GSE168155
A plant-like mechanism coupling m6A reading to polyadenylation safeguards transcriptome integrity and developmental gene partitioning in Toxoplasma
In this study, we report on our findings in T. gondii of an m6A-dependent 3'end polyadenylation serving as a transcriptional barrier at certain loci such as stage-specific genes. 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. We bring Nanopore direct RNA sequencing-based evidence of 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. Overall design: Methods: RNA profiles were generated from HFF cells infected by RHDku80 CPSF4-mAID-HA T. gondii strain for untreated conditions, auxin induced KD at 7h, 24h, and 48h of CPSF4. Total RNAs were extracted and purified using TRIzol (Invitrogen, Carlsbad, CA, USA) and RNeasy Plus Mini Kit (Qiagen). RNA-sequencing was performed by GENEWIZ (South Plainfield, NJ, USA). RNA quantity and integrity were determined using the Qubit® Fluorometer and the Fragment Analyzer system with the PROSize 3.0 software (Agilent Technologies, Palo Alto, California, USA). The RQN were ranging from 8.8 to 10 for all samples, which was considered sufficient. Illumina TruSEQ RNA library prep and sequencing reagents were used following the manufacturer's recommendations using polyA-selected transcripts (Illumina, San Diego, CA, USA). The samples were sequenced on the Illumina NovaSeq platform (2 x 150 bp, single index) and generated ~20 million paired-end reads for each sample.