Co-transcriptional splicing regulates 3′ end cleavage during mammalian erythropoiesis

Pre-mRNA processing steps are tightly coordinated with transcription in many organisms. To determine how co-transcriptional splicing is integrated with transcription elongation and 3′ end formation in mammalian cells, we performed long-read sequencing of individual nascent RNAs and precision run-on sequencing (PRO-seq) during mouse erythropoiesis. Splicing was not accompanied by transcriptional pausing and was detected when RNA polymerase II (Pol II) was within 75–300 nucleotides of 3′ splice sites (3′SSs), often during transcription of the downstream exon. Interestingly, several hundred introns displayed abundant splicing intermediates, suggesting that splicing delays can take place between the two catalytic steps. Overall, splicing efficiencies were correlated among introns within the same transcript, and intron retention was associated with inefficient 3′ end cleavage. Remarkably, a thalassemia patient-derived mutation introducing a cryptic 3′SS improved both splicing and 3′ end cleavage of individual β-globin transcripts, demonstrating functional coupling between the two co-transcriptional processes as a determinant of productive gene output. [Display omitted] •Most introns in differentiating murine erythroblasts are spliced co-transcriptionally•Long-read sequencing of nascent RNA reveals coordinated removal of multiple introns•Pol II pausing is not significantly detected at splice sites•Co-transcriptional splicing efficiency influences 3′ end cleavage efficiency Does rapid splicing after intron transcription promote mammalian gene expression? Reimer et al. report that most introns are removed when Pol II is located within the downstream exon, suggesting that proximity to the spliceosome supports regulation. Introns within single transcripts were coordinately spliced, and efficient splicing promoted 3′ end cleavage.