Article | Published:

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

Nature Structural & Molecular Biology volume 13, pages 973980 (2006) | Download Citation

Subjects

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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    , , , & In situ transcription and splicing in the Balbiani ring 3 gene. EMBO J. 20, 2564–2574 (2001).

  2. 2.

    , & RNA polymerase II carboxy-terminal domain phosphorylation is required for cotranscriptional pre-mRNA splicing and 3′-end formation. Mol. Cell. Biol. 24, 8963–8969 (2004).

  3. 3.

    et al. Functional coupling of RNAP II transcription to spliceosome assembly. Genes Dev. 20, 1100–1109 (2006).

  4. 4.

    , , , & Multiple links between transcription and splicing. RNA 10, 1489–1498 (2004).

  5. 5.

    & A genomic view of alternative splicing. Nat. Genet. 30, 13–19 (2002).

  6. 6.

    et al. Genome-wide survey of human alternative pre-mRNA splicing with exon junction microarrays. Science 302, 2141–2144 (2003).

  7. 7.

    et al. Novel RNAs identified from an in-depth analysis of the transcriptome of human chromosomes 21 and 22. Genome Res. 14, 331–342 (2004).

  8. 8.

    et al. A polar mechanism coordinates different regions of alternative splicing within a single gene. Mol. Cell 19, 393–404 (2005).

  9. 9.

    , & Distal regulation of alternative splicing by splicing enhancer in equine beta-casein intron 1. RNA 12, 498–507 (2006).

  10. 10.

    & The splicing factor, Prp40, binds the phosphorylated carboxyl-terminal domain of RNA polymerase II. J. Biol. Chem. 275, 39935–39943 (2000).

  11. 11.

    , , , & Protein-interaction modules that organize nuclear function: FF domains of CA150 bind the phosphoCTD of RNA polymerase II. Proc. Natl. Acad. Sci. USA 97, 9015–9020 (2000).

  12. 12.

    , , & Splicing factors associate with hyperphosphorylated RNA polymerase II in the absence of pre-mRNA. J. Cell Biol. 136, 19–28 (1997).

  13. 13.

    et al. A hyperphosphorylated form of the large subunit of RNA polymerase II is associated with splicing complexes and the nuclear matrix. Proc. Natl. Acad. Sci. USA 93, 8253–8257 (1996).

  14. 14.

    et al. The C-terminal domain of the largest subunit of RNA polymerase II interacts with a novel set of serine/arginine-rich proteins. Proc. Natl. Acad. Sci. USA 93, 6975–6980 (1996).

  15. 15.

    et al. The C-terminal domain of RNA polymerase II couples mRNA processing to transcription. Nature 385, 357–361 (1997).

  16. 16.

    & RNA polymerase II targets pre-mRNA splicing factors to transcription sites in vivo. Mol. Cell 3, 697–705 (1999).

  17. 17.

    & A functional interaction between the carboxy-terminal domain of RNA polymerase II and pre-mRNA splicing. J. Cell Biol. 136, 5–18 (1997).

  18. 18.

    , , , & Co-transcriptional commitment to alternative splice site selection. Nucleic Acids Res. 26, 5568–5572 (1998).

  19. 19.

    , & Perturbation of transcription elongation influences the fidelity of internal exon inclusion in Saccharomyces cerevisiae. RNA 9, 993–1006 (2003).

  20. 20.

    et al. Antagonistic effects of T-Ag and VP16 reveal a role for RNA pol II elongation on alternative splicing. EMBO J. 20, 5759–5768 (2001).

  21. 21.

    , , , & Transcriptional activators differ in their abilities to control alternative splicing. J. Biol. Chem. 277, 43110–43114 (2002).

  22. 22.

    et al. A slow RNA polymerase II affects alternative splicing in vivo. Mol. Cell 12, 525–532 (2003).

  23. 23.

    & Specific transcriptional pausing activates polyadenylation in a coupled in vitro system. Mol. Cell 3, 593–600 (1999).

  24. 24.

    , & Cotranscriptional coupling of splicing factor recruitment and precursor messenger RNA splicing in mammalian cells. Nat. Struct. Mol. Biol. 13, 815–822 (2006).

  25. 25.

    Rules of engagement: co-transcriptional recruitment of pre-mRNA processing factors. Curr. Opin. Cell Biol. 17, 251–256 (2005).

  26. 26.

    & Investigating RNA polymerase II carboxyl-terminal domain (CTD) phosphorylation. Eur. J. Biochem. 270, 3859–3870 (2003).

  27. 27.

    & Regulation of transcription elongation by phosphorylation. Biochim. Biophys. Acta 1577, 261–275 (2002).

  28. 28.

    The CTD code. Nat. Struct. Biol. 10, 679–680 (2003).

  29. 29.

    & Analysis of the requirement for RNA polymerase II CTD heptapeptide repeats in pre-mRNA splicing and 3′-end cleavage. RNA 10, 581–589 (2004).

  30. 30.

    , , & Genetic analysis of the repetitive carboxyl-terminal domain of the largest subunit of mouse RNA polymerase II. Mol. Cell. Biol. 8, 330–339 (1988).

  31. 31.

    , , & Functional association between promoter structure and transcript alternative splicing. Proc. Natl. Acad. Sci. USA 94, 11456–11460 (1997).

  32. 32.

    , , & Conditional expression of RNA polymerase II in mammalian cells. Deletion of the carboxyl-terminal domain of the large subunit affects early steps in transcription. J. Biol. Chem. 275, 24375–24382 (2000).

  33. 33.

    et al. RNA polymerase II C-terminal domain required for enhancer-driven transcription. Nature 374, 660–662 (1995).

  34. 34.

    & Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc. Natl. Acad. Sci. USA 89, 5547–5551 (1992).

  35. 35.

    et al. Three functional classes of transcriptional activation domain. Mol. Cell. Biol. 16, 2044–2055 (1996).

  36. 36.

    , , & A monoclonal antibody against 2,2,7-trimethylguanosine that reacts with intact, class U, small nuclear ribonucleoproteins as well as with 7-methylguanosine-capped RNAs. Eur. J. Biochem. 168, 461–467 (1987).

  37. 37.

    , , , & The last CTD repeat of the mammalian RNA polymerase II large subunit is important for its stability. Nucleic Acids Res. 32, 35–44 (2004).

  38. 38.

    , , & A 10 residue motif at the C-terminus of the RNA pol II CTD is required for transcription, splicing and 3′ end processing. EMBO J. 22, 4274–4282 (2003).

  39. 39.

    , , , & RNA editing and alternative splicing: the importance of co-transcriptional coordination. EMBO Rep. 7, 303–307 (2006).

  40. 40.

    , & Role of the mammalian RNA polymerase II C-terminal domain (CTD) nonconsensus repeats in CTD stability and cell proliferation. Mol. Cell. Biol. 25, 7665–7674 (2005).

  41. 41.

    et al. Coupling of transcription with alternative splicing: RNA pol II promoters modulate SF2/ASF and 9G8 effects on an exonic splicing enhancer. Mol. Cell 4, 251–258 (1999).

  42. 42.

    & Participation of the C-terminal domain of RNA polymerase II in exon definition during pre-mRNA splicing. Mol. Cell. Biol. 20, 8290–8301 (2000).

  43. 43.

    & Capping, splicing, and 3′ processing are independently stimulated by RNA polymerase II: different functions for different segments of the CTD. Genes Dev. 15, 1783–1795 (2001).

  44. 44.

    , & Different phosphorylated forms of RNA polymerase II and associated mRNA processing factors during transcription. Genes Dev. 14, 2452–2460 (2000).

  45. 45.

    et al. Transition from initiation to promoter proximal pausing requires the CTD of RNA polymerase II. Nucleic Acids Res. 33, 5139–5144 (2005).

  46. 46.

    , , & Requirements of the RNA polymerase II C-terminal domain for reconstituting pre-mRNA 3′ cleavage. Mol. Cell. Biol. 22, 1684–1692 (2002).

  47. 47.

    , , & Regulation of alternative splicing by a transcriptional enhancer through RNA pol II elongation. Proc. Natl. Acad. Sci. USA 99, 8185–8190 (2002).

  48. 48.

    , & The two steps of poly(A)-dependent termination, pausing and release, can be uncoupled by truncation of the RNA polymerase II carboxyl-terminal repeat domain. Mol. Cell. Biol. 24, 4092–4103 (2004).

  49. 49.

    et al. Role of RNA polymerase II carboxy-terminal domain in coordinating transcription with RNA processing. Cold Spring Harb. Symp. Quant. Biol. 63, 301–309 (1998).

  50. 50.

    et al. A set of consensus mammalian mediator subunits identified by multidimensional protein identification technology. Mol. Cell 14, 685–691 (2004).

  51. 51.

    & Distribution of pre-mRNA splicing factors at sites of RNA polymerase II transcription. Genes Dev. 11, 1148–1159 (1997).

  52. 52.

    & Differential recruitment of pre-mRNA splicing factors to alternatively spliced transcripts in vivo. PLoS Biol. 3, e374 (2005).

  53. 53.

    & Alternative pre-mRNA splicing: the logic of combinatorial control. Trends Biochem. Sci. 25, 381–388 (2000).

  54. 54.

    , & Understanding alternative splicing: towards a cellular code. Nat. Rev. Mol. Cell Biol. 6, 386–398 (2005).

  55. 55.

    et al. A novel bipartite splicing enhancer modulates the differential processing of the human fibronectin EDA exon. Nucleic Acids Res. 22, 1018–1022 (1994).

  56. 56.

    et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498 (2001).

  57. 57.

    , , , & Regulated tissue-specific expression of antagonistic pre-mRNA splicing factors. RNA 4, 430–444 (1998).

Download references

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.

Author information

Affiliations

  1. Laboratorio de Fisiología y Biología Molecular, Departamento de Fisiología, 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

Authors

  1. Search for Manuel de la Mata in:

  2. Search for Alberto R Kornblihtt in:

Contributions

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.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Alberto R Kornblihtt.

Supplementary information

PDF files

  1. 1.

    Supplementary Fig. 1

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

  2. 2.

    Supplementary Fig. 2

    CTD sequences of RPB1 clones.

  3. 3.

    Supplementary Methods

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nsmb1155

Further reading