Regulation of pre-mRNA splicing by m6A through the low-complexity protein hnRNPG and co-transcriptional pausing

Co-transcriptional processing of nascent transcripts is coupled with transcription through the carboxy-terminal domain (CTD) of RNA polymerase II (RNAPII). The mRNA modification N6-methyladenosine (m6A) occurs co-transcriptionally and impacts pre-mRNA processing. How alternative splicing of pre-mRNA is co-transcriptionally regulated in an m6A-dependent manner is not well understood. Furthermore, splicing regulation through RNAPII pausing has been frequently suggested but the underlying control mechanism remains unclear. Here, we show that the m6A reader protein hnRNPG directly binds to the phosphorylated CTD of RNAPII using Arg-Gly-Gly (RGG) motifs in its low-complexity region. This RGG-phospho-CTD interaction and nascent RNA binding by hnRNPG enables its co-transcriptional association with RNAPII, resulting in transcriptome-wide regulation of alternative splicing and transcript abundance. m6A sites near exon splice sites in nascent mRNA further modulate hnRNPG binding and exon splicing. Exon inclusion is associated with RNAPII pausing downstream of the m6A site. Our results reveal an integrated nuclear mechanism of m6A-mediated gene regulation, in which an m6A reader protein regulates gene expression by using RGG motifs to co-transcriptionally interact with both RNAPII and m6A-modified nascent pre-mRNA, while the interplay between hnRNPG binding and RNAPII pausing modulates alternative splicing regulation. HEK293T cells were transfected with hnRNPG siRNA and either pCMV3-Flag plasmid (NCV) or wild-type or mutant pCMV3-Flag-hnRNPG plasmid (WT, RRMmut, RGG1mut, or RGG2mut), with 3 biological replicates for each of the 5 different plasmid transfections. mRNA sequencing libraries were prepared using the Tru-Seq Stranded mRNA LT Sample Prep Kit (RS-122-9005DOC, Illumina). The WT replicates were used as the reference when analyzing for changes in transcript abundance or exon expression. To investigate the hnRNPG effect on alternative splicing through its binding to nascent mRNA, we performed hnRNPG PAR-CLIP with the chromatin fraction to sequence only the chromatin-bound mRNAs that were crosslinked to hnRNPG. To investigate whether hnRNPG affects RNAPII pausing, we performed quantitative chromatin-immunoprecipitation followed by sequencing (ChIP-seq). We first knocked down hnRNPG protein using two different siRNA, followed by building ChIP-seq libraries (Figure S7E) using individual antibodies for RNAPII, the S2-phosphorylated CTD (S2P), and the S5-phosphorylated CTD (S5P). Each sample was spiked-in with a same amount of the Drosophila chromatin and Drosophila-specific antibody to enable cross-sample comparison of RNAPII densities.