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

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.

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

The degradation of eukaryotic mRNAs can be initiated by deadenylation, decapping, or endonuclease cleavage. This is followed by 5'-3' degradation by homologs of Xrn1, and/or 3'-5' degradation by the exosome. We previously reported that, in African trypanosome Trypanosoma brucei, most mRNAs are deadenylated prior to degradation, and that depletion of the major 5'-3' exoribonuclease XRNA preferentially stabilizes unstable mRNAs. We now show that depletion of either CAF1 or CNOT10, two components of the principal deadenylation complex, strongly inhibits degradation of most mRNAs. RNAi targeting another deadenylase, PAN2, or RRP45, a core component of the exosome, preferentially stabilized mRNAs with intermediate half-lives. RRP45 depletion resulted in a 5' bias of mRNA sequences, suggesting action by a distributive 3'-5' exoribonuclease. Results suggested that the exosome is involved in the processing of trypanosome snoRNAs. There was no correlation between effects on half-lives and on mRNA abundance.

Trypanosome CNOT10 is essential for the integrity of the NOT deadenylase complex and for degradation of many mRNAs.

The degradation of most eukaryotic mRNAs is initiated by removal of the poly(A) tail, and the major deadenylase activity is associated with the CCR4/CAF1/NOT complex (NOT complex). We here study the role of CNOT10, a protein that is found in human and trypanosome, but not in yeast, NOT complexes. Trypanosome (Tb) CNOT10 is essential for growth. TbCNOT10 interacted with the deadenylase TbCAF1 and the scaffold protein TbNOT1; TbCAF1 also interacted with TbNOT1 in a yeast two-hybrid assay. In both trypanosomes and human embryonic kidney cells, approximately half of CAF1 was associated with the NOT complex. Depletion of CNOT10 from human cells did not affect this association. In contrast, depletion of TbCNOT10 in trypanosomes caused a decrease in the level of TbNOT1, detachment of TbCAF1 from the complex and pronounced stabilization of most trypanosome mRNAs. Artificial tethering of TbCAF1 to a reporter mRNA in vivo resulted in mRNA degradation, and this was not affected by TbCNOT10 depletion. We conclude that in trypanosomes, TbCNOT10 may stabilize the interaction between TbCAF1 and the NOT complex. The results further suggest that TbCAF1 is only able to deadenylate mRNA in vivo if it is recruited to the mRNA through other NOT complex components.