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Spliceosome assembly is coupled to RNA polymerase II dynamics at the 3′ end of human genes

Nature Structural & Molecular Biology volume 18, pages 1115–1123 (2011)Cite this article

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Abstract

In the nucleus of higher eukaryotes, maturation of mRNA precursors involves an orderly sequence of transcription-coupled interdependent steps. Transcription is well known to influence splicing, but how splicing may affect transcription remains unclear. Here we show that a splicing mutation that prevents recruitment of spliceosomal snRNPs to nascent transcripts causes co-transcriptional retention of unprocessed RNAs that remain associated with polymerases stalled predominantly at the 3′ end of the gene. In contrast, treatment with spliceostatin A, which allows early spliceosome formation but destabilizes subsequent assembly of the catalytic complex, abolishes 3′ end pausing of polymerases and induces leakage of unspliced transcripts to the nucleoplasm. Taken together, the data suggest that recruitment of splicing factors and correct assembly of the spliceosome are coupled to transcription termination, and this might ensure a proofreading mechanism that slows down release of unprocessed transcripts from the transcription site.

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Figure 1: Imaging β-globin transcription sites in vivo.
Figure 2: Characterization of U2OS-βWT cells.
Figure 3: Imaging β-globin transcription dynamics.
Figure 4: Effect of intron 2 deletion on β-globin RNA splicing and polymerase dynamics.
Figure 5: Effect of SSA on β-globin RNA splicing and polymerase dynamics.
Figure 6: SSA reduces RNAPII density downstream of the poly(A) site in both β-globin and endogenous genes.
Figure 7: Coupling spliceosome assembly to leakage of unspliced transcripts to the nucleoplasm.
Figure 8: Schematic model illustrating the main conclusions from this study.

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Acknowledgements

We are grateful to R. Singer from Albert Einstein College of Medicine, E. Bertrand from the Institute of Molecular Genetics of Montpellier (IGMM) and Y. Shav-Tal from Bar-Ilan University for insightful discussions and technical advice. We also thank E. Bertrand (IGMM), J. Lykke-Andersen (University of California, San Diego), N. Gehring (Heidelberg University), R. Chapman (Helmholtz Institute Munich), B. Blencowe (University of Toronto) and M. Antoniou (King's College London) for kindly providing reagents. This work was supported by Fundação para a Ciência e Tecnologia (PTDC/BIA-BCM/101575/2008 to M.C.-F., SFRH/BPD/42523/2007 to J.R. and SFRH/BPD/34679/2007 to S.F.d.A.) and the European Commission (EURASNET, LSHG-CT-2005-518238 to M.C.-F.).

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  1. Sandra Bento Martins and José Rino: These authors contributed equally to this work.

Authors and Affiliations

  1. Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal

    Sandra Bento Martins, José Rino, Teresa Carvalho, Célia Carvalho, Jasmim Mona Klose, Sérgio Fernandes de Almeida & Maria Carmo-Fonseca

  2. Chemical Genetics Laboratory, RIKEN Advanced Science Institute, Saitama, Japan

    Minoru Yoshida

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Contributions

S.B.M. and J.R. designed and conducted the experiments and analyzed the data. J.R. was responsible for the microscopy. T.C. and C.C. generated and characterized the cell lines. J.M.K. carried out ChIP experiments and S.F.d.A. designed, conducted and analyzed biochemical experiments. M.Y. synthesized SSA. M.C.-F. conceived and supervised the project. S.B.M., J.R. and M.C.-F. wrote the paper.

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Correspondence to Maria Carmo-Fonseca.

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Martins, S., Rino, J., Carvalho, T. et al. Spliceosome assembly is coupled to RNA polymerase II dynamics at the 3′ end of human genes. Nat Struct Mol Biol 18, 1115–1123 (2011). https://doi.org/10.1038/nsmb.2124

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