Transcript architecture sets the m 6 A landscape: CSTF2 and CSTF2T reshape m 6 A through cleavage-dependent and -independent mechanisms.
Alternative RNA processing generates extensive transcript diversity, yet how transcript architecture influences selective m 6 A deposition is incompletely understood. Exon-junction-based models explain where m 6 A is excluded, but a positive determinant of m 6 A accumulation remains undefined. Here, we leverage Zika virus-induced changes in m 6 A 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 m 6 A dynamics during infection. We identify over 2,000 dynamic m 6 A sites, many arising from changes in transcript architecture, and pinpoint proximal polyadenylation sites as positive determinants of m 6 A 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 m 6 A landscape.
