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N 6 -methyladenosine (m 6 A) modification occurs co-transcriptionally and impacts pre-mRNA processing; however, the mechanism of co-transcriptional m 6 A-dependent alternative splicing regulation is still poorly understood. Heterogeneous nuclear ribonucleoprotein G (hnRNPG) is an m 6 A 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. m 6 A 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 m 6 A-mediated splicing regulation, in which an m 6 A reader protein uses RGG motifs to co-transcriptionally interact with both RNAPII and m 6 A-modified nascent pre-mRNA to modulate RNAPII occupancy and alternative splicing., (Copyright © 2019 Elsevier Inc. All rights reserved.)

N6-methyladenosine (m6A) is the most abundant internal modification in eukaryotic messenger RNA (mRNA), and affects almost every stage of the mRNA life cycle. The YTH-domain proteins can specifically recognize m6A modification to control mRNA maturation, translation and decay. m6A can also alter RNA structures to affect RNA-protein interactions in cells. Here, we show that m6A increases the accessibility of its surrounding RNA sequence to bind heterogeneous nuclear ribonucleoprotein G (HNRNPG). Furthermore, HNRNPG binds m6A-methylated RNAs through its C-terminal low-complexity region, which self-assembles into large particles in vitro. The Arg-Gly-Gly repeats within the low-complexity region are required for binding to the RNA motif exposed by m6A methylation. We identified 13,191 m6A sites in the transcriptome that regulate RNA-HNRNPG interaction and thereby alter the expression and alternative splicing pattern of target mRNAs. Low-complexity regions are pervasive among mRNA binding proteins. Our results show that m6A-dependent RNA structural alterations can promote direct binding of m6A-modified RNAs to low-complexity regions in RNA binding proteins., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)