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. 1994 Feb 15;13(4):879–887. doi: 10.1002/j.1460-2075.1994.tb06331.x

A dominant negative mutation in the conserved RNA helicase motif 'SAT' causes splicing factor PRP2 to stall in spliceosomes.

M Plumpton 1, M McGarvey 1, J D Beggs 1
PMCID: PMC394887  PMID: 8112301

Abstract

To characterize sequences in the RNA helicase-like PRP2 protein of Saccharomyces cerevisiae that are essential for its function in pre-mRNA splicing, a pool of random PRP2 mutants was generated. A dominant negative allele was isolated which, when overexpressed in a wild-type yeast strain, inhibited cell growth by causing a defect in pre-mRNA splicing. This defect was partially alleviated by simultaneous co-overexpression of wild-type PRP2. The dominant negative PRP2 protein inhibited splicing in vitro and caused the accumulation of stalled splicing complexes. Immunoprecipitation with anti-PRP2 antibodies confirmed that dominant negative PRP2 protein competed with its wild-type counterpart for interaction with spliceosomes, with which the mutant protein remained associated. The PRP2-dn1 mutation led to a single amino acid change within the conserved SAT motif that in the prototype helicase eIF-4A is required for RNA unwinding. Purified dominant negative PRP2 protein had approximately 40% of the wild-type level of RNA-stimulated ATPase activity. As ATPase activity was reduced only slightly, but splicing activity was abolished, we propose that the dominant negative phenotype is due primarily to a defect in the putative RNA helicase activity of PRP2 protein.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Baldari C., Murray J. A., Ghiara P., Cesareni G., Galeotti C. L. A novel leader peptide which allows efficient secretion of a fragment of human interleukin 1 beta in Saccharomyces cerevisiae. EMBO J. 1987 Jan;6(1):229–234. doi: 10.1002/j.1460-2075.1987.tb04743.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bonneaud N., Ozier-Kalogeropoulos O., Li G. Y., Labouesse M., Minvielle-Sebastia L., Lacroute F. A family of low and high copy replicative, integrative and single-stranded S. cerevisiae/E. coli shuttle vectors. Yeast. 1991 Aug-Sep;7(6):609–615. doi: 10.1002/yea.320070609. [DOI] [PubMed] [Google Scholar]
  3. 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. 1993 Jul 2;73(7):1377–1391. doi: 10.1016/0092-8674(93)90363-u. [DOI] [PubMed] [Google Scholar]
  4. Burgess S., Couto J. R., Guthrie C. A putative ATP binding protein influences the fidelity of branchpoint recognition in yeast splicing. Cell. 1990 Mar 9;60(5):705–717. doi: 10.1016/0092-8674(90)90086-t. [DOI] [PubMed] [Google Scholar]
  5. Chen J. H., Lin R. J. The yeast PRP2 protein, a putative RNA-dependent ATPase, shares extensive sequence homology with two other pre-mRNA splicing factors. Nucleic Acids Res. 1990 Nov 11;18(21):6447–6447. doi: 10.1093/nar/18.21.6447. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Company M., Arenas J., Abelson J. Requirement of the RNA helicase-like protein PRP22 for release of messenger RNA from spliceosomes. Nature. 1991 Feb 7;349(6309):487–493. doi: 10.1038/349487a0. [DOI] [PubMed] [Google Scholar]
  7. Deschavanne P. J., Harosh I. The Rad3 protein from Saccharomyces cerevisiae: a DNA and DNA:RNA helicase with putative RNA helicase activity. Mol Microbiol. 1993 Mar;7(6):831–835. doi: 10.1111/j.1365-2958.1993.tb01173.x. [DOI] [PubMed] [Google Scholar]
  8. Feig L. A., Cooper G. M. Inhibition of NIH 3T3 cell proliferation by a mutant ras protein with preferential affinity for GDP. Mol Cell Biol. 1988 Aug;8(8):3235–3243. doi: 10.1128/mcb.8.8.3235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Feinberg A. P., Vogelstein B. "A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity". Addendum. Anal Biochem. 1984 Feb;137(1):266–267. doi: 10.1016/0003-2697(84)90381-6. [DOI] [PubMed] [Google Scholar]
  10. Fleig U. N., Gould K. L., Nurse P. A dominant negative allele of p34cdc2 shows altered phosphoamino acid content and sequesters p56cdc13 cyclin. Mol Cell Biol. 1992 May;12(5):2295–2301. doi: 10.1128/mcb.12.5.2295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fuller-Pace F. V., Nicol S. M., Reid A. D., Lane D. P. DbpA: a DEAD box protein specifically activated by 23s rRNA. EMBO J. 1993 Sep;12(9):3619–3626. doi: 10.1002/j.1460-2075.1993.tb06035.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gorbalenya A. E., Koonin E. V., Donchenko A. P., Blinov V. M. Two related superfamilies of putative helicases involved in replication, recombination, repair and expression of DNA and RNA genomes. Nucleic Acids Res. 1989 Jun 26;17(12):4713–4730. doi: 10.1093/nar/17.12.4713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Green M. R. Biochemical mechanisms of constitutive and regulated pre-mRNA splicing. Annu Rev Cell Biol. 1991;7:559–599. doi: 10.1146/annurev.cb.07.110191.003015. [DOI] [PubMed] [Google Scholar]
  14. Guthrie C. Messenger RNA splicing in yeast: clues to why the spliceosome is a ribonucleoprotein. Science. 1991 Jul 12;253(5016):157–163. doi: 10.1126/science.1853200. [DOI] [PubMed] [Google Scholar]
  15. Herskowitz I. Functional inactivation of genes by dominant negative mutations. Nature. 1987 Sep 17;329(6136):219–222. doi: 10.1038/329219a0. [DOI] [PubMed] [Google Scholar]
  16. Hirling H., Scheffner M., Restle T., Stahl H. RNA helicase activity associated with the human p68 protein. Nature. 1989 Jun 15;339(6225):562–564. doi: 10.1038/339562a0. [DOI] [PubMed] [Google Scholar]
  17. Hoffmann A., Roeder R. G. Purification of his-tagged proteins in non-denaturing conditions suggests a convenient method for protein interaction studies. Nucleic Acids Res. 1991 Nov 25;19(22):6337–6338. doi: 10.1093/nar/19.22.6337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hopper A. K., Banks F. A yeast mutant which accumulates precursor tRNAs. Cell. 1978 Jun;14(2):211–219. doi: 10.1016/0092-8674(78)90108-3. [DOI] [PubMed] [Google Scholar]
  19. Iggo R. D., Lane D. P. Nuclear protein p68 is an RNA-dependent ATPase. EMBO J. 1989 Jun;8(6):1827–1831. doi: 10.1002/j.1460-2075.1989.tb03577.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Jackson S. P., Lossky M., Beggs J. D. Cloning of the RNA8 gene of Saccharomyces cerevisiae, detection of the RNA8 protein, and demonstration that it is essential for nuclear pre-mRNA splicing. Mol Cell Biol. 1988 Mar;8(3):1067–1075. doi: 10.1128/mcb.8.3.1067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. King D. S., Beggs J. D. Interactions of PRP2 protein with pre-mRNA splicing complexes in Saccharomyces cerevisiae. Nucleic Acids Res. 1990 Nov 25;18(22):6559–6564. doi: 10.1093/nar/18.22.6559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Konarska M. M., Grabowski P. J., Padgett R. A., Sharp P. A. Characterization of the branch site in lariat RNAs produced by splicing of mRNA precursors. Nature. 1985 Feb 14;313(6003):552–557. doi: 10.1038/313552a0. [DOI] [PubMed] [Google Scholar]
  24. Koonin E. V. Similarities in RNA helicases. Nature. 1991 Jul 25;352(6333):290–290. doi: 10.1038/352290c0. [DOI] [PubMed] [Google Scholar]
  25. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  26. Laín S., Riechmann J. L., García J. A. RNA helicase: a novel activity associated with a protein encoded by a positive strand RNA virus. Nucleic Acids Res. 1990 Dec 11;18(23):7003–7006. doi: 10.1093/nar/18.23.7003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Leberer E., Dignard D., Hougan L., Thomas D. Y., Whiteway M. Dominant-negative mutants of a yeast G-protein beta subunit identify two functional regions involved in pheromone signalling. EMBO J. 1992 Dec;11(13):4805–4813. doi: 10.1002/j.1460-2075.1992.tb05586.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lee C. G., Hurwitz J. Human RNA helicase A is homologous to the maleless protein of Drosophila. J Biol Chem. 1993 Aug 5;268(22):16822–16830. [PubMed] [Google Scholar]
  29. Lee M. G., Young R. A., Beggs J. D. Cloning of the RNA2 gene of Saccharomyces cerevisiae. EMBO J. 1984 Dec 1;3(12):2825–2830. doi: 10.1002/j.1460-2075.1984.tb02215.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lin R. J., Lustig A. J., Abelson J. Splicing of yeast nuclear pre-mRNA in vitro requires a functional 40S spliceosome and several extrinsic factors. Genes Dev. 1987 Mar;1(1):7–18. doi: 10.1101/gad.1.1.7. [DOI] [PubMed] [Google Scholar]
  31. Lin R. J., Newman A. J., Cheng S. C., Abelson J. Yeast mRNA splicing in vitro. J Biol Chem. 1985 Nov 25;260(27):14780–14792. [PubMed] [Google Scholar]
  32. Linder P., Lasko P. F., Ashburner M., Leroy P., Nielsen P. J., Nishi K., Schnier J., Slonimski P. P. Birth of the D-E-A-D box. Nature. 1989 Jan 12;337(6203):121–122. doi: 10.1038/337121a0. [DOI] [PubMed] [Google Scholar]
  33. Mendenhall M. D., Richardson H. E., Reed S. I. Dominant negative protein kinase mutations that confer a G1 arrest phenotype. Proc Natl Acad Sci U S A. 1988 Jun;85(12):4426–4430. doi: 10.1073/pnas.85.12.4426. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Messing J., Gronenborn B., Müller-Hill B., Hans Hopschneider P. Filamentous coliphage M13 as a cloning vehicle: insertion of a HindII fragment of the lac regulatory region in M13 replicative form in vitro. Proc Natl Acad Sci U S A. 1977 Sep;74(9):3642–3646. doi: 10.1073/pnas.74.9.3642. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Miraglia L., Seiwert S., Igel A. H., Ares M., Jr Limited functional equivalence of phylogenetic variation in small nuclear RNA: yeast U2 RNA with altered branchpoint complementarity inhibits splicing and produces a dominant lethal phenotype. Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):7061–7065. doi: 10.1073/pnas.88.16.7061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Naumovski L., Friedberg E. C. Analysis of the essential and excision repair functions of the RAD3 gene of Saccharomyces cerevisiae by mutagenesis. Mol Cell Biol. 1986 Apr;6(4):1218–1227. doi: 10.1128/mcb.6.4.1218. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Padgett R. A., Konarska M. M., Grabowski P. J., Hardy S. F., Sharp P. A. Lariat RNA's as intermediates and products in the splicing of messenger RNA precursors. Science. 1984 Aug 31;225(4665):898–903. doi: 10.1126/science.6206566. [DOI] [PubMed] [Google Scholar]
  38. Padgett R. A., Konarska M. M., Grabowski P. J., Hardy S. F., Sharp P. A. Lariat RNA's as intermediates and products in the splicing of messenger RNA precursors. Science. 1984 Aug 31;225(4665):898–903. doi: 10.1126/science.6206566. [DOI] [PubMed] [Google Scholar]
  39. Pause A., Sonenberg N. Mutational analysis of a DEAD box RNA helicase: the mammalian translation initiation factor eIF-4A. EMBO J. 1992 Jul;11(7):2643–2654. doi: 10.1002/j.1460-2075.1992.tb05330.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Pause A., Sonenberg N. Mutational analysis of a DEAD box RNA helicase: the mammalian translation initiation factor eIF-4A. EMBO J. 1992 Jul;11(7):2643–2654. doi: 10.1002/j.1460-2075.1992.tb05330.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Pikielny C. W., Rymond B. C., Rosbash M. Electrophoresis of ribonucleoproteins reveals an ordered assembly pathway of yeast splicing complexes. 1986 Nov 27-Dec 3Nature. 324(6095):341–345. doi: 10.1038/324341a0. [DOI] [PubMed] [Google Scholar]
  42. Ray B. K., Lawson T. G., Kramer J. C., Cladaras M. H., Grifo J. A., Abramson R. D., Merrick W. C., Thach R. E. ATP-dependent unwinding of messenger RNA structure by eukaryotic initiation factors. J Biol Chem. 1985 Jun 25;260(12):7651–7658. [PubMed] [Google Scholar]
  43. Rozen F., Edery I., Meerovitch K., Dever T. E., Merrick W. C., Sonenberg N. Bidirectional RNA helicase activity of eucaryotic translation initiation factors 4A and 4F. Mol Cell Biol. 1990 Mar;10(3):1134–1144. doi: 10.1128/mcb.10.3.1134. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Schmid S. R., Linder P. D-E-A-D protein family of putative RNA helicases. Mol Microbiol. 1992 Feb;6(3):283–291. doi: 10.1111/j.1365-2958.1992.tb01470.x. [DOI] [PubMed] [Google Scholar]
  45. Schwer B., Guthrie C. A dominant negative mutation in a spliceosomal ATPase affects ATP hydrolysis but not binding to the spliceosome. Mol Cell Biol. 1992 Aug;12(8):3540–3547. doi: 10.1128/mcb.12.8.3540. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Schwer B., Guthrie C. PRP16 is an RNA-dependent ATPase that interacts transiently with the spliceosome. Nature. 1991 Feb 7;349(6309):494–499. doi: 10.1038/349494a0. [DOI] [PubMed] [Google Scholar]
  47. Sharp P. A. On the origin of RNA splicing and introns. Cell. 1985 Sep;42(2):397–400. doi: 10.1016/0092-8674(85)90092-3. [DOI] [PubMed] [Google Scholar]
  48. Strauss E. J., Guthrie C. A cold-sensitive mRNA splicing mutant is a member of the RNA helicase gene family. Genes Dev. 1991 Apr;5(4):629–641. doi: 10.1101/gad.5.4.629. [DOI] [PubMed] [Google Scholar]
  49. Teigelkamp S., McGarvey M., Plumpton M., Beggs J. D. The splicing factor PRP2, a putative RNA helicase, interacts directly with pre-mRNA. EMBO J. 1994 Feb 15;13(4):888–897. doi: 10.1002/j.1460-2075.1994.tb06332.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Walker J. E., Saraste M., Runswick M. J., Gay N. J. Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J. 1982;1(8):945–951. doi: 10.1002/j.1460-2075.1982.tb01276.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Wassarman D. A., Steitz J. A. RNA splicing. Alive with DEAD proteins. Nature. 1991 Feb 7;349(6309):463–464. doi: 10.1038/349463a0. [DOI] [PubMed] [Google Scholar]
  52. Whittaker E., Beggs J. D. The yeast PRP8 protein interacts directly with pre-mRNA. Nucleic Acids Res. 1991 Oct 25;19(20):5483–5489. doi: 10.1093/nar/19.20.5483. [DOI] [PMC free article] [PubMed] [Google Scholar]

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