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 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 take place between the two catalytic steps. We also observed gene-specific differences in co-transcriptional splicing efficiency, and intron retention correlated with inefficient 3' end cleavage. Remarkably, a thalassemia patient-derived mutation introducing a cryptic 3'SS improves both splicing and 3' end cleavage of individual beta-globin transcripts, revealing that functional coupling between the two co-transcriptional processes is a determinant of productive gene output. Nascent RNA was purified from murine erythroleukemia (MEL) cells before and after erythroid differentiation and sequenced for long read sequencing (LRS) using PacBio RSII and Sequel I flowcells in two biological replicates. PRO-seq was performed on MEL cells before and after erythroid differentiation in three biological replicates.