Regulation of Co-transcriptional Pre-mRNA Splicing by m6A through the Low-Complexity Protein hnRNPG

N6-methyladenosine (m6A) modification occurs co-transcriptionally and impacts pre-mRNA processing; however, the mechanism of co-transcriptional m6A-dependent alternative splicing regulation is still poorly understood. Heterogeneous nuclear ribonucleoprotein G (hnRNPG) is an m6A reader protein that binds RNA through RRM and Arg-Gly-Gly (RGG) motifs. Here, we show that hnRNPG directly binds to the phosphorylated carboxy-terminal domain (CTD) of RNA polymerase II (RNAPII) using RGG motifs in its low-complexity region. Through interactions with the phosphorylated CTD and nascent RNA, hnRNPG associates co-transcriptionally with RNAPII and regulates alternative splicing transcriptome-wide. m6A near splice sites in nascent pre-mRNA modulates hnRNPG binding, which influences RNAPII occupancy patterns and promotes exon inclusion. Our results reveal an integrated mechanism of co-transcriptional m6A-mediated splicing regulation, in which an m6A reader protein uses RGG motifs to co-transcriptionally interact with both RNAPII and m6A-modified nascent pre-mRNA to modulate RNAPII occupancy and alternative splicing. [Display omitted] •The m6A reader protein hnRNPG interacts with RNA polymerase II using an RGG region•hnRNPG binds to nascent m6A-modified pre-mRNA and regulates alternative splicing•m6A is enriched in exonic regions near splice sites of hnRNPG-regulated exons•hnRNPG affects RNA polymerase II occupancy around hnRNPG-regulated exons Zhou et al. show that the m6A reader protein hnRNPG interacts with m6A-modified nascent pre-mRNA and the phosphorylated C-terminal domain of RNA polymerase II to regulate alternative splicing. These interactions depend on an RGG region in the low-complexity region of hnRNPG.