A plant-like mechanism coupling m6A reading to polyadenylation safeguards transcriptome integrity and developmental gene partitioning in Toxoplasma.

Dayana C Farhat,Caroline Mas,Thierry Lagrange,Alexandre Bougdour,Christopher Swale,Guillaume Communie,Mohamed-Ali Hakimi,Dominique Pontier,Matthew W Bowler,Charlotte Corrao,Lucid Belmudes,Yohann Couté

doi:10.7554/elife.68312

Abstract

Correct 3'end processing of mRNAs is one of the regulatory cornerstones of gene expression. In a parasite that must adapt to the regulatory requirements of its multi-host life style, there is a need to adopt additional means to partition the distinct transcriptional signatures of the closely and tandemly arranged stage-specific genes. In this study, we report our findings in T. gondii of an m6A-dependent 3'end polyadenylation serving as a transcriptional barrier at these loci. We identify the core polyadenylation complex within T. gondii and establish CPSF4 as a reader for m6A-modified mRNAs, via a YTH domain within its C-terminus, a feature which is shared with plants. We bring evidence of the specificity of this interaction both biochemically, and by determining the crystal structure at high resolution of the T. gondii CPSF4-YTH in complex with an m6A-modified RNA. We show that the loss of m6A, both at the level of its deposition or its recognition is associated with an increase in aberrantly elongated chimeric mRNAs emanating from impaired transcriptional termination, a phenotype previously noticed in the plant model Arabidopsis thaliana. Nanopore direct RNA sequencing shows the occurrence of transcriptional read-through breaching into downstream repressed stage-specific genes, in the absence of either CPSF4 or the m6A RNA methylase components in both T. gondii and A. thaliana. Taken together, our results shed light on an essential regulatory mechanism coupling the pathways of m6A metabolism directly to the cleavage and polyadenylation processes, one that interestingly seem to serve, in both T. gondii and A. thaliana, as a guardian against aberrant transcriptional read-throughs.

Highlights

In view of the unique architecture of this protein, combining the conserved 3’end processing zinc fingers, with the YTH that we structurally demonstrated as an m6A reader, it seemed only logical to tackle the weight of this RNA modification on the termination defects that we observed in the context of the CPSF4 KD
The correct processing of the 5’ and 3’ ends of mRNA is paramount to the effective expression of any functional gene for all eukaryotes
3’ end processing, and notably cleavage and polyadenylation, are emerging as attractive targets for chemical inhibition (Bellini et al, 2020; Palencia et al, 2017; Swale et al, 2019) as these highly replicative cells strongly depend on consistent mRNA production

Summary

Introduction

The parasite faces the challenge of limiting and coordinating its transcriptional potentials in a way to swiftly adjust gene expression to its corresponding developmental requirements The paucity both in numbers and variety of specific transcription factors (Bozdech et al, 2003), relatively to the high number of protein-encoding genes, leaves the possibility of alternative mechanisms open. A large part of the gene silencing machinery seems to be governed by the action of chromatin shapers, which were assigned significant roles in directing developmental trajectories and sexual commitment (Farhat et al, 2020; Waldman et al, 2020). Along these lines, epigenetic changes are acknowledged as driving regulators of gene expression at the level of transcription.

Methods

Results

Conclusion