Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Opposing classes of prp8 alleles modulate the transition between the catalytic steps of pre-mRNA splicing

Nature Structural & Molecular Biology volume 14, pages 519–526 (2007)Cite this article

Abstract

The spliceosome is thought to undergo a conformational change between the two catalytic steps of precursor messenger RNA splicing, although the specific events in this transition are poorly understood. We previously proposed a two-state model of splicing in which the conformations required for the first and second steps are in competition. Here, we identify and characterize a class of prp8 mutants that suppress first-step splicing defects and oppose the action of the previously described prp8 suppressors of second-step defects; these opposing effects parallel those of ribosomal 'ram' and 'restrictive' mutants, which alter fidelity of transfer RNA decoding. On the basis of genetic interactions, we propose that prp8-mediated substrate repositioning during the transition occurs between catalytic-center opening and closure mediated by the U6 small nuclear RNA and the DExH/D ATPase gene prp16. Modulation of these events alters splice-site selection and splicing fidelity.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: The prp8-R1753K allele suppresses first-step defects.
Figure 2: First-step prp8 alleles improve the first step (when limiting) and inhibit the second step.
Figure 3: Combination of first- and second-step suppressor mutations in the same Prp8 polypeptide restores a splicing phenotype similar to that of WT Prp8.
Figure 4: prp8 and U6-U57C alleles affect splicing at distinct steps.
Figure 5: prp8 and prp16 alleles affect splicing at distinct steps.
Figure 6: Proposed contributions of Prp8, U6 snRNA, Prp16 and Prp22 to distinct steps in spliceosomal transitions.

Similar content being viewed by others

References

  1. Burge, C.B., Tuschl, T.H. & Sharp, P.A. Splicing of precursors to mRNAs by the spliceosomes. in The RNA World second edn. (eds. Gesteland, R.F., Cech, T.R. & Atkins, J.F.) 525–560 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1999).

    Google Scholar 

  2. Jurica, M.S. & Moore, M.J. Pre-mRNA splicing: awash in a sea of proteins. Mol. Cell 12, 5–14 (2003).

    Article  CAS  Google Scholar 

  3. Nilsen, T.W. RNA-RNA interactions in nuclear pre-mRNA splicing. in RNA Structure and Function (eds. Simons, R.W. & Grunberg-Manago, M.) 279–307 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1998).

    Google Scholar 

  4. Staley, J.P. & Guthrie, C. Mechanical devices of the spliceosome: motors, clocks, springs, and things. Cell 92, 315–326 (1998).

    Article  CAS  Google Scholar 

  5. Grainger, R.J. & Beggs, J.D. Prp8 protein: at the heart of the spliceosome. RNA 11, 533–557 (2005).

    Article  CAS  Google Scholar 

  6. Query, C.C. & Konarska, M.M. Suppression of multiple substrate mutations by spliceosomal prp8 alleles suggests functional correlations with ribosomal ambiguity mutants. Mol. Cell 14, 343–354 (2004).

    Article  CAS  Google Scholar 

  7. Schneider, S., Campodonico, E. & Schwer, B. Motifs IV and V in the DEAH box splicing factor Prp22 are important for RNA unwinding, and helicase-defective Prp22 mutants are suppressed by Prp8. J. Biol. Chem. 279, 8617–8626 (2004).

    Article  CAS  Google Scholar 

  8. Umen, J.G. & Guthrie, C. A novel role for a U5 snRNP protein in 3′ splice site selection. Genes Dev. 9, 855–868 (1995).

    Article  CAS  Google Scholar 

  9. Ben-Yehuda, S., Russell, C.S., Dix, I., Beggs, J.D. & Kupiec, M. Extensive genetic interactions between PRP8 and PRP17/CDC40, two yeast genes involved in pre-mRNA splicing and cell cycle progression. Genetics 154, 61–71 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Siatecka, M., Reyes, J.L. & Konarska, M.M. Functional interactions of Prp8 with both splice sites at the spliceosomal catalytic center. Genes Dev. 13, 1983–1993 (1999).

    Article  CAS  Google Scholar 

  11. Lesser, C.F. & Guthrie, C. Mutational analysis of pre-mRNA splicing in Saccharomyces cerevisiae using a sensitive new reporter gene, CUP1. Genetics 133, 851–863 (1993).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Burgess, S.M. & Guthrie, C. A mechanism to enhance mRNA splicing fidelity: the RNA-dependent ATPase Prp16 governs usage of a discard pathway for aberrant lariat intermediates. Cell 73, 1377–1391 (1993).

    Article  CAS  Google Scholar 

  13. Collins, C.A. & Guthrie, C. Allele-specific genetic interactions between Prp8 and RNA active site residues suggest a function for Prp8 at the catalytic core of the spliceosome. Genes Dev. 13, 1970–1982 (1999).

    Article  CAS  Google Scholar 

  14. McPheeters, D.S. Interactions of the yeast U6 RNA with the pre-mRNA branch site. RNA 2, 1110–1123 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Konarska, M.M., Vilardell, J. & Query, C.C. Repositioning of the reaction intermediate within the catalytic center of the spliceosome. Mol. Cell 21, 543–553 (2006).

    Article  CAS  Google Scholar 

  16. Villa, T. & Guthrie, C. The Isy1p component of the NineTeen complex interacts with the ATPase Prp16p to regulate the fidelity of pre-mRNA splicing. Genes Dev. 19, 1894–1904 (2005).

    Article  CAS  Google Scholar 

  17. Ogle, J.M., Carter, A.P. & Ramakrishnan, V. Insights into the decoding mechanism from recent ribosome structures. Trends Biochem. Sci. 28, 259–266 (2003).

    Article  CAS  Google Scholar 

  18. Kurland, C.G., Hughes, D. & Ehrenberg, M. Limitations of translational accuracy. in E. coli and S. typhimurium: Cellular and Molecular Biology Vol. 1 (eds. Neidhardt, F.C. et al.) 979–1004 (ASM Press, Washington, DC, 1996).

    Google Scholar 

  19. Piepersberg, W., Noseda, V. & Bock, A. Bacterial ribosomes with two ambiguity mutations: effects of translational fidelity, on the response to aminoglycosides and on the rate of protein synthesis. Mol. Gen. Genet. 171, 23–34 (1979).

    Article  CAS  Google Scholar 

  20. Rosset, R. & Gorini, L. A ribosomal ambiguity mutation. J. Mol. Biol. 39, 95–112 (1969).

    Article  CAS  Google Scholar 

  21. Umen, J.G. & Guthrie, C. Mutagenesis of the yeast gene PRP8 reveals domains governing the specificity and fidelity of 3′ splice site selection. Genetics 143, 723–739 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Schwer, B. & Guthrie, C. PRP16 is an RNA-dependent ATPase that interacts transiently with the spliceosome. Nature 349, 494–499 (1991).

    Article  CAS  Google Scholar 

  23. Mayas, R.M., Maita, H. & Staley, J.P. Exon ligation is proofread by the DExD/H-box ATPase Prp22p. Nat. Struct. Mol. Biol. 13, 482–490 (2006).

    Article  CAS  Google Scholar 

  24. Schwer, B. & Gross, C.H. Prp22, a DExH-box RNA helicase, plays two distinct roles in yeast pre-mRNA splicing. EMBO J. 17, 2086–2094 (1998).

    Article  CAS  Google Scholar 

  25. Wagner, J.D., Jankowsky, E., Company, M., Pyle, A.M. & Abelson, J.N. The DEAH-box protein PRP22 is an ATPase that mediates ATP-dependent mRNA release from the spliceosome and unwinds RNA duplexes. EMBO J. 17, 2926–2937 (1998).

    Article  CAS  Google Scholar 

  26. Umen, J.G. & Guthrie, C. Prp16p, Slu7p, and Prp8p interact with the 3′ splice site in two distinct stages during the second catalytic step of pre-mRNA splicing. RNA 1, 584–597 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Bellare, P., Kutach, A.K., Rines, A.K., Guthrie, C. & Sontheimer, E.J. Ubiquitin binding by a variant Jab1/MPN domain in the essential pre-mRNA splicing factor Prp8p. RNA 12, 292–302 (2006).

    Article  CAS  Google Scholar 

  28. Pena, V., Liu, S., Bujnicki, J.M., Lührmann, R. & Wahl, M.C. Structure of a multipartite protein-protein interaction domain in splicing factor prp8 and its link to retinitis pigmentosa. Mol. Cell 25, 615–624 (2007).

    Article  CAS  Google Scholar 

  29. Reyes, J.L., Gustafson, E.H., Luo, H.R., Moore, M.J. & Konarska, M.M. The C-terminal region of hPrp8 interacts with the conserved GU dinucleotide at the 5′ splice site. RNA 5, 167–179 (1999).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank B. Schwer (Weill Cornell Medical College) for prp16 and prp22 mutant plasmids, C. Guthrie (University of California, San Francisco) for prp8-101 plasmid, D. McPheeters (Case Western Reserve University) for U6-U57 mutant plasmids, and D. Smith, J. Warner and members of our laboratories for helpful discussions and critical readings of the manuscript. This work was supported by US National Institutes of Health grants GM49044 to M.M.K. and GM57829 to C.C.Q., and by a Cancer Center Support (core) grant from the National Cancer Institute to Albert Einstein College of Medicine.

Author information

Authors and Affiliations

  1. The Rockefeller University, New York, 10021, New York, USA

    Li Liu & Maria M Konarska

  2. Albert Einstein College of Medicine, Bronx, 10461, New York, USA

    Charles C Query

Authors
  1. Li Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Charles C Query
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Maria M Konarska
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Charles C Query or Maria M Konarska.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Liu, L., Query, C. & Konarska, M. Opposing classes of prp8 alleles modulate the transition between the catalytic steps of pre-mRNA splicing. Nat Struct Mol Biol 14, 519–526 (2007). https://doi.org/10.1038/nsmb1240

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb1240

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing