Zhou KI, Shi H, Lyu R, Wylder AC, Matuszek Ż, Pan JN, He C, Parisien M, Pan T
N-methyladenosine (mA) modification occurs co-transcriptionally and impacts pre-mRNA processing; however, the mechanism of co-transcriptional mA-dependent alternative splicing regulation is still poorly understood. Heterogeneous nuclear ribonucleoprotein G (hnRNPG) is an mA 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. mA 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 mA-mediated splicing regulation, in which an mA reader protein uses RGG motifs to co-transcriptionally interact with both RNAPII and mA-modified nascent pre-mRNA to modulate RNAPII occupancy and alternative splicing.