomes. We conclude from these data that let-7 depletes its targets lin-41 and daf-12 from bona fide polysomes, consistent with blocking translation initiation on these mRNAs. Translational repression requires let-7 complementary sites in the lin-41 3′UTR The lin-41 3′ untranslated region (3′UTR) is necessary and sufficient to confer let-7-mediated regulation on an unrelated reporter gene (Slack et al, 2000). To verify that let-7 impaired lin-41 translation by binding to the lin-41 3′UTR, we employed transgenic animals expressing a lacZ reporter gene fused to the lin-41 3′UTR or a mutant variant thereof lacking the let-7-binding sites (Figure 2A). We expressed the transgene from the col-10 promoter to accumulate it specifically in the seam cells, where let-7 mediates lin-41 repression (Slack et al, 2000; Johnson et al, 2003). Figure 2.lin-41 translational repression is mediated through let-7-binding sites. (A) Schematic representation of the reporter strains. Black square, WT N2; Is[col-10∷lacZ∷lin-41], white square, let-7(n2853); Is[col-10∷lacZ∷lin-41], grey square, WT N2; xeIs11[col-10∷lacZ∷lin-41-ΔLCS]. The vertical lines in the lin-41 3′UTR represent let-7 complementary sequences (LCSs). (B) lacZ and act-1 mRNA distributions, determined by RT–qPCR, across polysome profiles. (C) Polysomal fraction of lacZ, lin-41 and act-1 mRNAs as a percentage of the total. (D) Average number of ribosomes on lacZ and act-1 mRNA (*P<0.05, **P<0.01). Synchronized late L4 reporter animals were used. Note that the repression of endogenous lin-41, carrying the full-length 3′UTR, is maintained in the wild-type strain expressing the truncated col-10∷lacZ∷lin-41-ΔLCS transgene. The difference between endogenous lin-41 translational repression in let-7(n2853) and wild-type animals is no longer statistically significant (P=0.067) in transgenic animals, possibly due to sequestering of endogenous let-7 by reporter transgenes in wild-type animals. Download figure Download PowerPoint Consistent with inhibition of translation initiation, we observed that only 40% of the reporter mRNA was associated with polysomes in wild-type animals, whereas this level reached almost 70% in let-7 mutant animals. Deletion of the validated let-7-binding sites in the reporter 3′UTR (Vella et al, 2004) relieved translational repression to the same extent in wild-type animals (Figure 2B and C). Consequently, let-7 mutation or deletion of its binding sites increased the average number of ribosomes per lacZ mRNA by more than two-fold relative to the wild-type situation, whereas the average number of ribosomes per act-1 mRNA stayed constant (Figure 2D; see Materials and methods for details on the calculation). This result shows that the interaction between let-7 and its binding sites in the lin-41 3′UTR mediates significant translational repression of the target mRNAs. This is confirmed by our finding that loss of let-7 regulation causes a ⩾5-fold derepression of the lacZ reporter (Supplementary Figure S4), although mRNA levels change less than two-fold (see below). mRNA degradation does not correlate with translational repression Bagga et al (2005) observed dramatic reduction of target mRNA levels in the presence of their cognate miRNAs in C. elegans. By RT–qPCR, we determined mRNA levels of the let-7 targets in total RNA that we prepared from aliquots of the same whole animal lysates that were used for the polysome profile experiments (Figure 3A). At the L3 stage, lin-41 and daf-12 mRNA levels are similar in wild-type and let-7(n2853) animals. However, at the late L4 stage, lin-41 mRNA is six-fold and daf-12 two-fold more abundant in let-7(n2853) relative to wild-type animals. Similar ratios were obtained when summing up the amounts of these mRNAs across all fractions of the sucrose gradients, further confirming that RNA extracted from the gradients is representative of total cellular full-length mRNAs. It is to be noted that the levels of lin-41 and daf-12 mRNAs are reduced by two-fold even in the let-7 mutant animals between the L3 and L4 stages. Figure 3.let-7 mediates target mRNA degradation. (A) Analysis of daf-12, lin-41, ama-1 and act-1 total mRNA levels by RT–qPCR. Data are normalized for the average of ama-1 and act-1 values (**P<0.01). (B) Northern blot analysis of lin-41 and act-1 mRNA levels using 20 μg of total RNA. Numbers indicate lin-41 mRNA levels normalized for act-1. Wild-type and let-7(n2853) samples are presented in separated panels for clarity; however, RNA samples were assessed on the same membrane and exposition time is identical. (C) Analysis of lacZ, lin-41, act-1 and ama-1 total mRNA levels in L4 stage reporter animals by RT–qPCR, data normalized for the average of act-1 and ama-1 values. Download figure Download PowerPoint For lin-41, our results are in agreement with those seen by Bagga et al (2005), and northern blot analysis of total RNA using a probe against lin-41 identified a single band, the intensity of which mirrored the signal obtained by RT–qPCR in the same backgrounds (Figure 3B). Although lin-41 mRNA levels in individual sucrose gradient fractions were below the limit of detection by northern blot analysis, these results essentially exclude the possibility that accumulation of lin-41 mRNA degradation products could bias our RT–qPCR results and confirm that we reliably quantify full-length mRNAs. For daf-12 mRNA, its low abundance prevents detection by northern blotting even in unfractionated, total RNA without prior selection of polyadenylated mRNA (Snow and Larsen, 2000). Therefore, to confirm that our RT–qPCR assay similarly measures the levels of full-length daf-12 mRNA, we tested a second set of primers, and obtained comparable results as expected (Supplementary Figure S5A). Finally, we examined the expression levels of both daf-12 and lin-41 using cDNA obtained through oligo-dT-primed RT. Again, we obtained comparable results (Supplementary Figure S5B and C), arguing against the detection of a stable degradation product and suggesting that any residual poly(A) tail on these mRNAs is sufficient to support priming through oligo-dT oligonucleotides. In summary, we confirm by several independent methods that our assays quantify full-length mRNAs, and we find that the daf-12 and lin-41 mRNAs are not only translationally repressed by let-7 but also subject to degradation. Translational repression of daf-12 is at least equal to that of lin-41 but the decrease of daf-12 mRNA levels is more modest, suggesting that translational repression and transcript degradation may not be directly linked. Indeed, although the lacZ∷lin-41 reporter mRNA is very efficiently repressed translationally, mRNA levels differed by less than two-fold in wild-type relative to let-7(n2853) animals (Figure 3C). Although these findings strongly argue against a scenario where lower abundance of an mRNA diminishes its access to the translational machinery, we wished to exclude the possibility further that the translational effects that we observed were due to altered mRNA levels. ugt-63 and vit-1 are differentially expressed in synchronized late L4 wild-type and let-7(n2853) animals but are not direct targets of let-7 (B Hurschler and HG, unpublished data). Although vit-1 was four-fold less abundant in let-7(n2853) than in wild-type animals, and ugt-63 was two-fold more abundant, the translational profiles of both genes were similar in wild-type and let-7(n2853) (Supplementary Figure S6). Thus, altered mRNA levels per se do not appear to influence the efficiency of translation initiation. Multiple miRNAs function by preventing translation initiation The finding that let-7 mediates repression of translation initiation on its targets in C. elegans was unexpected, as C. elegans lin-4 was previously reported to repress these mRNAs at a step downstream of translation initiation (Olsen and Ambros, 1999; Seggerson et al, 2002). To determine whether repression of translation initiation is specific for let-7 or a more general mechanism, we tested whether lin-4 repressed translation initiation of lin-14 and lin-28, two experimentally validated targets (Wightman et al, 1993; Moss et al, 1997). lin-4 is first expressed in the mid-L1 stage and represses lin-14 by late L1/early L2 and lin-28 one stage later (Olsen and Ambros, 1999; Seggerson et al, 2002). When we compared extracts from late L2 stage wild-type and lin-4(e912) mutant animals, we observed that both mRNAs were shifted into the polysomal fraction in the mutant (Figure 4A and B). This shift is particularly pronounced for lin-28, where the effect is highly statistically significant (Figure 4A). By contrast, polysome association of the control mRNAs act-1 and ama-1 and the let-7 target daf-12 is identical in lin-4(e912) and wild-type animals (Figure 4A and B). We conclude that lin-4, similar to let-7, can repress its target at the level of translation initiation. Figure 4.lin-4 inhibits translation initiation of lin-14 and lin-28. (A) Polysomal fraction of lin-14, lin-28, act-1, ama-1 and daf-12 in synchronized late L2 wild-type and lin-4(e912) animals as a percentage of the total (*P<0.05, **P<0.01). (B) lin-14, lin-28 and daf-12 distribution across polysome profiles from synchronized late L2 wild-type and lin-4(e912) animals. (C) Analysis of total mRNA levels in synchronized late L2 wild-type and lin-4(e912) animals by RT–qPCR, data are normalized for the average of the control gene values. Download figure Download PowerPoint lin-14 and lin-28 transcript levels are increased in lin-4 mutants compared with wild-type animals, whereas daf-12, act-1 and ama-1 mRNA levels remain unchanged (Figure 4C). The observation that lin-4 induces a stronger translational blockade of lin-28 than of lin-14 and conversely a more pronounced degradation of lin-14 than of lin-28 further suggests that translational repression and target mRNA degradation are not directly linked mechanisms. Translational repression and degradation of miRNA targets require the GW182 proteins AIN-1 and AIN-2 Having established that miRNAs mediate both target mRNA degradation and translational repression in vivo, we sought to identify the factors mediating these mechanisms. Good candidates were the GW182 homologues AIN-1 and AIN-2 (Ding et al, 2005; Zhang et al, 2007), as depletion of GW182 causes upregulation of miRNA target genes in various systems (Ding and Han, 2007). However, although mRNA degradation is readily prevented upon GW182 depletion, derepression of those miRNA targets that are not strongly regulated by degradation is typically well below that seen with AGO depletion (Behm-Ansmant et al, 2006; Eulalio et al, 2007), consistent with the proposal that GW182 proteins might enhance miRNA activity by targeting repressed mRNAs to P-bodies for degradation (Ding et al, 2005). To determine whether depletion of the GW182 family members AIN-1 and AIN-2 permitted uncoupling of translational repression and degradation of miRNA targets, we performed polysome profile analyses on L4 stage wild-type and ain-2(RNAi); ain-1(ku322) animals and analysed various targets of multiple miRNAs: the let-7 targets daf-12 and lin-41, the lin-4 targets lin-14 and lin-28, the lsy-6 targets cog-1 (Johnston and Hobert, 2003) and hbl-1, which is targeted by mir-48, mir-84, mir-241, let-7 and lin-4 (Abrahante et al, 2003; Lin et al, 2003; Abbott et al, 2005). As predicted, depletion of AIN-1/-2 increased lin-41 and daf-12 transcript levels (Figure 5A). However, to our surprise, translational repression of both let-7 targets was also efficiently relieved (Figure 5B and C). In fact, the relief of both modes of let-7 target repression was more extensive in ain-2; ain-1 than in let-7(n2853) mutant animals, possibly suggesting that remaining let-7 activity or distinct miRNAs, perhaps of the let-7 family, contribute to residual repression of lin-41 and daf-12 in let-7(n2853) animals. Consistent with this idea, mRNA levels of the two let-7 targets daf-12 and lin-41 are upregulated in miR-48 miR-241; miR-84 triple mutant animals (Supplementary Figure S7). Figure 5.AIN-1 and AIN-2 mediate translational repression and degradation of miRNA target mRNAs. (A) Analysis of total mRNA levels in synchronized late L4 wild-type and ain-2(RNAi); ain-1(ku322) animals by RT–qPCR, data are normalized for the average of the control gene values. (B) lin-41, lin-14 and tbb-2 distribution across polysome profiles from synchronized late L4 wild-type and ain-2(RNAi); ain-1(ku322) animals. (C) Polysomal fraction of several miRNA targets and control genes in synchronized late L4 wild-type and ain-2(RNAi); ain-1(ku322) animals as a percentage of the total (*P<0.05). Download figure Download PowerPoint Translational repression was also relieved for lin-14, lin-28, cog-1 and hbl-1, although the results for lin-28 and hbl-1 narrowly missed statistical significance (lin-28, P=0.053; hbl-1, P=0.056). We also analysed genes not known to be miRNA targets (act-1, tbb-2, ama-1 and eft-2). We observed no effect on total mRNA levels and no consistent trend of translational upregulation in response to AIN-1/-2 depletion (Figure 5). Low abundance of the investigated miRNA target mRNAs in late L4 wild-type animals (see Figure 3B) prevented us from performing northern blot analysis on polysome profile fractions. However, consistent results were obtained by RT–qPCR with multiple lin-14 primer pairs (Supplementary Figure S8) and by semiquantitative classical RT–PCR (Supplementary Figure S9) confirming our observation that translational repression of miRNA target is relieved in AIN-1/-2 depleted animals. Taken together, these data reveal that translational control is a mechanism that is widely used by miRNAs in vivo. Equally significant, our results show that AIN-1/-2 have a general and important function in the process. Notably, although transcript levels of lin-14, lin-28 and hbl-1 increased in ain-2; ain-1 mutant relative to wild-type animals, cog-1 mRNA levels remained unchanged (Figure 5C), demonstrating that translational repression can occur independently of target mRNA degradation. Discussion We report here that endogenous daf-12 and lin-41 mRNAs are t