The role of deadenylation in the degradation of unstable mRNAs in trypanosomes

Abstract

Removal of the poly(A) tail is the first step in the degradation of many eukaryotic mRNAs. In metazoans and yeast, the Ccr4/Caf1/Not complex has the predominant deadenylase activity, while the Pan2/Pan3 complex may trim poly(A) tails to the correct size, or initiate deadenylation. In trypanosomes, turnover of several constitutively-expressed or long-lived mRNAs is not affected by depletion of the 5′–3′ exoribonuclease XRNA, but is almost completely inhibited by depletion of the deadenylase CAF1. In contrast, two highly unstable mRNAs, encoding EP procyclin and a phosphoglycerate kinase, PGKB, accumulate when XRNA levels are reduced. We here show that degradation of EP mRNA was partially inhibited after CAF1 depletion. RNAi-targeting trypanosome PAN2 had a mild effect on global deadenylation, and on degradation of a few mRNAs including EP. By amplifying and sequencing degradation intermediates, we demonstrated that a reduction in XRNA had no effect on degradation of a stable mRNA encoding a ribosomal protein, but caused accumulation of EP mRNA fragments that had lost substantial portions of the 5′ and 3′ ends. The results support a model in which trypanosome mRNAs can be degraded by at least two different, partially independent, cytoplasmic degradation pathways attacking both ends of the mRNA.

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

“…Several studies revealed a biphasic decay pattern for EP1 mRNA in bloodstream forms and identified the nucleases involved [27][28][29]46]. In one of our experiments, a decay pattern reminiscent of biphasic decay kinetics was found for GPEET mRNA in late PF (with only 0.4% GPEET-positive cells) (Fig.…”

“…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].…”

“…The stability of bulk procyclin mRNA was previously measured in bloodstream forms and PF [24,25], and turnover of EP1 mRNA in bloodstream forms was determined by means of reporter mRNAs [26][27][28][29][30]. However, these studies either did not discriminate between GPEET and EP, or lacked data on GPEET mRNA in general; in addition, procyclin mRNA stability has never been assessed during the transition from early to late PF.…”

The role of the 5′–3′ exoribonuclease XRNA in transcriptome-wide mRNA degradation

“…Trypanosomes are therefore an ideal model system for the analysis of mRNA turnover. They have homologs of deadenylases (Milone et al 2004;Schwede et al 2008Schwede et al , 2009) and the exosome (Clayton and Estevez 2010) and three potential homologs of Xrn1, XRNA, XRNB, and XRNC (Li et al 2006). Of these, XRNA was shown to be essential for the growth of both life cycle stage forms of T. brucei and for the degradation of two highly unstable developmentally regulated mRNAs (Li et al 2006).…”

“…Of these, XRNA was shown to be essential for the growth of both life cycle stage forms of T. brucei and for the degradation of two highly unstable developmentally regulated mRNAs (Li et al 2006). More detailed analysis revealed that the unstable EP mRNA was degraded from both the 59 and 39 ends (Schwede et al 2009). …”