Transcript architecture sets the m6A landscape: CSTF2 and CSTF2T reshape m6A through cleavage-dependent and -independent mechanisms

Abstract

Alternative RNA processing generates extensive transcript diversity, yet how transcript architecture influences selective m6A deposition is incompletely understood. Exon-junction-based models explain where m6A is excluded, but a positive determinant of m6A accumulation remains undefined. Here, we leverage Zika virus-induced changes in m6A deposition to uncover determinants of transcript-selective methylation. By integrating GLORI-seq, native METTL3 RNA immunoprecipitation, and nanopore direct RNA sequencing, we generate a single-nucleotide, isoform-resolved map of m6A dynamics during infection. We identify over 2,000 dynamic m6A sites, many arising from changes in transcript architecture, and pinpoint proximal polyadenylation sites as positive determinants of m6A accumulation. The cleavage stimulation factors CSTF2 and CSTF2T drive this remodeling through two routes: redundant induction of intronic polyadenylation, which converts internal exons into terminal exons that expose DRACH motifs to METTL3, and non-redundant, cleavage-independent recruitment of METTL3 near proximal polyadenylation sites, establishing alternative polyadenylation as a key architectural determinant of the m6A landscape.