microRNAs (miRNAs) are ~22 nt regulatory RNAs that in animals typically bind with partial complementarity to sequences in the 3′ untranslated (UTR) regions of target mRNAs, to induce a decrease in the production of the encoded protein. The relative contributions of translational inhibition of intact mRNAs and degradation of mRNAs caused by binding of the miRNA vary; for many genetically validated miRNA targets, translational repression has been implicated, whereas some analyses of other miRNA targets have revealed only modest translational repression and more significant mRNA destabilization. In Caenorhabditis elegans, the lin-4 miRNA accumulates during early larval development, binds to target elements in the lin-14 mRNA, and causes a sharp decrease in the abundance of LIN-14 protein. Here, we monitor the dynamics of lin-14 mRNA and protein as well as lin-4 miRNA levels in finely staged animals during early larval development. We find complex regulation of lin-14, with the abundance of lin-14 mRNA initially modestly declining followed by fluctuation but little further decline of lin-14 mRNA levels accompanied by continuing and more dramatic decline in LIN-14 protein abundance. We show that the translational inhibition of lin-14 is dependent on binding of the lin-4 miRNA to multiple lin-4 complementary sites in the lin-14 3′UTR. Our results point to the importance of translational inhibition in silencing of lin-14 by the lin-4 miRNA.
References
[1]
Lewis BP, Shih IH, Jones-Rhoades MW, Bartel DP, Burge CB (2003) Prediction of mammalian microRNA targets. Cell 115: 787–798.
[2]
Djuranovic S, Nahvi A, Green R (2011) A parsimonious model for gene regulation by miRNAs. Science 331: 550–553.
[3]
Huntzinger E, Izaurralde E (2011) Gene silencing by microRNAs: contributions of translational repression and mRNA decay. Nat Rev Genet 12: 99–110.
[4]
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.
[5]
Ding XC, Grosshans H (2009) Repression of C. elegans microRNA targets at the initiation level of translation requires GW182 proteins. EMBO J 28: 213–222.
[6]
Petersen CP, Bordeleau ME, Pelletier J, Sharp PA (2006) Short RNAs repress translation after initiation in mammalian cells. Mol Cell 21: 533–542.
Zdanowicz A, Thermann R, Kowalska J, Jemielity J, Duncan K, et al. (2009) Drosophila miR2 primarily targets the m7GpppN cap structure for translational repression. Mol Cell 35: 881–888.
[9]
Fabian MR, Mathonnet G, Sundermeier T, Mathys H, Zipprich JT, et al. (2009) Mammalian miRNA RISC recruits CAF1 and PABP to affect PABP-dependent deadenylation. Mol Cell 35: 868–880.
[10]
Djuranovic S, Nahvi A, Green R (2012) miRNA-mediated gene silencing by translational repression followed by mRNA deadenylation and decay. Science 336: 237–240.
[11]
Bazzini AA, Lee MT, Giraldez AJ (2012) Ribosome profiling shows that miR-430 reduces translation before causing mRNA decay in zebrafish. Science 336: 233–237.
[12]
Selbach M, Schwanhausser B, Thierfelder N, Fang Z, Khanin R, et al. (2008) Widespread changes in protein synthesis induced by microRNAs. Nature 455: 58–63.
[13]
Lee RC, Feinbaum RL, Ambros V (1993) The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75: 843–854.
[14]
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.
[15]
Ha I, Wightman B, Ruvkun G (1996) A bulged lin-4/lin-14 RNA duplex is sufficient for Caenorhabditis elegans lin-14 temporal gradient formation. Genes Dev 10: 3041–3050.
[16]
Ambros V, Horvitz HR (1987) The lin-14 locus of Caenorhabditis elegans controls the time of expression of specific postembryonic developmental events. Genes Dev 1: 398–414.
[17]
Wightman B, Burglin TR, Gatto J, Arasu P, Ruvkun G (1991) Negative regulatory sequences in the lin-14 3′-untranslated region are necessary to generate a temporal switch during Caenorhabditis elegans development. Genes Dev 5: 1813–1824.
[18]
Reinhart BJ, Ruvkun G (2001) Isoform-specific mutations in the Caenorhabditis elegans heterochronic gene lin-14 affect stage-specific patterning. Genetics 157: 199–209.
[19]
Stoeckius M, Maaskola J, Colombo T, Rahn HP, Friedlander MR, et al. (2009) Large-scale sorting of C. elegans embryos reveals the dynamics of small RNA expression. Nat Methods 6: 745–751.
[20]
Feinbaum R, Ambros V (1999) The timing of lin-4 RNA accumulation controls the timing of postembryonic developmental events in Caenorhabditis elegans. Dev Biol 210: 87–95.
[21]
Holtz J, Pasquinelli AE (2009) Uncoupling of lin-14 mRNA and protein repression by nutrient deprivation in Caenorhabditis elegans. RNA 15: 400–405.
[22]
Chatterjee S, Grosshans H (2009) Active turnover modulates mature microRNA activity in Caenorhabditis elegans. Nature 461: 546–549.
[23]
Chatterjee S, Fasler M, Bussing I, Grosshans H (2011) Target-Mediated Protection of Endogenous MicroRNAs in C. elegans. Dev Cell 20: 388–396.
[24]
Ruvkun G, Ambros V, Coulson A, Waterston R, Sulston J, et al. (1989) Molecular genetics of the Caenorhabditis elegans heterochronic gene lin-14. Genetics 121: 501–516.
[25]
Stadler M, Artiles K, Pak J, Fire A (2012) Contributions of mRNA abundance, ribosome loading, and post- or peri-translational effects to temporal repression of C. elegans heterochronic miRNA targets. Genome Res.
[26]
Bagga S, Bracht J, Hunter S, Massirer K, Holtz J, et al. (2005) Regulation by let-7 and lin-4 miRNAs results in target mRNA degradation. Cell 122: 553–563.
