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Exon ligation is proofread by the DExD/H-box ATPase Prp22p

Nature Structural & Molecular Biology volume 13, pages 482–490 (2006)Cite this article

Abstract

To produce messenger RNA, the spliceosome excises introns from precursor (pre)-mRNA and splices the flanking exons. To establish fidelity, the spliceosome discriminates against aberrant introns, but current understanding of such fidelity mechanisms is limited. Here we show that an ATP-dependent activity represses formation of mRNA from aberrant intermediates having mutations in any of the intronic consensus sequences. This proofreading activity is disabled by mutations that impair the ATPase or RNA unwindase activity of Prp22p, a conserved spliceosomal DExD/H-box ATPase. Further, cold-sensitive prp22 mutants permit aberrant mRNA formation from a mutated 3′ splice-site intermediate in vivo. We conclude that Prp22p generally represses splicing of aberrant intermediates, in addition to its known ATP-dependent role in promoting release of genuine mRNA. This dual function for Prp22p validates a general model in which fidelity can be enhanced by a DExD/H-box ATPase.

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Figure 1: An ATP-dependent mechanism represses exon ligation at near-consensus 3′ splice sites.
Figure 2: An ATP-dependent mechanism represses exon ligation at a mutated, but not a wild-type, 3′ splice site.
Figure 3: Repression of aberrant exon ligation at mutated 3′ splice sites is compromised in vitro and in vivo by ATPase- and RNA unwindase–deficient variants of the DEAH-box ATPase Prp22p.
Figure 4: An ATP- and Prp22p-dependent mechanism also represses aberrant exon ligation of intermediates having mutations of the branch site or 5′ splice site.
Figure 5: A dual role for Prp22p in fidelity and mRNA release suggests a proofreading mechanism7,8,9 for exon ligation.

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References

  1. Will, C.L. & Lührmann, R. Spliceosome structure and function. in The RNA World 3rd edn. (eds. Gesteland, R.F., Cech, T.R. & Atkins, J.F.) 369–400 (Cold Spring Harbor Laboratory Press, New York, 2006).

    Google Scholar 

  2. Valadkhan, S. snRNAs as the catalysts of pre-mRNA splicing. Curr. Opin. Chem. Biol. 9, 603–608 (2005).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  4. Cordin, O., Banroques, J., Tanner, N.K. & Linder, P. The DEAD-box protein family of RNA helicases. Gene 367, 17–37 (2005).

    Article  PubMed  Google Scholar 

  5. 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  PubMed  Google Scholar 

  6. Schwer, B. & Guthrie, C. A conformational rearrangement in the spliceosome is dependent on PRP16 and ATP hydrolysis. EMBO J. 11, 5033–5039 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Burgess, S.M. & Guthrie, C. Beat the clock: paradigms for NTPases in the maintenance of biological fidelity. Trends Biochem. Sci. 18, 381–384 (1993).

    Article  CAS  PubMed  Google Scholar 

  8. Hopfield, J.J. Kinetic proofreading: a new mechanism for reducing errors in biosynthetic processes requiring high specificity. Proc. Natl. Acad. Sci. USA 71, 4135–4139 (1974).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Yarus, M. Proofreading, NTPases and translation: constraints on accurate biochemistry. Trends Biochem. Sci. 17, 130–133 (1992).

    Article  CAS  PubMed  Google Scholar 

  10. Newman, A.J. & Norman, C. U5 snRNA interacts with exon sequences at 5′ and 3′ splice sites. Cell 68, 743–754 (1992).

    Article  CAS  PubMed  Google Scholar 

  11. Lesser, C.F. & Guthrie, C. Mutations in U6 snRNA that alter splice site specificity: implications for the active site. Science 262, 1982–1988 (1993).

    Article  CAS  PubMed  Google Scholar 

  12. Madhani, H.D. & Guthrie, C. Randomization-selection analysis of snRNAs in vivo: evidence for a tertiary interaction in the spliceosome. Genes Dev. 8, 1071–1086 (1994).

    Article  CAS  PubMed  Google Scholar 

  13. 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 

  14. 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  PubMed  PubMed Central  Google Scholar 

  15. Chang, J.S. & McPheeters, D.S. Identification of a U2/U6 helix la mutant that influences 3′ splice site selection during nuclear pre-mRNA splicing. RNA 6, 1120–1130 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. 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 

  17. Dagher, S.F. & Fu, X.-D. Evidence for a role of Sky1p-mediated phosphorylation in 3′ splice site recognition involving both Prp8 and Prp17/Slu4. RNA 7, 1284–1297 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. 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  PubMed  Google Scholar 

  19. 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  PubMed  PubMed Central  Google Scholar 

  20. Konarska, M.M. & Query, C.C. Insights into the mechanisms of splicing: more lessons from the ribosome. Genes Dev. 19, 2255–2260 (2005).

    Article  CAS  PubMed  Google Scholar 

  21. Vijayraghavan, U. et al. Mutations in conserved intron sequences affect multiple steps in the yeast splicing pathway, particularly assembly of the spliceosome. EMBO J. 5, 1683–1695 (1986).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Rymond, B.C. & Rosbash, M. Cleavage of 5′ splice site and lariat formation are independent of 3′ splice site in yeast mRNA splicing. Nature 317, 735–737 (1985).

    Article  CAS  PubMed  Google Scholar 

  23. Reed, R. The organization of 3′ splice-site sequences in mammalian introns. Genes Dev. 3, 2113–2123 (1989).

    Article  CAS  PubMed  Google Scholar 

  24. Fouser, L.A. & Friesen, J.D. Effects on mRNA splicing of mutations in the 3′ region of the Saccharomyces cerevisiae actin intron. Mol. Cell. Biol. 7, 225–230 (1987).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Luukkonen, B.G. & Séraphin, B. The role of branchpoint-3′ splice site spacing and interaction between intron terminal nucleotides in 3′ splice site selection in Saccharomyces cerevisiae. EMBO J. 16, 779–792 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. 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  PubMed  Google Scholar 

  27. Frank, D. & Guthrie, C. An essential splicing factor, SLU7, mediates 3′ splice site choice in yeast. Genes Dev. 6, 2112–2124 (1992).

    Article  CAS  PubMed  Google Scholar 

  28. Chua, K. & Reed, R. The RNA splicing factor hSlu7 is required for correct 3′ splice-site choice. Nature 402, 207–210 (1999).

    Article  CAS  PubMed  Google Scholar 

  29. 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  PubMed  PubMed Central  Google Scholar 

  30. 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  PubMed  PubMed Central  Google Scholar 

  31. Bousquet-Antonelli, C., Presutti, C. & Tollervey, D. Identification of a regulated pathway for nuclear pre-mRNA turnover. Cell 102, 765–775 (2000).

    Article  CAS  PubMed  Google Scholar 

  32. Hilleren, P.J. & Parker, R. Cytoplasmic degradation of splice-defective pre-mRNAs and intermediates. Mol. Cell 12, 1453–1465 (2003).

    Article  CAS  PubMed  Google Scholar 

  33. Aroian, R.V. et al. Splicing in Caenorhabditis elegans does not require an AG at the 3′ splice acceptor site. Mol. Cell. Biol. 13, 626–637 (1993).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Dominski, Z. & Kole, R. Identification and characterization by antisense oligonucleotides of exon and intron sequences required for splicing. Mol. Cell. Biol. 14, 7445–7454 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Frilander, M.J. & Steitz, J.A. Dynamic exchanges of RNA interactions leading to catalytic core formation in the U12-dependent spliceosome. Mol. Cell 7, 217–226 (2001).

    Article  CAS  PubMed  Google Scholar 

  36. Lingner, J. & Cech, T.R. Purification of telomerase from Euplotes aediculatus: requirement of a primer 3′ overhang. Proc. Natl. Acad. Sci. USA 93, 10712–10717 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Kim, S.H., Smith, J., Claude, A. & Lin, R.-J. The purified yeast pre-mRNA splicing factor PRP2 is an RNA-dependent NTPase. EMBO J. 11, 2319–2326 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Tanaka, N. & Schwer, B. Characterization of the NTPase, RNA-binding, and RNA helicase activities of the DEAH-box splicing factor Prp22. Biochemistry 44, 9795–9803 (2005).

    Article  CAS  PubMed  Google Scholar 

  39. McPheeters, D.S. & Muhlenkamp, P. Spatial organization of protein-RNA interactions in the branch site-3′ splice site region during pre-mRNA splicing in yeast. Mol. Cell. Biol. 23, 4174–4186 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Horowitz, D.S. & Abelson, J. Stages in the second reaction of pre-mRNA splicing: the final step is ATP independent. Genes Dev. 7, 320–329 (1993).

    Article  CAS  PubMed  Google Scholar 

  41. James, S.A., Turner, W. & Schwer, B. How Slu7 and Prp18 cooperate in the second step of yeast pre-mRNA splicing. RNA 8, 1068–1077 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Schwer, B. & Meszaros, T. RNA helicase dynamics in pre-mRNA splicing. EMBO J. 19, 6582–6591 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Schneider, S., Hotz, H.R. & Schwer, B. Characterization of dominant-negative mutants of the DEAH-box splicing factors Prp22 and Prp16. J. Biol. Chem. 277, 15452–15458 (2002).

    Article  CAS  PubMed  Google Scholar 

  44. 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  PubMed  Google Scholar 

  45. Martin, A., Schneider, S. & Schwer, B. Prp43 is an essential RNA-dependent ATPase required for release of lariat-intron from the spliceosome. J. Biol. Chem. 277, 17743–17750 (2002).

    Article  CAS  PubMed  Google Scholar 

  46. Arenas, J.E. & Abelson, J.N. Prp43: An RNA helicase-like factor involved in spliceosome disassembly. Proc. Natl. Acad. Sci. USA 94, 11798–11802 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Leeds, N.B., Small, E.C., Hiley, S.L., Hughes, T.R. & Staley, J.P. The splicing factor Prp43p, a DEAH box ATPase, functions in ribosome biogenesis. Mol. Cell. Biol. 26, 513–522 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. 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 

  49. Fouser, L.A. & Friesen, J.D. Mutations in a yeast intron demonstrate the importance of specific conserved nucleotides for the two stages of nuclear mRNA splicing. Cell 45, 81–93 (1986).

    Article  CAS  PubMed  Google Scholar 

  50. Query, C.C., Strobel, S.A. & Sharp, P.A. Three recognition events at the branch-site adenine. EMBO J. 15, 1392–1402 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Parker, R. & Siliciano, P.G. Evidence for an essential non-Watson-Crick interaction between the first and last nucleotides of a nuclear pre-mRNA intron. Nature 361, 660–662 (1993).

    Article  CAS  PubMed  Google Scholar 

  52. Campodonico, E. & Schwer, B. ATP-dependent remodeling of the spliceosome: intragenic suppressors of release-defective mutants of Saccharomyces cerevisiae Prp22. Genetics 160, 407–415 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Ohno, M. & Shimura, Y. A human RNA helicase-like protein, HRH1, facilitates nuclear export of spliced mRNA by releasing the RNA from the spliceosome. Genes Dev. 10, 997–1007 (1996).

    Article  CAS  PubMed  Google Scholar 

  54. van Nues, R.W. & Beggs, J.D. Functional contacts with a range of splicing proteins suggest a central role for Brr2p in the dynamic control of the order of events in spliceosomes of Saccharomyces cerevisiae. Genetics 157, 1451–1467 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Cochella, L. & Green, R. Fidelity in protein synthesis. Curr. Biol. 15, R536–R540 (2005).

    Article  CAS  PubMed  Google Scholar 

  56. Mohr, S., Stryker, J.M. & Lambowitz, A.M. A DEAD-box protein functions as an ATP-dependent RNA chaperone in group I intron splicing. Cell 109, 769–779 (2002).

    Article  CAS  PubMed  Google Scholar 

  57. Ballut, L. et al. The exon junction core complex is locked onto RNA by inhibition of eIF4AIII ATPase activity. Nat. Struct. Mol. Biol. 12, 861–869 (2005).

    Article  CAS  PubMed  Google Scholar 

  58. Brys, A. & Schwer, B. Requirement for SLU7 in yeast pre-mRNA splicing is dictated by the distance between the branchpoint and the 3′ splice site. RNA 2, 707–717 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. Stevens, S.W. & Abelson, J. Yeast pre-mRNA splicing: methods, mechanisms, and machinery. Methods Enzymol. 351, 200–220 (2002).

    Article  CAS  PubMed  Google Scholar 

  60. Rigaut, G. et al. A generic protein purification method for protein complex characterization and proteome exploration. Nat. Biotechnol. 17, 1030–1032 (1999).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank S.-C. Cheng (Academia Sinica) for the gift of antibodies to Ntc20; D. Bishop, L. Cochella, B. Glick, R. Green, J. Piccirilli, H. Singh, E. Sontheimer and members of the Staley laboratory for critical reading of the manuscript; and C. Jordan, V. Shaw and M. Norman for technical assistance. This research was supported by a predoctoral fellowship from the Ford Foundation to R.M.M. and by grants from the US National Institutes of Health and the Packard Foundation to J.P.S.

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  1. Hiroshi Maita

    Present address: Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo, 060-0812, Japan

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, The University of Chicago, Cummings Life Science Center 817, 920 E. 58th Street, Chicago, 60637, Illinois, USA

    Rabiah M Mayas

  2. Department of Molecular Genetics and Cell Biology, The University of Chicago, Cummings Life Science Center 817, 920 E. 58th Street, Chicago, 60637, Illinois, USA

    Hiroshi Maita & Jonathan P Staley

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Supplementary information

Supplementary Fig. 1

A prp43 mutant does not permit exon ligation at an aberrant 3′ splice site (PDF 721 kb)

Supplementary Methods

Oligonucleotides, plasmids, strains and RT-PCR (PDF 106 kb)

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Mayas, R., Maita, H. & Staley, J. Exon ligation is proofread by the DExD/H-box ATPase Prp22p. Nat Struct Mol Biol 13, 482–490 (2006). https://doi.org/10.1038/nsmb1093

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