Azizi Hiva, Dumas Carole, Papadopoulou Barbara
Research Center in Infectious Diseases, CHU de Quebec Research Center-Laval University, Quebec, QC, G1V 4G2 Canada.
Department of Microbiology, Infectious Diseases and Immunology, Faculty of Medicine, Laval University, Quebec, QC, G1V 0A6 Canada.
RNA. 2017 Dec;23(12):1874-1885. doi: 10.1261/rna.062950.117. Epub 2017 Sep 6.
and other trypanosomatid protozoa lack control at the level of transcription initiation and regulate gene expression exclusively post-transcriptionally. We have reported previously that harbors a unique class of hort nterspersed generate etroposons (SIDERs) that are predominantly located within 3'UTRs and play a major role in post-transcriptional control. We have shown that members of the SIDER2 subfamily initiate mRNA decay through endonucleolytic cleavage within the second conserved 79-nt signature sequence of SIDER2 retroposons. Here, we have developed an optimized MS2 coat protein tethering system to capture -acting factor(s) regulating SIDER2-mediated mRNA decay. Tethering of the MS2 coat protein to a reporter RNA harboring two MS2 stem-loop aptamers and the cognate SIDER2-containing 3'UTR in combination with immunoprecipitation and mass spectrometry analysis led to the identification of RNA-binding proteins with known functions in mRNA decay. Among the candidate SIDER2-interacting proteins that were individually tethered to a SIDER2 reporter RNA, the Pumilio-domain protein PUF6 was shown to enhance degradation and reduce transcript half-life. Furthermore, we showed that PUF6 binds to SIDER2 sequences that include the regulatory 79-nt signature motif, hence contributing to the mRNA decay process. Consistent with a role of PUF6 in SIDER2-mediated decay, genetic inactivation of PUF6 resulted in increased accumulation and higher stability of endogenous SIDER2-bearing transcripts. Overall, these studies provide new insights into regulated mRNA decay pathways in controlled by SIDER2 retroposons and propose a broader role for PUF proteins in mRNA decay within the eukaryotic kingdom.
而其他锥虫原生动物在转录起始水平缺乏调控，仅在转录后调控基因表达。我们之前报道过， 含有一类独特的hort散布产生的反转录转座子（SIDERs），它们主要位于3'UTR内，并在转录后调控中起主要作用。我们已经表明，SIDER2亚家族的成员通过在SIDER2反转录转座子的第二个保守的79个核苷酸特征序列内进行内切核酸酶切割来启动mRNA降解。在这里，我们开发了一种优化的MS2外壳蛋白拴系系统，以捕获调节SIDER2介导的mRNA降解的作用因子。将MS2外壳蛋白拴系到含有两个MS2茎环适体和同源含SIDER2的3'UTR的报告RNA上，结合免疫沉淀和质谱分析，导致鉴定出在mRNA降解中具有已知功能的RNA结合蛋白。在单独拴系到SIDER2报告RNA的候选SIDER2相互作用蛋白中，Pumilio结构域蛋白PUF6被证明可增强降解并缩短转录本半衰期。此外，我们表明PUF6与包含调控性79个核苷酸特征基序的SIDER2序列结合，从而促进mRNA降解过程。与PUF6在SIDER2介导的降解中的作用一致，PUF6的基因失活导致内源性含SIDER2转录本的积累增加和稳定性提高。总体而言，这些研究为SIDER2反转录转座子控制的 中受调控的mRNA降解途径提供了新见解，并提出了PUF蛋白在真核生物界mRNA降解中的更广泛作用。
