Cotranscriptional coupling of splicing factor recruitment and precursor messenger RNA splicing in mammalian cells

Nat Struct Mol Biol. 2006 Sep;13(9):815-22. doi: 10.1038/nsmb1135. Epub 2006 Aug 20.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Camptothecin / pharmacology
  • Chromatin Immunoprecipitation
  • Gene Expression Regulation / drug effects
  • HSP70 Heat-Shock Proteins / metabolism
  • Heterogeneous Nuclear Ribonucleoprotein A1
  • Heterogeneous-Nuclear Ribonucleoprotein Group A-B / metabolism*
  • Humans
  • Introns / genetics
  • Proto-Oncogene Proteins c-fos / metabolism
  • RNA Cap-Binding Proteins / metabolism
  • RNA Polymerase II / metabolism
  • RNA Precursors / genetics
  • RNA Precursors / metabolism*
  • RNA Splicing / drug effects
  • RNA Splicing / genetics*
  • Transcription, Genetic* / drug effects
  • Tumor Cells, Cultured

Substances

  • HSP70 Heat-Shock Proteins
  • Heterogeneous Nuclear Ribonucleoprotein A1
  • Heterogeneous-Nuclear Ribonucleoprotein Group A-B
  • Proto-Oncogene Proteins c-fos
  • RNA Cap-Binding Proteins
  • RNA Precursors
  • RNA Polymerase II
  • Camptothecin