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.

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