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 5'-3' 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.

FIGURE 1.

Most ORFs are represented as less than three mRNAs per cell. Each bar represents the number of distinct ORFs with an mRNA abundance within the indicated range.

FIGURE 2.

Transcriptome-wide mRNA half-lives. (A) Half-lives for total mRNA from WT (dark gray bars) and XRNA RNAi (black bars) cells and for poly(A)+ mRNA (light gray bars) from WT cells. For each ORF, we calculated the arithmetic mean half-life (WT total, three measurements; others, two measurements). Negative half-lives were classified as >60 min, ORFs with <10 rpkm at time = 0 are removed. The number of mRNAs in each half-life category was counted. (B) Scatter plot showing half-lives for individual ORFs, total mRNA, and poly(A)+ mRNA. Data were filtered as in A; half-lives were removed if the mean was less than twice the SD (Supplemental Table S4). Only half-lives between 5 min and 120 min are shown. The correlation coefficient was calculated in KaleidaGraph; the dashed line indicates the line that would be obtained from a perfect 1:1 correlation. (C) As in B but using a log₂ scale.

FIGURE 3.

Relationship between mRNA abundance and half-life. Data were filtered as for Figure 2B and Supplemental Table S4. For each individual ORF, the copy number–normalized rpkm was plotted against the half-life. Results are shown for total RNA (A,B) and poly(A)+ RNA (C,D). Plots B and D use log scales; regression lines were plotted in KaleidaGraph. With control by degradation alone, using the log scale, the slope should be 1 (dotted line).

FIGURE 4.

XRNA depletion preferentially affects less stable mRNAs. (A) Abundance data for total RNA of WT and XRNA-depleted cells, filtered as in Figure 2B, and for half-lives under 120 min only; regression line plotted using KaleidaGraph, and dotted line shows 1:1 correlation. (B) As in A, but showing half-life results. (C) The fold effect of XRNA depletion was calculated (half-life in XRNA-depleted cells divided by WT half-life) and then plotted (y-axis) against the WT half-life (x-axis). (D) mRNAs were divided into classes according to the fold effect of XRNA depletion; bars indicate the mean ± SD. The number of ORFs in each class is indicated above the bars.