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.

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.

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.

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.

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.

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).