In Brief Zhou et al. show that the m 6 A reader protein hnRNPG interacts with m 6 Amodified nascent pre-mRNA and the phosphorylated C-terminal domain of RNA polymerase II to regulate alternative splicing. These interactions depend on an RGG region in the low-complexity region of hnRNPG.

Transcription is a highly dynamic process that generates single-stranded DNA (ssDNA) in the genome as ‘transcription bubbles’. Here we describe a k ethoxal- a ssisted s ingle-stranded DNA seq uencing (KAS-seq) approach, based on the fast and specific reaction between N 3 -kethoxal and guanines in ssDNA in live cells and mouse tissues. KAS-seq enables rapid (within 5 min), sensitive, and genome-wide capture and mapping of ssDNA produced by transcriptionally active RNA polymerases or other processes in situ by using as few as 1,000 cells. KAS-seq defines a group of enhancers that are single-stranded, which enrich unique sequence motifs and are associated with specific transcription factor binding and more enhancer-promotor interactions. Under protein condensation inhibition conditions, KAS-seq uncovers a rapid release of RNA polymerase II (Pol II) from a group of promotors. KAS-seq thus facilitates fast, comprehensive, and accurate analysis of transcription dynamics and enhancer activities simultaneously in a low input and high-throughput manner.

N 6 –methyladenosine (m 6 A) is the most abundant mRNA modification and plays crucial roles in diverse physiological processes. Utilizing a Massively Parallel Assay for m 6 A (MPm 6 A), we discover that m 6 A specificity is globally regulated by “suppressors” that prevent m 6 A deposition in unmethylated transcriptome regions. We identify Exon Junction Complexes (EJCs) as m 6 A suppressors that protect exon junction-proximal RNA within coding sequences from methylation and regulate mRNA stability through m 6 A suppression. EJC suppression of m 6 A underlies multiple global characteristics of mRNA m 6 A specificity, with the local range of EJC protection sufficient to suppress m 6 A deposition in average-length internal exons, but not in long internal and terminal exons. EJC-suppressed methylation sites co-localize with EJC-suppressed splice sites, suggesting that exon architecture broadly determines local mRNA accessibility to regulatory complexes.