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.
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Acknowledgements
Special thanks to F. Stewart for his suggestions regarding FOS induction and the use of camptothecin. We are grateful to D. Black, M. Rosbash, C. Eckmann, T. Langenberg, D. Stanek and members of our laboratory for helpful discussions and comments on the manuscript. This research was funded by support from the Max Planck Gesellschaft.
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K.M.N. and I.L. conceived and designed the experiments. I.L. and A.K.S. performed the experiments. I.L. analyzed the data. K.M.N. wrote the paper.
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Supplementary information
Supplementary Fig. 1
Sodium arsenite treatment does not alter the nuclear localization of U1-70K, U2AF65 and snRNAs. (PDF 110 kb)
Supplementary Fig. 2
Distribution of acetylated histone H4 along the induced FOS gene with and without camptothecin. (PDF 88 kb)
Supplementary Fig. 3
Characterization of the ChRIP assay. (PDF 257 kb)
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Listerman, I., Sapra, A. & Neugebauer, K. Cotranscriptional coupling of splicing factor recruitment and precursor messenger RNA splicing in mammalian cells. Nat Struct Mol Biol 13, 815–822 (2006). https://doi.org/10.1038/nsmb1135
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DOI: https://doi.org/10.1038/nsmb1135
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