biostudies-literatureReimer KANCATS NIH HHSNIDDK NIH HHSNHLBI NIH HHSNational Institutes of HealthNIGMS NIH HHS998-1012.e7https://www.ebi.ac.uk/biostudies/studies/S-EPMC8038867biostudies-literatureUnknown81(5)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.Molecular cellCo-transcriptional splicing regulates 3' end cleavage during mammalian erythropoiesis.PMC8038867R21 HL150642R01 GM112766U54 DK106857UL1 TR001863Neugebauer KMReimer KAMimoso CAAdelman KfalseCo-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.2021-01-01T00:00:00Z2021 Mar2026-05-31T05:00:18.237Z2026-04-08T08:10:07.684ZS-EPMC80388673344016910.1016/j.molcel.2020.12.018