Cotranscriptional coupling of splicing factor recruitment and precursor messenger RNA splicing in mammalian cells.
Coupling between transcription and RNA processing is a key gene regulatory mechanism. Here we use chromatin immunoprecipitation to detect transcription-dependent accumulation of the precursor mRNA (pre-mRNA) splicing factors hnRNP A1, U2AF65 and U1 and U5 snRNPs on the intron-containing human FOS gene. These factors were poorly detected on intronless heat-shock and histone genes, a result that opposes direct recruitment by RNA polymerase II (Pol II) or the cap-binding complex in vivo. However, an observed RNA-dependent interaction between U2AF65 and active forms of Pol II may stabilize U2AF65 binding to intron-containing nascent RNA. We establish chromatin-RNA immunoprecipitation and show that FOS pre-mRNA is cotranscriptionally spliced. Notably, the topoisomerase I inhibitor camptothecin, which stalls elongating Pol II, increased cotranscriptional splicing factor accumulation and splicing in parallel. This provides direct evidence for a kinetic link between transcription, splicing factor recruitment and splicing catalysis.
Listerman
Imke
I
Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany.
Sapra
Aparna K
AK
Neugebauer
Karla M
KM
D002166:Camptothecin / Q000494:pharmacology; D047369:Chromatin Immunoprecipitation; D005786:Gene Expression Regulation / Q000187:drug effects; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B / Q000378:metabolism*; D006801:Humans; D007438:Introns / Q000235:genetics; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism; D039381:RNA Cap-Binding Proteins / Q000378:metabolism; D012319:RNA Polymerase II / Q000378:metabolism; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism*; D012326:RNA Splicing / Q000187:drug effects / Q000235:genetics*; D014158:Transcription, Genetic / Q000187:drug effects; D014407:Tumor Cells, Cultured
D018840:HSP70 Heat-Shock Proteins; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D016760:Proto-Oncogene Proteins c-fos; D039381:RNA Cap-Binding Proteins; D012322:RNA Precursors; D012319:RNA Polymerase II; D002166:Camptothecin