Transcriptome‐wide analysis of trypanosome mRNA decay reveals complex degradation kinetics and suggests a role for co‐transcriptional degradation in determining mRNA levels

Abstract

African trypanosomes are an excellent system for quantitative modelling of post-transcriptional mRNA control. Transcription is constitutive and polycistronic; individual mRNAs are excised by trans splicing and polyadenylation. We here measure mRNA decay kinetics in two life cycle stages, bloodstream and procyclic forms, by transcription inhibition and RNASeq. Messenger RNAs with short half-lives tend to show initial fast degradation, followed by a slower phase; they are often stabilized by depletion of the 5′–3′ exoribonuclease XRNA. Many longer-lived mRNAs show initial slow degradation followed by rapid destruction: we suggest that the slow phase reflects gradual deadenylation. Developmentally regulated mRNAs often show regulated decay, and switch their decay pattern. Rates of mRNA decay are good predictors of steady state levels for short mRNAs, but mRNAs longer than 3 kb show unexpectedly low abundances. Modelling shows that variations in splicing and polyadenylation rates can contribute to steady-state mRNA levels, but this is completely dependent on competition between processing and co-transcriptional mRNA precursor destruction.

Simultaneous detection of mRNA transcription and decay intermediates by dual colour single mRNA FISH on subcellular resolution

“…Yellow spots decreased to 27% within 15 min, indicating that the mRNA has a half-life of less than 15 min – shorter than the mean half-life of 20 min measured for trypanosome mRNAs (18). The number of green spots gradually decreased to 34% within 30 min and to almost 0 within 60 min.…”

The zinc finger proteins ZC3H20 and ZC3H21 stabilise mRNAs encoding membrane proteins and mitochondrial proteins in insect‐form Trypanosoma brucei

“…RNAi to deplete both proteins in Lister 427 procyclic forms was induced for 36 hr. (a) Correlation between the effects of ZC3H20 and ZC3H21 RNAi in procyclic forms, and developmental regulation of mRNA (RNA amounts in bloodstream forms divided by the amount in procyclic forms (Fadda et al, )). Some of the strongly decreased mRNAs are indicated, and colour‐coded according to their binding to ZC3H20 and/or ZC3H21 (key on Figure).…”

“…“Bind Z20” is the average of bound/unbound for the mRNA; the equivalent values are given for ZC3H21 and RBP10 (Mugo & Clayton, ). "RNA PC/BS" is regulation at the RNA level (Fadda et al, ) and Ribosomes PC/BS" shows the ratios of normalised total ribosomal reads (Antwi et al, ). #Results for META domain protein family, which is absent from the “unique” gene list, were calculated from the raw data.…”

“…The 3′‐UTRs of these mRNAs have, respectively, two, five and seven copies of the RBP10 consensus recognition motif (Figure a). All three mRNAs are more abundant, and considerably better translated, in procyclic forms than in growing bloodstream forms (Antwi et al, ; Dejung et al, ; Fadda et al, ; Jensen et al, ), and their expression is also lower in metacyclic forms than in procyclic forms (Christiano et al, ). When ZC3H20 or ZC3H21 are tethered (via a lambdaN‐peptide–boxB interaction) to a reporter mRNA in bloodstream forms, expression is stimulated, whereas tethering of ZC3H22 leads to repression (Erben, Fadda, Lueong, Hoheisel, & Clayton, ; Lueong, Merce, Fischer, Hoheisel, & Erben, ).…”

GT ‐rich promoters can drive RNA pol II transcription and deposition of H2A.Z in African trypanosomes

“…The definition of the first gene of a PTU is based on a previous study (Kolev et al , ) and genome version Tb927v24. Total nucleosome occupancy is plotted relative to the ATG and averaged across all genes except the first gene of a PTU ( n = 12,220). Nucleosome occupancy is plotted relative to the splice acceptor sites and averaged across the 25% of genes containing the highest RNA levels (left panel, n = 690), the 25% of genes containing intermediate RNA levels (middle panel, n = 690), and the 25% of genes containing the lowest RNA levels (lower panel, n = 690). RNA levels were determined previously (Fadda et al , ).…”