Transcription Start Site Choice Regulates m⁶A Stoichiometry in Cap-Proximal Regions

Background/Objectives:N6-methyladenosine (m⁶A) is the most prevalent and functionally significant internal modification within eukaryotic mRNA. While m⁶A is known to be regulated at internal sites by factors such as splice junctions, the mechanisms governing deposition within the cap-proximal region remain poorly understood. This study aims to determine the patterns of m⁶A stoichiometry in cap-proximal regions and to investigate whether the choice of the specific transcription start site (TSS) can affect m⁶A stoichiometry. Methods: We re-analyzed our published single-nucleotide-resolution CROWN-seq data to quantify m⁶A stoichiometry across transcript isoforms with different TSSs, and assessed the relationship between specific TSSs and specific m⁶A sites ("TSS-m⁶A-site pairs"). Results: We established the first single-nucleotide-resolution dataset of m⁶A stoichiometry across the transcriptome in cap proximal regions, including stoichiometry of m⁶A across 5' isoforms for each gene. We found that m⁶A deposition is markedly inhibited within a narrow cap-proximal region in a distance-sensitive manner. m⁶A sites located close to both 5' and 3' exon ends exhibit low methylation due to the overlap between the cap-proximal and 3' exon-end exclusion zones. Conclusions: We find that the first exon contains a narrow m⁶A exclusion zone at its 5' end. As a result, cap-proximal m⁶A sites can have different stoichiometries depending on the TSS choice. As the m⁶A site is positioned farther from the TSS, m⁶A stoichiometry increases. These results reveal that TSS switching is a regulatory mechanism for m⁶A stoichiometry in cap-proximal regions and provide a mechanism for fine-tuning gene expression and mRNA fate through isoform-specific m⁶A modification stoichiometry.