Rapid decay of unstable Leishmania mRNAs bearing a conserved retroposon signature 3′-UTR motif is initiated by a site-specific endonucleolytic cleavage without prior deadenylation

Abstract

We have previously shown that the Leishmania genome possess two widespread families of extinct retroposons termed Short Interspersed DEgenerated Retroposons (SIDER1/2) that play a role in post-transcriptional regulation. Moreover, we have demonstrated that SIDER2 retroposons promote mRNA degradation. Here we provide new insights into the mechanism by which unstable Leishmania mRNAs harboring a SIDER2 retroposon in their 3′-untranslated region are degraded. We show that, unlike most eukaryotic transcripts, SIDER2-bearing mRNAs do not undergo poly(A) tail shortening prior to rapid turnover, but instead, they are targeted for degradation by a site-specific endonucleolytic cleavage. The main cleavage site was mapped in two randomly selected SIDER2-containing mRNAs in vivo between an AU dinucleotide at the 5′-end of the second 79-nt signature (signature II), which represents the most conserved sequence amongst SIDER2 retroposons. Deletion of signature II abolished endonucleolytic cleavage and deadenylation-independent decay and increased mRNA stability. Interestingly, we show that overexpression of SIDER2 anti-sense RNA can increase sense transcript abundance and stability, and that complementarity to the cleavage region is required for protecting SIDER2-containing transcripts from degradation. These results establish a new paradigm for how unstable mRNAs are degraded in Leishmania and could serve as the basis for a better understanding of mRNA decay pathways in general.

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

“…As we have reported previously, SIDER2 retroposon promotes rapid RNA decay, whereas deletion of SIDER2 from the 3 ′ UTR blocks degradation and causes accumulation of the LUC reporter mRNA ( Fig. 1D; Bringaud et al 2007;Müller et al 2010b). Here, we show that addition of two MS2 hairpins in the reporter RNA and binding to MCP does not alter degradation by SIDER2 (Fig.…”

“…In addition, gene expression has been shown to change dramatically throughout the complex life cycles of these parasites (Haile and Papadopoulou 2007;Rochette et al 2008;Nilsson et al 2010;Siegel et al 2010). 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 thus 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). Several RBPs, such as zinc finger proteins, Alba-domain proteins, and Pumilio (PUF) proteins, have been found to regulate mRNA stability in trypanosomatids (Clayton 2013;Dupé et al 2014).…”

“…Members of the two major SIDER subfamilies were shown to regulate mRNA turnover in a stage-and species-specific manner (SIDER2 subfamily) (Bringaud et al 2007;Rochette et al 2008;Müller and Papadopoulou 2010;Müller et al 2010a,b) or mRNA translation (SIDER1 subfamily) (Boucher et al 2002;McNicoll et al 2005), and possibly to form RNA regulons (Ouellette and Papadopoulou 2009;Smith et al 2009). 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). Cleavage was mapped at AU/CU dinucleotides within the second tandem 79-nt hallmark sequence (signature II) (Müller et al 2010a;Azizi et al 2017b) that is conserved at the 5 ′ -end of all SIDER2 retroposons but not SIDER1 (Bringaud et al 2007;Smith et al 2009).…”

Insights into the regulation of GPEET procyclin during differentiation from early to late procyclic forms of Trypanosoma brucei

“…An identical mechanism was considered for the fast decay of EP1 mRNA in bloodstream form T. brucei [26], but this was not confirmed by a subsequent study [28]. However, degradation of unstable Leishmania transcripts bearing a retroposon-derived instability motif in their 3 UTR appears to be initiated by endonucleolytic cleavages at discrete sites [48]. We identified 9 different 3 ends within the GPEET 3 UTR (Fig.…”

Regulatory mechanisms of Leishmania Aquaglyceroporin AQP1

“…Instead, these protein-coding genes are constitutively transcribed from large gene clusters, and steady state levels of their mature mRNAs are regulated post-transcriptionally by trans-splicing and polyadenylation (Kramer 2012 ;Haile and Papadopoulou 2007). Several studies reported that posttranscriptional regulation of developmentally expressed transcripts in Leishmania species involves sequences present mainly in the 3'-UTR (Muller et al, 2010 ;Boucher et al, 2002;McNicoll et al, 2005;Bringaud et al, 2007), but also in the 5'UTR Lamontagne and Papadopoulou 1999) and in intergenic regions between tandemly repeated genes (Ramamoorthy et al, 1995;Beetham et al, 1997). Logarithmic-stationary gene regulation is also regulated by 3'-UTR (Myung et al, 2002).…”