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RNA polymerase II C-terminal domain mediates regulation of alternative splicing by SRp20

Nature Structural & Molecular Biology volume 13, pages 973–980 (2006)Cite this article

Abstract

Previous studies have linked the C-terminal domain (CTD) of RNA polymerase II (pol II) with cotranscriptional precursor messenger RNA processing, but little is known about the CTD's function in regulating alternative splicing. We have examined this function using α-amanitin–resistant pol II CTD mutants and fibronectin reporter minigenes. We found that the CTD is required for the inhibitory action of the serine/arginine-rich (SR) protein SRp20 on the inclusion of a fibronectin cassette exon in the mature mRNA. CTD phosphorylation controls transcription elongation, which is a major contributor to alternative splicing regulation. However, the effect of SRp20 is still observed when transcription elongation is reduced. These results suggest that the CTD promotes exon skipping by recruiting SRp20 and that this contributes independently of elongation to the transcriptional control of alternative splicing.

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Figure 1: ΔCTD pol II increases EDI inclusion levels independently of the promoter.
Figure 2: Results from an inducible system show that the CTD deletion's effect on alternative splicing is dependent on transcription by ΔCTD pol II.
Figure 3: The CTD's effect on EDI inclusion depends on its length but not its heptad composition.
Figure 4: SRp20 promotes EDI skipping via the CTD.
Figure 5: Unresponsiveness to siSRp20 is independent of the promoter used and is not a consequence of elevated basal EDI inclusion levels.

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Acknowledgements

We thank P. Cramer and D. Bentley for their initial contributions to this work and N. Fong, V. Buggiano, J.P. Fededa, M.J. Muñoz, M. Blaustein, F. Pelisch, E. Petrillo, M. Alló, A. Srebrow and I. Schor for their help and discussions. Special thanks to R. Chapman for helpful criticism as well as for providing reagents. This work was supported by grants from the Fundación Antorchas, the Agencia Nacional de Promoción de Ciencia y Tecnología of Argentina, the European Union Network of Excellence on Alternative Splicing and the University of Buenos Aires. M.d.l.M. is recipient of a fellowship from the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) of Argentina. A.R.K. is a Howard Hughes Medical Institute international research scholar and a career investigator of the CONICET.

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  1. Departamento de Fisiología, Laboratorio de Fisiología y Biología Molecular, Biología Molecular y Celular, IFIBYNE-CONICET, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabellón 2, (C1428EHA), Buenos Aires, Argentina

    Manuel de la Mata & Alberto R Kornblihtt

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  1. Manuel de la Mata
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M.d.l.M. designed, performed and interpreted the experiments. A.R.K. discussed the design and interpretation of experiments and supervised the whole project. The manuscript was cowritten by both authors.

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Correspondence to Alberto R Kornblihtt.

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Supplementary information

Supplementary Fig. 1

The effect of the CTD on alternative splicing is independent of its influence on capping and 3′-end processing. (PDF 619 kb)

Supplementary Fig. 2

CTD sequences of RPB1 clones. (PDF 59 kb)

Supplementary Methods (PDF 46 kb)

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de la Mata, M., Kornblihtt, A. RNA polymerase II C-terminal domain mediates regulation of alternative splicing by SRp20. Nat Struct Mol Biol 13, 973–980 (2006). https://doi.org/10.1038/nsmb1155

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