Evolution and post-transcriptional regulation insights of m⁶A writers, erasers, and readers in plant epitranscriptome

As a dynamic and reversible post-transcriptional marker, N⁶-methyladenosine (m⁶A) plays an important role in the regulation of biological functions, which are mediated by m⁶A pathway components including writers (MT-A70, FIP37, VIR and HAKAI family), erasers (ALKBH family) and readers (YTH family). There is an urgent need for a comprehensive analysis of m⁶A pathway components across species at evolutionary levels. In this study, we identified 4062 m⁶A pathway components from 154 plant species including green algae, utilizing large-scale phylogenetic to explore their origin and evolution. We discovered that the copy number of writers was conserved among different plant lineages, with notable expansions in the ALKBH and YTH families. Synteny network analysis revealed conserved genomic contexts and lineage-specific transpositions. Furthermore, we used Direct RNA Sequencing (DRS) to reveal the Poly(A) length (PAL) and m⁶A ratio profiles in six angiosperms species, with a particular focus on the m⁶A pathway components. The ECT1/2-Poeaece4 sub-branches (YTH family) with unique genomic contexts exhibited significantly higher expression level than genes of other ECT1/2 poeaece sub-branches (ECT1/2-Poeaece1-3), accompanied by lower m⁶A modification and PAL. Besides, conserved m⁶A sites distributed in CDS and 3'UTR were detected in the ECT1/2-Poaceae4, and the dual-luciferase assay further demonstrated that these conserved m⁶A sites in the 3'UTR negatively regulated the expression of Firefly luciferase (LUC) gene. Finally, we developed transcription factor regulatory networks for m⁶A pathway components, using yeast one-hybrid assay demonstrated that PheBPC1 could interact with the PheECT1/2-5 promoter. Overall, this study presents a comprehensive evolutionary and functional analysis of m⁶A pathway components and their modifications in plants, providing a valuable resource for future functional analysis in this field.