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The Pumilio-domain protein PUF6 contributes to SIDER2 retroposon-mediated mRNA decay in .

RNA; 23(12): 1874-1885, 2017 Dec.
Article in English | MEDLINE | ID: mdl-28877997
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.