• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of embojLink to Publisher's site
EMBO J. Feb 4, 2009; 28(3): 213–222.
Published online Jan 8, 2009. doi:  10.1038/emboj.2008.275
PMCID: PMC2637332

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

Abstract

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.

Keywords: AIN-1, let-7, lin-4, microRNA, translational repression

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 ...

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] ...

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 [gt-or-equal, slanted]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 ...

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, ** ...

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 ...

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 translationally controlled by let-7 in vivo. Polysomal shifts can even be seen in whole worm lysates, despite the fact that let-7 regulates these targets only in a subset of those tissues where they are expressed (Antebi et al, 2000; Slack et al, 2000; Johnson et al, 2003). Nonetheless, the degree of spatial and temporal co-expression of the miRNA and its targets limits the sensitivity of our assay, as demonstrated for pha-4, a third experimentally validated let-7 target (Grosshans et al, 2005). let-7 regulates pha-4 in the intestine (Grosshans et al, 2005), but not in the pharynx, where pha-4 expression is particularly strong, and where let-7 is not co-expressed (Azzaria et al, 1996; Johnson et al, 2003). Under these conditions, we can neither observe polysomal shifts (Supplementary Figure S1) nor pha-4 mRNA accumulation (data not shown) in let-7 mutant relative to wild-type animals. By contrast, the magnitude of repression of translation initiation can be well appreciated for the col-10::lacZ::lin-41 reporter mRNA, which is exclusively expressed in the seam cells where let-7 is also active.

We subsequently expanded our studies to mRNAs targeted by other miRNAs, including the lin-4 targets lin-14 and lin-28 and found that these, too, were repressed at the level of translation initiation. These findings resonate well with results from cell-free and a subset of cell culture-based assays (Eulalio et al, 2008a; Filipowicz et al, 2008), and extend these studies by demonstrating such function under physiological conditions. Equally important, most published evidence for translation initiation has been obtained through the use of transfected target reporter genes, the mode of repression of which appears to be susceptible to transfection conditions (Lytle et al, 2007) and promoter choice (Kong et al, 2008). To our knowledge, only a single endogenous target gene was demonstrated to be repressed by this mechanism (Bhattacharyya et al, 2006), whereas this has been ruled out for others (Olsen and Ambros, 1999; Seggerson et al, 2002; Kong et al, 2008). Our study now demonstrates that repression of translation initiation is nonetheless widespread for endogenous miRNA targets, and different miRNAs.

Our finding that lin-4 represses its targets, at least in part, at the level of translation initiation contrasts with earlier experiments that had revealed largely unchanged polysomal distributions of C. elegans lin-14 and lin-28 before (L1 stage) and after (L2 stage) the onset of expression of their cognate miRNA, lin-4 (Olsen and Ambros, 1999; Seggerson et al, 2002). The reason for this discrepancy is currently unclear, but as lin-4 mutant animals were not compared with wild-type animals in the earlier studies, it is possible that the resulting translational profiles might also have reflected developmental, lin-4-independent effects such as potential repression of lin-28 by LIN-66 (Morita and Han, 2006). We also note that at least one of the earlier publications (Olsen and Ambros, 1999) displayed a—statistically nonsignificant—trend of lin-14 shifting to the (sub-)monosomal fraction at the L2 stage, when lin-4 expression is high.

Other miRNAs, in addition to lin-4, possibly its ‘sister' mir-237, or even let-7 (Reinhart et al, 2000; Slack et al, 2000; Esquela-Kerscher et al, 2005; Grosshans et al, 2005; Morita and Han, 2006), may also regulate lin-14 or lin-28, and contribute to the polysomal shift observed in ain-2; ain-1 mutant animals. However, although this remains to be tested for lin-28, we did not detect any change in lin-14 mRNA levels or translation in response to the let-7(n2853) mutation (data not shown). Irrespective of this possibility, our experiments using lin-4 mutant animals clearly demonstrate that this miRNA can mediate repression of translation initiation.

In most instances, we observed significant amounts of cognate mRNA degradation alongside translational repression, but the extent of degradation varied by target. Moreover, there was no clear correlation between the extent of translational repression and target mRNA degradation, for example, we observed more degradation for endogenous lin-41 than for the reporter mRNA, although less translational silencing is apparent for the endogenous transcript. Although we cannot formally rule out the possibility that this specific case reflects differences between endogenous targets and targets expressed from transgenes or that transcriptional effects mediated by the lin-41 promoter may contribute to these differences, we favour the idea that translational repression and target mRNA degradation may be independent mechanisms and that seam cells favour translational repression. This is consistent with an earlier study showing that different types of cultured cells evoke different responses to identical target mRNA reporters, with degradation dominating in some cell lines and translational blockade in others (Schmitter et al, 2006). Indeed, the observation that cog-1 is regulated translationally, but not at the level of mRNA degradation, further supports our conclusion that mRNA degradation and translational repression are two distinct mechanisms in vivo that may, however, frequently act together on the same miRNA targets.

It is to be noted that our results cannot rule out that the two mechanisms might function sequentially in that translational repression precedes mRNA degradation (e.g. Selbach et al, 2008). Indeed, we note that repression of translation initiation by lin-4 appears more prominent for lin-28 than lin-14. As lin-14 is repressed at an earlier developmental stage than lin-28 (Olsen and Ambros, 1999; Seggerson et al, 2002), it is tempting to speculate that increased lin-14 mRNA degradation might deplete the monosomal pool of translationally repressed mRNA, effectively reducing the apparent polysomal shift. Analysis of polysome profiles at increased temporal resolution might help to address this possibility in the future.

Early reports on GW182 suggested a more auxiliary function in miRNA activity, with a greater importance in repressing targets susceptible to mRNA degradation (Ding et al, 2005; Liu et al, 2005; Behm-Ansmant et al, 2006; Eulalio et al, 2007). However, as AIN-1/-2 appear rather distantly related to GW182 proteins (Behm-Ansmant et al, 2006), it was unknown whether their functions can be inferred from GW182 activity in other organisms. Moreover, recent work on Drosophila GW182 has shown that degradation-independent, possibly translational, repressive mechanisms may also crucially involve GW182 (Eulalio et al, 2008b), possibly in an miRNA- and/or target-specific manner. We find that depletion of the GW182 proteins AIN-1/-2 severely impairs both cognate mRNA degradation and translational control in vivo, for a number of different miRNA targets and miRNAs, supporting the notion that these proteins are widely used, essential effectors of miRNA activities. This conclusion is also consistent with the alg-1/2-like phenotypes observed in ain-2; ain-1 double mutant animals (Zhang et al, 2007). In view of the fact that both mRNA degradation and translational repression require AIN-1/-2, we speculate that GW182 proteins may coordinate these two activities, possibly through interaction with distinct mediators or effectors, the identities of which remain to be elucidated.

Taken together, our study provides insights into miRNA function in an animal model and establishes C. elegans let-7 as a model for the genetic dissection of miRNA-mediated repression of translation initiation, complementing available biochemical systems. The fact that translational repression in vivo may be substantial at least for a subset of targets, and possibly occur even without any degradation altogether, suggests that the identification of targets of this important miRNA will benefit greatly from recently established proteomics approaches (Baek et al, 2008; Selbach et al, 2008).

Materials and methods

C. elegans strains and RNAi

Wild-type N2, MT7626: let-7(n2853), MH2385: ain-1(ku322) and DR721: lin-4(e912) strains were provided by the CGC; CT5a: N2;Is[goa-1::gfp; col-10::lacZ::lin-41] (Caudy et al, 2003) by R Plasterk; VT1066: mir-48 mir-241(nDf51); mir-84(n4037) (Abbott et al, 2005) by V Ambros. The HW211: let-7(n2853);Is[goa-1::gfp; col-10::lacZ::lin-41] strain was obtained by crossing CT5a with MT7626. The HW390: N2;xeIs11[rol-6(su1006); col-10::lacZ::lin-41ΔLCS 1-3] strain was generated by genomic integration of an extrachromosomal array made of pFS1031 and rol-6(su1006) (Vella et al, 2004), followed by several rounds of backcrossing to N2. As ain-2(tm1863); ain-1(ku322) (Zhang et al, 2007) double mutant animals grow very poorly, we exposed ain-1(ku322) animals to ain-2(RNAi), starting with L1 larvae and using a published RNAi feeding construct and protocol (Kamath et al, 2003; Grosshans et al, 2005). Enhancement of the alae defect phenotype from 21% penetrant in the ain-1(ku322) single mutant to 100% in the double mutant confirmed efficient ain-2 knockdown (n[gt-or-equal, slanted]17 each). To circumvent reduced brood size associated with the lin-4(e912) allele and obtain a sufficiently large synchronized population, lin-4(e912) animals were expanded at 20°C on lin-14(RNAi). Following synchronization by hatching in M9 buffer, animals were shifted back to standard non-RNAi food (OP50) and grown to the desired stage for extract preparation. The reappearance of the lin-4(e912) phenotypes (long, egg-laying defective) on these animals excluded the possibility that lin-14(RNAi) had prolonged effects.

Polysome profile analysis

A detailed description can be found in the Supplementary data. Briefly, lysates of synchronized worms were layered on linear sucrose gradients (15–60% w/v) and centrifuged for 3 h at 39 000 r.p.m., 4°C, using a SW-40 rotor and an Optima L-80 XP Ultracentrifuge (Beckman Coulter). The gradients were fractionated in 12 fractions of equal volume while absorbance at 254 nm was recorded. The entire gradient was fractionated so that any pelleted material would be recovered with the last fraction. However, we typically found very little RNA in this fraction, suggesting that RNAs did not substantially occur in heavy particles or compartments under our experimental conditions. After adjusting sucrose concentration in each fraction to 30% (w/v), RNA was extracted using TRIzol (Invitrogen) and RNA integrity confirmed on ethidium bromide-stained agarose gels before proceeding to RT. RNA recovery was quantitative under these conditions reaching up to 89% of input RNA. See Results section for a discussion of control experiments confirming that RNA extracted from the lysate is representative of the composition of total cellular RNA.

RT–qPCR

RNA RT was performed using the ImProm-II™ Reverse Transcription System (Promega) with random hexamer primers, according to the manufacturer's recommendations using equal amounts of RNA (400 or 800 ng) for each sample to avoid saturating the RT reactions in fractions with high concentrations of RNA. For polysome profile fractions, relative transcript levels quantified by qPCR (below) were subsequently corrected for the total amount of RNA extracted from each fraction and expressed as a percentage of the total amount recovered for the gradients. Identical results were obtained using oligo-(dT)15 primers and equal volumes of RNA without applying any correction, validating the method and establishing that qPCR following RT by random hexamer is unlikely to detect stable degradation fragments. This was also directly examined for lin-14 and daf-12 using distinct qPCR primer sets, which yielded comparable results to the original primers.

qPCR reactions were performed in technical duplicate using the ABsolute™ QPCR SYBR® Green ROX Mix (Thermo Fisher Scientific), according to the manufacturer's recommendations, on an ABI Prism 7000 Sequence Detection System coupled to ABI Prism 7000 SDS 1.0 Software (Applied Biosystems). Relative transcript levels were calculated using the 2−Δ Ct method (Livak and Schmittgen, 2001). For all primer pairs (Supplementary data), amplification efficiencies were determined to be equal or superior to 1.8. Control reactions lacking either the reverse transcriptase or template mRNA confirmed specificity of the amplification reaction.

Northern blot

RNA electrophoresis and transfer were performed as described earlier (Bagga et al, 2005). UV crosslinked membranes were hybridized using ULTRAhyb hybridization buffer (Ambion) according to the manufacturer's recommendations with randomly radiolabelled probes prepared from PCR-amplified DNA (see Supplementary data for oligonucleotide sequences). Radioactive signals were detected and quantified using a Storage Phosphor Screen and a Typhoon 9400 with the Imagequant TL software (all GE Healthcare).

Calculation of average number of ribosomes per mRNA

To calculate the average number of ribosomes per mRNA, each gradient fraction was assigned an average number of ribosomes by counting the peaks of the polysome profile at 254 nm. This number was multiplied with relative amount of the mRNA detected in this specific fraction. The sum of this product over all the fractions yielded the average number of ribosomes per mRNA.

Statistical methods

All statistical significances were calculated using the paired one-tailed Student's t-test. Note that the statistical significance in this stringent test not only depends on the average and standard deviation of the data sets but also on the variation of the difference between the paired values so that error bars will not fully reflect the statistical significance obtained through this test.

Supplementary Material

Supplementary Information

Acknowledgments

We thank V Ambros, M Han, R Plasterk and the Caenorhabditis Genetics Center, which is funded by the NIH National Center for Research Resources, for providing nematode strains. We are grateful to M Bühler, W Filipowicz, F Meins Jr, and F Slack for a critical reading of earlier versions of our manuscript. XCD was supported by a PhD student fellowship from the Boehringer Ingelheim Foundation. Research in HG's lab is funded by the Novartis Research Foundation and the Swiss National Science Foundation.

References

  • Abbott AL, Alvarez-Saavedra E, Miska EA, Lau NC, Bartel DP, Horvitz HR, Ambros V (2005) The let-7 microRNA family members mir-48, mir-84, and mir-241 function together to regulate developmental timing in Caenorhabditis elegans. Dev Cell 9: 403–414 [PMC free article] [PubMed]
  • Abrahante JE, Daul AL, Li M, Volk ML, Tennessen JM, Miller EA, Rougvie AE (2003) The Caenorhabditis elegans hunchback-like gene lin-57/hbl-1 controls developmental time and is regulated by microRNAs. Dev Cell 4: 625–637 [PubMed]
  • Antebi A, Yeh WH, Tait D, Hedgecock EM, Riddle DL (2000) daf-12 encodes a nuclear receptor that regulates the dauer diapause and developmental age in C. elegans. Genes Dev 14: 1512–1527 [PMC free article] [PubMed]
  • Azzaria M, Goszczynski B, Chung MA, Kalb JM, McGhee JD (1996) A fork head/HNF-3 homolog expressed in the pharynx and intestine of the Caenorhabditis elegans embryo. Dev Biol 178: 289–303 [PubMed]
  • Baek D, Villén J, Shin C, Camargo FD, Gygi SP, Bartel DP (2008) The impact of microRNAs on protein output. Nature 455: 64–71 [PMC free article] [PubMed]
  • Bagga S, Bracht J, Hunter S, Massirer K, Holtz J, Eachus R, Pasquinelli AE (2005) Regulation by let-7 and lin-4 miRNAs results in target mRNA degradation. Cell 122: 553–563 [PubMed]
  • Behm-Ansmant I, Rehwinkel J, Doerks T, Stark A, Bork P, Izaurralde E (2006) mRNA degradation by miRNAs and GW182 requires both CCR4:NOT deadenylase and DCP1:DCP2 decapping complexes. Genes Dev 20: 1885–1898 [PMC free article] [PubMed]
  • Bhattacharyya SN, Habermacher R, Martine U, Closs EI, Filipowicz W (2006) Relief of microRNA-mediated translational repression in human cells subjected to stress. Cell 125: 1111–1124 [PubMed]
  • Bushati N, Cohen SM (2007) microRNA functions. Annu Rev Cell Dev Biol 23: 175–205 [PubMed]
  • Büssing I, Slack FJ, Großhans H (2008) let-7 microRNAs in development, stem cells and cancer. Trends Mol Med 14: 400–409 [PubMed]
  • Caudy AA, Ketting RF, Hammond SM, Denli AM, Bathoorn AMP, Tops BBJ, Silva JM, Myers MM, Hannon GJ, Plasterk RHA (2003) A micrococcal nuclease homologue in RNAi effector complexes. Nature 425: 411–414 [PubMed]
  • Chendrimada TP, Finn KJ, Ji X, Baillat D, Gregory RI, Liebhaber SA, Pasquinelli AE, Shiekhattar R (2007) MicroRNA silencing through RISC recruitment of eIF6. Nature 447: 823–828 [PubMed]
  • Ding L, Han M (2007) GW182 family proteins are crucial for microRNA-mediated gene silencing. Trends Cell Biol 17: 411–416 [PubMed]
  • Ding L, Spencer A, Morita K, Han M (2005) The developmental timing regulator AIN-1 interacts with miRISCs and may target the Argonaute protein ALG-1 to cytoplasmic P bodies in C. elegans. Mol Cell 19: 437–447 [PubMed]
  • Ding XC, Slack FJ, Großhans H (2008) The let-7 microRNA interfaces extensively with the translation machinery to regulate cell differentiation. Cell Cycle 7: 3083–3090 [PMC free article] [PubMed]
  • Esquela-Kerscher A, Johnson SM, Bai L, Saito K, Partridge J, Reinert KL, Slack FJ (2005) Post-embryonic expression of C. elegans microRNAs belonging to the lin-4 and let-7 families in the hypodermis and the reproductive system. Dev Dyn 234: 868–877 [PMC free article] [PubMed]
  • Eulalio A, Huntzinger E, Izaurralde E (2008a) Getting to the root of miRNA-mediated gene silencing. Cell 132: 9–14 [PubMed]
  • Eulalio A, Huntzinger E, Izaurralde E (2008b) GW182 interaction with Argonaute is essential for miRNA-mediated translational repression and mRNA decay. Nat Struct Mol Biol 15: 346–353 [PubMed]
  • Eulalio A, Rehwinkel J, Stricker M, Huntzinger E, Yang S, Doerks T, Dorner S, Bork P, Boutros M, Izaurralde E (2007) Target-specific requirements for enhancers of decapping in miRNA-mediated gene silencing. Genes Dev 21: 2558–2570 [PMC free article] [PubMed]
  • Filipowicz W, Bhattacharyya SN, Sonenberg N (2008) Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight? Nat Rev Genet 9: 102–114 [PubMed]
  • Giraldez AJ, Mishima Y, Rihel J, Grocock RJ, Dongen SV, Inoue K, Enright AJ, Schier AF (2006) Zebrafish MiR-430 promotes deadenylation and clearance of maternal mRNAs. Science 312: 75–79 [PubMed]
  • Grosshans H, Johnson T, Reinert KL, Gerstein M, Slack FJ (2005) The temporal patterning microRNA let-7 regulates several transcription factors at the larval to adult transition in C. elegans. Dev Cell 8: 321–330 [PubMed]
  • Johnson SM, Lin SY, Slack FJ (2003) The time of appearance of the C. elegans let-7 microRNA is transcriptionally controlled utilizing a temporal regulatory element in its promoter. Dev Biol 259: 364–379 [PubMed]
  • Johnston RJ, Hobert O (2003) A microRNA controlling left/right neuronal asymmetry in Caenorhabditis elegans. Nature 426: 845–849 [PubMed]
  • Jones-Rhoades MW, Bartel DP, Bartel B (2006) MicroRNAs and their regulatory roles in plants. Annu Rev Plant Biol 57: 19–53 [PubMed]
  • Kamath RS, Fraser AG, Dong Y, Poulin G, Durbin R, Gotta M, Kanapin A, Bot NL, Moreno S, Sohrmann M, Welchman DP, Zipperlen P, Ahringer J (2003) Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature 421: 231–237 [PubMed]
  • Kiriakidou M, Tan GS, Lamprinaki S, Planell-Saguer MD, Nelson PT, Mourelatos Z (2007) An mRNA m7G cap binding-like motif within human Ago2 represses translation. Cell 129: 1141–1151 [PubMed]
  • Kong YW, Cannell IG, de Moor CH, Hill K, Garside PG, Hamilton TL, Meijer HA, Dobbyn HC, Stoneley M, Spriggs KA, Willis AE, Bushell M (2008) The mechanism of micro-RNA-mediated translation repression is determined by the promoter of the target gene. Proc Natl Acad Sci USA 105: 8866–8871 [PMC free article] [PubMed]
  • Lagos-Quintana M, Rauhut R, Yalcin A, Meyer J, Lendeckel W, Tuschl T (2002) Identification of tissue-specific microRNAs from mouse. Curr Biol 12: 735–739 [PubMed]
  • Lall S, Grün D, Krek A, Chen K, Wang Y, Dewey CN, Sood P, Colombo T, Bray N, Macmenamin P, Kao H, Gunsalus KC, Pachter L, Piano F, Rajewsky N (2006) A genome-wide map of conserved microRNA targets in C. elegans. Curr Biol 16: 460–471 [PubMed]
  • Li M, Jones-Rhoades MW, Lau NC, Bartel DP, Rougvie AE (2005) Regulatory mutations of mir-48, a C. elegans let-7 family microRNA, cause developmental timing defects. Dev Cell 9: 415–422 [PubMed]
  • Lin S, Johnson SM, Abraham M, Vella MC, Pasquinelli A, Gamberi C, Gottlieb E, Slack FJ (2003) The C elegans hunchback homolog, hbl-1, controls temporal patterning and is a probable microRNA target. Dev Cell 4: 639–650 [PubMed]
  • Liu J, Rivas FV, Wohlschlegel J, Yates JR, Parker R, Hannon GJ (2005) A role for the P-body component GW182 in microRNA function. Nat Cell Biol 7: 1261–1266 [PMC free article] [PubMed]
  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(−delta delta C(T)) method. Methods 25: 402–408 [PubMed]
  • Lytle JR, Yario TA, Steitz JA (2007) Target mRNAs are repressed as efficiently by microRNA-binding sites in the 5′ UTR as in the 3′ UTR. Proc Natl Acad Sci USA 104: 9667–9672 [PMC free article] [PubMed]
  • Mathonnet G, Fabian MR, Svitkin YV, Parsyan A, Huck L, Murata T, Biffo S, Merrick WC, Darzynkiewicz E, Pillai RS, Filipowicz W, Duchaine TF, Sonenberg N (2007) MicroRNA inhibition of translation initiation in vitro by targeting the cap-binding complex eIF4F. Science 317: 1764–1767 [PubMed]
  • Morita K, Han M (2006) Multiple mechanisms are involved in regulating the expression of the developmental timing regulator lin-28 in Caenorhabditis elegans. EMBO J 25: 5794–5804 [PMC free article] [PubMed]
  • Moss EG, Lee RC, Ambros V (1997) The cold shock domain protein LIN-28 controls developmental timing in C. elegans and is regulated by the lin-4 RNA. Cell 88: 637–646 [PubMed]
  • Nottrott S, Simard MJ, Richter JD (2006) Human let-7a miRNA blocks protein production on actively translating polyribosomes. Nat Struct Mol Biol 13: 1108–1114 [PubMed]
  • Olsen PH, Ambros V (1999) The lin-4 regulatory RNA controls developmental timing in Caenorhabditis elegans by blocking LIN-14 protein synthesis after the initiation of translation. Dev Biol 216: 671–680 [PubMed]
  • Pasquinelli AE, Reinhart BJ, Slack F, Martindale MQ, Kuroda MI, Maller B, Hayward DC, Ball EE, Degnan B, Müller P, Spring J, Srinivasan A, Fishman M, Finnerty J, Corbo J, Levine M, Leahy P, Davidson E, Ruvkun G (2000) Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA. Nature 408: 86–89 [PubMed]
  • Pillai RS, Bhattacharyya SN, Artus CG, Zoller T, Cougot N, Basyuk E, Bertrand E, Filipowicz W (2005) Inhibition of translational initiation by Let-7 microRNA in human cells. Science 309: 1573–1576 [PubMed]
  • Reinhart BJ, Slack FJ, Basson M, Pasquinelli AE, Bettinger JC, Rougvie AE, Horvitz HR, Ruvkun G (2000) The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature 403: 901–906 [PubMed]
  • Schmitter D, Filkowski J, Sewer A, Pillai RS, Oakeley EJ, Zavolan M, Svoboda P, Filipowicz W (2006) Effects of Dicer and Argonaute down-regulation on mRNA levels in human HEK293 cells. Nucleic Acids Res 34: 4801–4815 [PMC free article] [PubMed]
  • Seggerson K, Tang L, Moss EG (2002) Two genetic circuits repress the Caenorhabditis elegans heterochronic gene lin-28 after translation initiation. Dev Biol 243: 215–225 [PubMed]
  • Selbach M, Schwanhäusser B, Thierfelder N, Fang Z, Khanin R, Rajewsky N (2008) Widespread changes in protein synthesis induced by microRNAs. Nature 455: 58–63 [PubMed]
  • Slack FJ, Basson M, Liu Z, Ambros V, Horvitz HR, Ruvkun G (2000) The lin-41 RBCC gene acts in the C. elegans heterochronic pathway between the let-7 regulatory RNA and the LIN-29 transcription factor. Mol Cell 5: 659–669 [PubMed]
  • Snow MI, Larsen PL (2000) Structure and expression of daf-12: a nuclear hormone receptor with three isoforms that are involved in development and aging in Caenorhabditis elegans. Biochim Biophys Acta 1494: 104–116 [PubMed]
  • Thermann R, Hentze MW (2007) Drosophila miR2 induces pseudo-polysomes and inhibits translation initiation. Nature 447: 875–878 [PubMed]
  • Vella MC, Choi E, Lin S, Reinert K, Slack FJ (2004) The C. elegans microRNA let-7 binds to imperfect let-7 complementary sites from the lin-41 3′UTR. Genes Dev 18: 132–137 [PMC free article] [PubMed]
  • Wakiyama M, Takimoto K, Ohara O, Yokoyama S (2007) Let-7 microRNA-mediated mRNA deadenylation and translational repression in a mammalian cell-free system. Genes Dev 21: 1857–1862 [PMC free article] [PubMed]
  • Wang B, Love TM, Call ME, Doench JG, Novina CD (2006) Recapitulation of short RNA-directed translational gene silencing in vitro. Mol Cell 22: 553–560 [PubMed]
  • Wightman B, Ha I, Ruvkun G (1993) Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans. Cell 75: 855–862 [PubMed]
  • Zhang L, Ding L, Cheung TH, Dong M, Chen J, Sewell AK, Liu X, Yates JR, Han M (2007) Systematic identification of C. elegans miRISC proteins, miRNAs, and mRNA targets by their interactions with GW182 proteins AIN-1 and AIN-2. Mol Cell 28: 598–613 [PMC free article] [PubMed]

Articles from The EMBO Journal are provided here courtesy of The European Molecular Biology Organization
PubReader format: click here to try

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

  • Gene
    Gene
    Gene links
  • GEO Profiles
    GEO Profiles
    Related GEO records
  • HomoloGene
    HomoloGene
    HomoloGene links
  • MedGen
    MedGen
    Related information in MedGen
  • Pathways + GO
    Pathways + GO
    Pathways, annotations and biological systems (BioSystems) that cite the current article.
  • PubMed
    PubMed
    PubMed citations for these articles
  • Taxonomy
    Taxonomy
    Related taxonomy entry
  • Taxonomy Tree
    Taxonomy Tree

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...