The highly intronic nature of protein coding genes in mammals necessitates a co-transcriptional splicing mechanism as revealed by mNET-seq analysis. Immunoprecipitation of MNase-digested chromatin with antibodies against RNA polymerase II (Pol II) shows that active spliceosomes (both snRNA and proteins) are complexed to Pol II S5P CTD during elongation and co-transcriptional splicing. Notably, elongating Pol II-spliceosome complexes form strong interactions with nascent transcripts, resulting in footprints of approximately 60 nucleotides. Also, splicing intermediates formed by cleavage at the 5′ splice site are associated with nearly all spliced exons. These spliceosome-bound intermediates are frequently ligated to upstream exons, implying a sequential, constitutive, and U12-dependent splicing process. Finally, lack of detectable spliced products connected to the Pol II active site in human HeLa or murine lymphoid cells suggests that splicing does not occur immediately following 3′ splice site synthesis. Our results imply that most mammalian splicing requires exon definition for completion. Nojima and colleagues demonstrate widespread co-transcriptional splicing of mammalian genes by complex formation between the active spliceosome and the RNA polymerase II S5P CTD isoform. RNA processing intermediates within this complex are invariably detected with the transcript cleaved at 5′ ss but already spliced to upstream exons, implying a sequential splicing process.
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cell Biology