N6-methyladenosine Recruits HNRNPG for Alternative Splicing Regulation

Understanding how RNA-binding proteins interact with RNA transcripts for splicing regulation is fundamental to human biology and disease. N6-methyladenosine (m6A), the most abundant and dynamic internal modification of eukaryotic messenger RNA (mRNA), is important for RNA splicing, but its detailed mechanism remains unclear. We find the splicing factor heterogeneous nuclear ribonucleoprotein G (HNRNPG) selectively binds m6A-modified RNA in vitro and in vivo. Transcriptome-wide identification of HNRNPG binding sites reveals the binding sequence specificity of HNRNPG (AGRAC, R=A/G), which embeds the m6A consensus motif. The m6A methylation makes the binding sequence more accessible to facilitate HNRNPG binding. In total, we identify 13,191 high-confidence m6A sites recruiting HNRNPG in HEK293T cells. Furthermore, m6A writers depletion decreases HNRNPG binding at m6A sites, and consequently cause alternative splicing changes similar as HNRNPG depletion on the m6A-containing RNA transcripts. These findings indicate that m6A RNA methylation facilitates HNRNPG binding to regulate mRNA alternative splicing. Overall design: Measure the m6A methylated HNRNPG binding sites transcriptome-wide by PAR-CLIP-MeRIP; measure the differential HNRNPG occupancies upon METTL3/METTL14 knockdown by PAR-CLIP; measure RNA abundance and splicing level changes upon HNRNPG, METTL3 and METTL14 knockdown