The m⁶A pathway protects the transcriptome integrity by restricting RNA chimera formation in plants

Abstract

Global, segmental, and gene duplication–related processes are driving genome size and complexity in plants. Despite their evolutionary potentials, those processes can also have adverse effects on genome regulation, thus implying the existence of specialized corrective mechanisms. Here, we report that an N6-methyladenosine (m6A)–assisted polyadenylation (m-ASP) pathway ensures transcriptome integrity in Arabidopsis thaliana. Efficient m-ASP pathway activity requires the m6A methyltransferase-associated factor FIP37 and CPSF30L, an m6A reader corresponding to an YT512-B Homology Domain-containing protein (YTHDC)-type domain containing isoform of the 30-kD subunit of cleavage and polyadenylation specificity factor. Targets of the m-ASP pathway are enriched in recently rearranged gene pairs, displayed an atypical chromatin signature, and showed transcriptional readthrough and mRNA chimera formation in FIP37- and CPSF30L-deficient plants. Furthermore, we showed that the m-ASP pathway can also restrict the formation of chimeric gene/transposable-element transcript, suggesting a possible implication of this pathway in the control of transposable elements at specific locus. Taken together, our results point to selective recognition of 3′-UTR m6A as a safeguard mechanism ensuring transcriptome integrity at rearranged genomic loci in plants.

Nanopore direct RNA sequencing maps an Arabidopsis N6 methyladenosine epitranscriptome

“…We detected 523 loci with increased levels of chimeric RNAs in vir-1 resulting from unterminated transcription proceeding into downstream genes on the same strand. Chimeric RNAs were recently detected in mutants affecting other components of the Arabidopsis m 6 A writer complex, MTA and FIP37 38 . However, only 33% of upstream genes forming the chimeric RNAs had detectable m 6 A sites in the VIR-complemented line with restored VIR activity.…”

“…Notably, alternative polyadenylation controls the expression of an Arabidopsis CPSF30 isoform that encodes a YT521-B homology (YTH) domain with the potential to bind and read m 6 A 43 . A recent study indicated that this YTH domain-containing isoform is required to supress chimera formation 38 . Consequently, in plants, m 6 A may also contribute to the recognition of specific RNA 3′ ends.…”

Occurrence and Functions of m6A and Other Covalent Modifications in Plant mRNA

“…However, the homology between FPA and RBM15A/B is confined to the RNA recognition motifs and the overall degree of identity is low. Although the possible involvement of FPA in m 6 A deposition remains uninvestigated, mutants in FPA and m 6 A pathway components share at least one molecular phenotype: partial loss of function causes transcriptional read-through and chimeric RNA formation (Hornyik et al, 2010;Duc et al, 2013;Pontier et al, 2019). Nonetheless, it is clear that the requirement of FPA for m 6 A writer function, if any, cannot be absolute, because in contrast to most knockout mutants in m 6 A writer components ( .…”

“…2) compared with five in humans. The 13 Arabidopsis YTH-domain proteins can be divided into 11 YTHDF proteins called EVOLU-TIONARILY CONSERVED C-TERMINAL REGION1 (ECT1) to ECT11, one classical YTHDC1-type protein (At4g11970), and one YTHDC protein, unusual in that it also contains additional folded domains: it is CPSF30, the 30-kD subunit of the cleavage and polyadenylation specificity factor involved in pre-mRNA cleavage and 39-end formation (Zhang et al, 2008;Thomas et al, 2012;Bruggeman et al, 2014;Pontier et al, 2019).…”

“…For example, m 6 A is required for sex determination in flies, because the sex determination pathway relies on alternative splicing of the sexlethal (sxl) pre-mRNA, a process that in turn depends critically on m 6 A deposition around the alternatively spliced sxl exon (Haussmann et al, 2016;Lence et al, 2016). In plants, a recent study has uncovered a special role of m 6 A in mRNA 39-end formation and transcription termination at recently duplicated genes: at such loci, read-through transcription into the downstream gene is observed in m 6 A-deficient plants and, interestingly, in plants containing point mutations to abrogate the aromatic cage in the YTH domain of CPSF30 (Pontier et al, 2019). It is possible that m 6 A-directed CPSF30 function stimulates pre-mRNA cleavage and RNA Polymerase II termination more generally but that the effect of losing this reinforcement only becomes visible at loci with recent gene duplications (Pontier et al, 2019).…”