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

Nature Structural & Molecular Biology volume 13, pages 815–822 (2006)Cite this article

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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Figure 1
Figure 2: Induction of FOS and HSPA1B transcription leads to downstream accumulation of RNA Pol II, CBP80 and hnRNP A1.
Figure 3: Splicing factors accumulate cotranscriptionally on the intron-containing FOS gene.
Figure 4: Splicing factors do not accumulate on the induced heat-shock gene, HSPA1B, in the presence or absence of camptothecin.
Figure 5: Coimmunoprecipitation of splicing factors with RNA Pol II without cross-linking.
Figure 6: Cotranscriptional FOS pre-mRNA splicing detected by ChRIP and enhanced by camptothecin.

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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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  1. Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, Dresden, 01307, Germany

    Imke Listerman, Aparna K Sapra & Karla M Neugebauer

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  1. Imke Listerman
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Contributions

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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Correspondence to Karla M Neugebauer.

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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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