When silencing mRNA, position matters

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Science Signaling  14 Feb 2017:
Vol. 10, Issue 466, eaam9537
DOI: 10.1126/scisignal.aam9537

The mechanism of posttranscriptional silencing by an RNA-binding protein depends on the position of the protein-binding sequence in the mRNA.

LIN14 is an RNA-binding protein in Caenorhabditis elegans. LIN14 and the mammalian homolog TRIM71are required for stem cell fate decisions. Mammalian TRIM71 has E3 ubiquitin ligase activity, but LIN41 and TRIM71 also contain a putative sequence-specific RNA-binding motif that may mediate mRNA silencing. Aeschimann et al. identified mRNA targets of LIN41 and showed that depending on the position of the LIN41-binding site in the transcript, the protein either targeted mRNA for degradation or repressed translation. Using ribosomal profiling and RNA-seq at different developmental stages in worms with increased or reduced abundance of LIN41, the authors identified gene expression changes regulated by LIN41. Analysis of patterns of transcriptional and translational activation and suppression in the different mutant lines identified six transcripts as potential LIN41 direct targets, and immunoprecipitation experiments confirmed that the mRNAs of four of these—mab-10, mab-3, dmd-3, and lin-29— bound LIN41. In worms with wild-type LIN41 or LIN41-knockdown worms each expressing a fluorescent reporter individually fused to the 3ʹ-untranslated regions (UTRs) of mab-10, mab-3, or dmd-3, the fluorescence of the reporter was higher in the LIN41-knockdown worms, consistent with these 3ʹ-UTRs containing sequences targeting the transcripts for LIN41-dependent degradation. In contrast, the expression of the reporter fused to the lin-29 3ʹ-UTR was similar in LIN41 wild-type and knockdown worms, despite FLAG-tagged LIN-29 exhibiting LIN41-dependent reduction in abundance. Depletion of LIN41 did not reduce the amount of lin-29 mRNA, and ribosomal profiling results suggested that LIN41 specifically impaired translation of the long isoform of the gene, but not of the short isoform, which lacks exons 1–4. LIN41 reduced fluorescence from reporters controlled by the lin-29 promoter or a universal promoter upstream of the lin-29 5ʹUTR. Analysis of LIN41-dependent reporter fluorescence in mutated or truncated lin-29 5ʹ-UTR sequences identified exon 1 as the region that mediated repression by LIN41. The lin-29 5ʹ-UTR did not affect the amount of reporter gene transcript in worms, suggesting that repression occurred at the level of translation. In contrast, the coupling the reporter to the 5ʹ UTR of mab-10 did not enable LIN41-mediated silencing. A reporter coupled to the 5ʹ UTR of lin-29 and the 3ʹ-UTR of mab-10 exhibited reduced transcript abundance, indicating that the mRNA degradation mechanism was engaged even in the presence of the sequence that enabled translational repression. Introduction of lin-29 exon 1 into the 3ʹ-UTR of another gene (unc-54) resulted in a LIN41-dependent decrease in fluorescence and the amount of the reporter mRNA transcript, suggesting that this LIN41-binding sequence in lin-29 engages different mechanisms of regulation depending on position within the gene sequence. Similarly, introduction of the mab-10 3ʹ-UTR sequence into the 5ʹ-UTR of the reporter gene resulted a in LIN41-dependent reduction in fluorescence without decreasing the amount of reporter transcript, suggesting that presence of the LIN41-binding motif in the 5ʹ-UTR position engages the translation-blocking mechanism of repression. Hand and Bazzini speculate that the location of a LIN-41 binding sequence may determine differential recruitment of cofactors that mediate silencing by either transcript degradation or translational suppression and discuss the possibility that sequence context of protein-binding mRNA sequences adds another dimension of posttranscriptional regulation.

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