Chromatin-bound U2AF2 splicing factor ensures exon inclusion

Most mRNA splicing occurs co-transcriptionally, but it is unclear how splicing factors accurately select exons for inclusion. Using CUT&RUN profiling in K562 cells, we demonstrate that three splicing factors-SF3B1, U2AF1, and U2AF2-bind near active promoters of intron-containing and intronless genes, implying their association with the general transcriptional machinery. RNase A treatment reduces factor binding at promoters, indicating that these proteins interact with nascent transcripts. Strikingly, the U2AF2 protein also accumulates throughout intron-containing gene bodies and requires histone H3-lysine36 trimethylation but not nascent transcripts or persistent RNA polymerase II. Chromatin-bound U2AF2 preferentially binds to exons of highly expressed, exon-dense genes, with greater occupancy at exons skipped after U2AF2 knockdown, suggesting that U2AF2 enhances exon selection accuracy. U2AF2-targeted genes include those encoding splicing factors, where it improves splicing accuracy and efficiency. Our findings provide a mechanistic basis for the homeostatic regulation of efficient co-transcriptional splicing by chromatin-bound U2AF2.