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

SUMMARY As a dynamic and reversible post‐transcriptional marker, N 6 ‐methyladenosine (m 6 A) plays an important role in the regulation of biological functions, which are mediated by m 6 A 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 m 6 A pathway components across species at evolutionary levels. In this study, we identified 4062 m 6 A 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 m 6 A ratio profiles in six angiosperms species, with a particular focus on the m 6 A 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 m 6 A modification and PAL. Besides, conserved m 6 A sites distributed in CDS and 3′UTR were detected in the ECT1/2‐Poaceae4, and the dual‐luciferase assay further demonstrated that these conserved m 6 A sites in the 3′UTR negatively regulated the expression of Firefly luciferase (LUC) gene. Finally, we developed transcription factor regulatory networks for m 6 A 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 m 6 A pathway components and their modifications in plants, providing a valuable resource for future functional analysis in this field.