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

Abstract

The steady-state level of each mRNA in a cell is a balance between synthesis and degradation. Here, we use high-throughput RNA sequencing (RNASeq) to determine the relationship between mRNA degradation and mRNA abundance on a transcriptome-wide scale. The model organism used was the bloodstream form of Trypanosoma brucei, a protist that lacks regulation of RNA polymerase II initiation. The mRNA half-lives varied over two orders of magnitude, with a median half-life of 13 min for total (rRNA-depleted) mRNA. Data for poly(A)+ RNA yielded shorter half-lives than for total RNA, indicating that removal of the poly(A) tail was usually the first step in degradation. Depletion of the major 59-39 exoribonuclease, XRNA, resulted in the stabilization of most mRNAs with half-lives under 30 min. Thus, on a transcriptome-wide scale, degradation of most mRNAs is initiated by deadenylation. Trypanosome mRNA levels are strongly influenced by gene copy number and mRNA half-life: Very abundant mRNAs that are required throughout the life-cycle may be encoded by multicopy genes and have intermediate-to-long half-lives; those encoding ribosomal proteins, with one to two gene copies, are exceptionally stable. Developmentally regulated transcripts with a lower abundance in the bloodstream forms than the procyclic forms had half-lives around the median, whereas those with a higher abundance in the bloodstream forms than the procyclic forms, such as those encoding glycolytic enzymes, had longer half-lives.

The roles of 3′-exoribonucleases and the exosome in trypanosome mRNA degradation

“…1) are listed in Table 1. The proportion of SL signal that remained after 30-min transcription inhibition in wildtype cells was 50%-60% (Table 1), which was approximately in agreement with our previous estimates based on the abundances of individual mRNAs (Haanstra et al 2008;Manful et al 2011). Supplemental Figure S1A shows the time course of disappearance of SL signal over a 2-h time course.…”

“…Transcriptome-wide analysis of trypanosome mRNA halflives in cells after XRNA RNAi demonstrated that XRNA depletion inhibited the degradation of unstable mRNAs, while some of the more stable mRNAs had shorter halflives (Manful et al 2011). We have now extended this to all the known factors involved in degradation from the 3 ′ end: PAN2, the exosome, and components of the CAF1/NOT complex.…”

“…Individual mRNAs are excised by 5 ′ trans splicing of a "spliced leader" (SL) and 3 ′ polyadenylation (Michaeli 2011). Since transcription of individual genes cannot be controlled, mRNA turnover plays an unusually dominant role in determining mRNA abundance (Ouellette and Papadopoulou 2009;Fernández-Moya and Estévez 2010;Kramer and Carrington 2011;Manful et al 2011). For most mRNAs, removal of the poly(A) tail precedes degradation of the remainder of the transcript (Manful et al 2011).…”

“…Since transcription of individual genes cannot be controlled, mRNA turnover plays an unusually dominant role in determining mRNA abundance (Ouellette and Papadopoulou 2009;Fernández-Moya and Estévez 2010;Kramer and Carrington 2011;Manful et al 2011). For most mRNAs, removal of the poly(A) tail precedes degradation of the remainder of the transcript (Manful et al 2011). A typical mRNA degradation machinery is present: an exosome (Estévez et al 2001; a scavenger decapping enzyme (Banerjee et al 2009); an Xrn1 homolog, XRNA (Li et al 2006); PAN2/PAN3 (Schwede et al 2009); PARN (Milone et al 2004;Utter et al 2011); a CAF1/NOT complex composed of CAF1, CAF40, NOT1, NOT2, NOT3/5 (Schwede et al 2008); a CNOT11 homolog (Schwede et al 2008;Mauxion et al 2013); and CNOT10 (Färber et al 2013).…”

“…Similarly, CNOT10 is required for the attachment of the deadenylase CAF1 to the CAF1/NOT complex (Färber et al 2013). The median mRNA half-life in bloodstream-form trypanosomes (the form that grows in mammals) is ∼15-20 min (Manful et al 2011). Depletion of XRNA (Li et al 2006), PAN2 (Schwede et al 2009), or the exosome (Haile et al 2003) delayed the degradation of the highly unstable developmentally regulated mRNA encoding EP procyclin, or reporter mRNAs bearing its 3 ′ UTR; ACT (actinA) mRNA, which has a half-life at the median, was also stabilized by RNAi targeting PAN2, and the developmentally regulated unstable mRNA encoding pyruvate phosphate decarboxylase was stabilized by RRP45 RNAi.…”