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

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 was 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.

m6A RNA methylation facilitates pre-mRNA 3’-end formation and is essential for viability of Toxoplasma gondii

“…While our manuscript was under review, a preprint entitled “A plant-like mechanisms coupling m6A reading to polyadenylation safeguards transcriptome integrity and developmental genes partitioning in Toxoplasma ” by Farhat et al was posted to the bioRxiv server [ 62 ]. As implied by the title, this study reached many of the same conclusions presented here, including the m6A writer and YTH1 complex constituents and the role of m6A in 3’-end formation.…”

“…All datasets from this work have been deposited in the NCBI GEO database accession number GSE178355 Differential expression of IAA-treated lines was determined following the same pipeline as described above without in silico depletion of rRNA and tRNA. De novo transcript assembly was performed with StringTie using default settings [62]. Transcript length and gene orientation were determined using tools available on the public Galaxy webserver and ToxoDB.org [64,69].…”

Altiratinib blocks Toxoplasma gondii and Plasmodium falciparum development by selectively targeting a spliceosome kinase

“…All datasets were subsequently basecalled with a Guppy version higher than 5.0.1 with a Q score cutoff of >7. Long read alignment was performed by Minimap2 as previously described ( 37 ). Alignments were converted and sorted using Samtools.…”

“Reading” a new chapter in protozoan parasite transcriptional regulation

“…More recently, the significance of mRNA methylation has been revealed in protozoans [ 59 – 66 ]. mRNA adenosines, particularly in the 3′ UTR, can be methylated at the N6 position (m6A) to regulate mRNA structure and recruit effector proteins.…”

“…Knockdown of PfYTH.2 revealed a role in translational repression that is essential for blood stage development [ 62 ]. Two YTH homologs have also been reported in Toxoplasma [ 59 , 61 ], one of which, TgYTH2, is also involved in repression of translation and is essential in the tachyzoite stage [ 59 , 61 ]. The divergence of these methyl-RNA readers from humans and their critical function in Apicomplexans hints at therapeutic potential for parasitic infections.…”