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

Hiva Azizi,Carole Dumas,Barbara Papadopoulou

doi:10.1261/rna.062950.117

Abstract

Leishmania and other trypanosomatid protozoa lack control at the level of transcription initiation and regulate gene expression exclusively post-transcriptionally. We have reported previously that Leishmania harbors a unique class of short interspersed degenerate retroposons (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 trans-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 Leishmania controlled by SIDER2 retroposons and propose a broader role for PUF proteins in mRNA decay within the eukaryotic kingdom.

Highlights

Leishmania spp. are unicellular eukaryotic pathogens causing a wide spectrum of pathologies in humans ranging from cutaneous to visceral infections (Desjeux 2004)
Regulation of mRNA decay rates through interactions of RNA-binding proteins (RBPs) with cis-acting sequences in 3′UTRs of trypanosomatid transcripts is central in determining the fate of mRNAs and fine-tuning developmental gene expression (McNicoll et al 2005; Bringaud et al 2007; Haile and Papadopoulou 2007; Haile et al 2008; Müller et al 2010b; Kramer 2012; Clayton 2013)
We showed that degradation of SIDER2-bearing transcripts is initiated by endonucleolytic cleavage (Müller et al 2010b; Papadopoulou et al 2015) as opposed to the default process in eukaryotes, which begins with poly(A) shortening by deadenylases followed by 5′ cap removal and 5′–3′ degradation by the XRN1 exoribonuclease or 3′–5′ degradation by the exosome (Schoenberg and Maquat 2012)

Summary

INTRODUCTION

Leishmania spp. are unicellular eukaryotic pathogens causing a wide spectrum of pathologies in humans ranging from cutaneous to visceral infections (Desjeux 2004). Regulation of mRNA decay rates through interactions of RNA-binding proteins (RBPs) with cis-acting sequences in 3′UTRs of trypanosomatid transcripts is central in determining the fate of mRNAs and fine-tuning developmental gene expression (McNicoll et al 2005; Bringaud et al 2007; Haile and Papadopoulou 2007; Haile et al 2008; Müller et al 2010b; Kramer 2012; Clayton 2013). We followed up on the mechanism of SIDER2-mediated mRNA decay by searching for trans-acting factors regulating this process To this end, we have developed a bipartite MS2 coat protein pull-down system to identify RNA-binding protein candidates bound to a SIDER2-containing reporter RNA. We showed that genetic inactivation of PUF6 leads to an increased stability of endogenous SIDER2-bearing transcripts, supporting a role of PUF6 protein in the SIDER2-mediated mRNA decay process

RESULTS

DISCUSSION

MATERIALS AND METHODS