Skip to main content
NCBI home page
RNA logoLink to RNA
. 2000 Aug;6(8):1106–1119. doi: 10.1017/s1355838200992483

Dominant negative mutants of the yeast splicing factor Prp2 map to a putative cleft region in the helicase domain of DExD/H-box proteins.

G Edwalds-Gilbert 1, D H Kim 1, S H Kim 1, Y H Tseng 1, Y Yu 1, R J Lin 1
PMCID: PMC1369985  PMID: 10943890

Abstract

The Prp2 protein of Saccharomyces cerevisiae is an RNA-dependent ATPase required before the first transesterification reaction in pre-mRNA splicing. Prp2 binds to the spliceosome in the absence of ATP and is released following ATP hydrolysis. We determined what regions in Prp2 are essential for release from the spliceosome by analyzing dominant negative mutants in vivo and in vitro. We made mutations in conserved motif II (DExH) and motif VI (QRxGR) of the helicase (H) domain. Mutations that inactivated PRP2 had a dominant negative phenotype when overexpressed in vivo. To test whether mutations outside of the H domain could confer a dominant negative phenotype, we mutagenized a GAL1-PRP2 construct and screened for mutants unable to grow on galactose-containing media. Five dominant negative mutants were characterized; three mapped within the H domain and two mapped downstream of motif VI, indicating that an extended helicase domain is required for release of Prp2 from the spliceosome. Most mutants stalled in the spliceosome in vitro. However, not all mutants that were dominant negative in vivo were dominant negative in vitro, indicating that multiple mechanisms may cause a dominant negative phenotype. Structural modeling of the H domain of Prp2 suggests that mutants map to a cleft region found in helicases of known structure.

Full Text

The Full Text of this article is available as a PDF (2.0 MB).

Selected References

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

  1. Arenas J. E., Abelson J. N. Prp43: An RNA helicase-like factor involved in spliceosome disassembly. Proc Natl Acad Sci U S A. 1997 Oct 28;94(22):11798–11802. doi: 10.1073/pnas.94.22.11798. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ares M., Jr, Weiser B. Rearrangement of snRNA structure during assembly and function of the spliceosome. Prog Nucleic Acid Res Mol Biol. 1995;50:131–159. doi: 10.1016/s0079-6603(08)60813-2. [DOI] [PubMed] [Google Scholar]
  3. Benz J., Trachsel H., Baumann U. Crystal structure of the ATPase domain of translation initiation factor 4A from Saccharomyces cerevisiae--the prototype of the DEAD box protein family. Structure. 1999 Jun 15;7(6):671–679. doi: 10.1016/s0969-2126(99)80088-4. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Cho H. S., Ha N. C., Kang L. W., Chung K. M., Back S. H., Jang S. K., Oh B. H. Crystal structure of RNA helicase from genotype 1b hepatitis C virus. A feasible mechanism of unwinding duplex RNA. J Biol Chem. 1998 Jun 12;273(24):15045–15052. doi: 10.1074/jbc.273.24.15045. [DOI] [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. Couto J. R., Tamm J., Parker R., Guthrie C. A trans-acting suppressor restores splicing of a yeast intron with a branch point mutation. Genes Dev. 1987 Jul;1(5):445–455. doi: 10.1101/gad.1.5.445. [DOI] [PubMed] [Google Scholar]
  8. Fuller-Pace F. V. RNA helicases: modulators of RNA structure. Trends Cell Biol. 1994 Aug;4(8):271–274. doi: 10.1016/0962-8924(94)90210-0. [DOI] [PubMed] [Google Scholar]
  9. Guex N., Diemand A., Peitsch M. C. Protein modelling for all. Trends Biochem Sci. 1999 Sep;24(9):364–367. doi: 10.1016/s0968-0004(99)01427-9. [DOI] [PubMed] [Google Scholar]
  10. Guex N., Peitsch M. C. SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis. 1997 Dec;18(15):2714–2723. doi: 10.1002/elps.1150181505. [DOI] [PubMed] [Google Scholar]
  11. Hamm J., Lamond A. I. Spliceosome assembly: the unwinding role of DEAD-box proteins. Curr Biol. 1998 Jul 16;8(15):R532–R534. doi: 10.1016/s0960-9822(07)00340-5. [DOI] [PubMed] [Google Scholar]
  12. Hotz H. R., Schwer B. Mutational analysis of the yeast DEAH-box splicing factor Prp16. Genetics. 1998 Jun;149(2):807–815. doi: 10.1093/genetics/149.2.807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kim D. H., Edwalds-Gilbert G., Ren C., Lin R. J. A mutation in a methionine tRNA gene suppresses the prp2-1 Ts mutation and causes a pre-mRNA splicing defect in Saccharomyces cerevisiae. Genetics. 1999 Nov;153(3):1105–1115. doi: 10.1093/genetics/153.3.1105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kim D. H., Rossi J. J. The first ATPase domain of the yeast 246-kDa protein is required for in vivo unwinding of the U4/U6 duplex. RNA. 1999 Jul;5(7):959–971. doi: 10.1017/s135583829999012x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kim J. L., Morgenstern K. A., Griffith J. P., Dwyer M. D., Thomson J. A., Murcko M. A., Lin C., Caron P. R. Hepatitis C virus NS3 RNA helicase domain with a bound oligonucleotide: the crystal structure provides insights into the mode of unwinding. Structure. 1998 Jan 15;6(1):89–100. doi: 10.1016/s0969-2126(98)00010-0. [DOI] [PubMed] [Google Scholar]
  16. Kim S. H., Lin R. J. Pre-mRNA splicing within an assembled yeast spliceosome requires an RNA-dependent ATPase and ATP hydrolysis. Proc Natl Acad Sci U S A. 1993 Feb 1;90(3):888–892. doi: 10.1073/pnas.90.3.888. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kim S. H., Lin R. J. Spliceosome activation by PRP2 ATPase prior to the first transesterification reaction of pre-mRNA splicing. Mol Cell Biol. 1996 Dec;16(12):6810–6819. doi: 10.1128/mcb.16.12.6810. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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. 1992 Jun;11(6):2319–2326. doi: 10.1002/j.1460-2075.1992.tb05291.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. Laggerbauer B., Achsel T., Lührmann R. The human U5-200kD DEXH-box protein unwinds U4/U6 RNA duplices in vitro. Proc Natl Acad Sci U S A. 1998 Apr 14;95(8):4188–4192. doi: 10.1073/pnas.95.8.4188. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lauber J., Fabrizio P., Teigelkamp S., Lane W. S., Hartmann E., Luhrmann R. The HeLa 200 kDa U5 snRNP-specific protein and its homologue in Saccharomyces cerevisiae are members of the DEXH-box protein family of putative RNA helicases. EMBO J. 1996 Aug 1;15(15):4001–4015. [PMC free article] [PubMed] [Google Scholar]
  23. Lin C., Kim J. L. Structure-based mutagenesis study of hepatitis C virus NS3 helicase. J Virol. 1999 Oct;73(10):8798–8807. doi: 10.1128/jvi.73.10.8798-8807.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lin J., Rossi J. J. Identification and characterization of yeast mutants that overcome an experimentally introduced block to splicing at the 3' splice site. RNA. 1996 Aug;2(8):835–848. [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Noble S. M., Guthrie C. Identification of novel genes required for yeast pre-mRNA splicing by means of cold-sensitive mutations. Genetics. 1996 May;143(1):67–80. doi: 10.1093/genetics/143.1.67. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. O'Day C. L., Dalbadie-McFarland G., Abelson J. The Saccharomyces cerevisiae Prp5 protein has RNA-dependent ATPase activity with specificity for U2 small nuclear RNA. J Biol Chem. 1996 Dec 27;271(52):33261–33267. doi: 10.1074/jbc.271.52.33261. [DOI] [PubMed] [Google Scholar]
  28. 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. 1996 Apr 15;10(8):997–1007. doi: 10.1101/gad.10.8.997. [DOI] [PubMed] [Google Scholar]
  29. Pause A., Méthot N., Sonenberg N. The HRIGRXXR region of the DEAD box RNA helicase eukaryotic translation initiation factor 4A is required for RNA binding and ATP hydrolysis. Mol Cell Biol. 1993 Nov;13(11):6789–6798. doi: 10.1128/mcb.13.11.6789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. Peitsch M. C. ProMod and Swiss-Model: Internet-based tools for automated comparative protein modelling. Biochem Soc Trans. 1996 Feb;24(1):274–279. doi: 10.1042/bst0240274. [DOI] [PubMed] [Google Scholar]
  32. Plumpton M., McGarvey M., Beggs J. D. A dominant negative mutation in the conserved RNA helicase motif 'SAT' causes splicing factor PRP2 to stall in spliceosomes. EMBO J. 1994 Feb 15;13(4):879–887. doi: 10.1002/j.1460-2075.1994.tb06331.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Raghunathan P. L., Guthrie C. RNA unwinding in U4/U6 snRNPs requires ATP hydrolysis and the DEIH-box splicing factor Brr2. Curr Biol. 1998 Jul 16;8(15):847–855. doi: 10.1016/s0960-9822(07)00345-4. [DOI] [PubMed] [Google Scholar]
  34. Roy J., Kim K., Maddock J. R., Anthony J. G., Woolford J. L., Jr The final stages of spliceosome maturation require Spp2p that can interact with the DEAH box protein Prp2p and promote step 1 of splicing. RNA. 1995 Jun;1(4):375–390. [PMC free article] [PubMed] [Google Scholar]
  35. Schmid S. R., Linder P. Translation initiation factor 4A from Saccharomyces cerevisiae: analysis of residues conserved in the D-E-A-D family of RNA helicases. Mol Cell Biol. 1991 Jul;11(7):3463–3471. doi: 10.1128/mcb.11.7.3463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Schwer B., Gross C. H. Prp22, a DExH-box RNA helicase, plays two distinct roles in yeast pre-mRNA splicing. EMBO J. 1998 Apr 1;17(7):2086–2094. doi: 10.1093/emboj/17.7.2086. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. 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]
  38. 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]
  39. Sikorski R. S., Boeke J. D. In vitro mutagenesis and plasmid shuffling: from cloned gene to mutant yeast. Methods Enzymol. 1991;194:302–318. doi: 10.1016/0076-6879(91)94023-6. [DOI] [PubMed] [Google Scholar]
  40. Staley J. P., Guthrie C. An RNA switch at the 5' splice site requires ATP and the DEAD box protein Prp28p. Mol Cell. 1999 Jan;3(1):55–64. doi: 10.1016/s1097-2765(00)80174-4. [DOI] [PubMed] [Google Scholar]
  41. Staley J. P., Guthrie C. Mechanical devices of the spliceosome: motors, clocks, springs, and things. Cell. 1998 Feb 6;92(3):315–326. doi: 10.1016/s0092-8674(00)80925-3. [DOI] [PubMed] [Google Scholar]
  42. 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]
  43. Subramanya H. S., Bird L. E., Brannigan J. A., Wigley D. B. Crystal structure of a DExx box DNA helicase. Nature. 1996 Nov 28;384(6607):379–383. doi: 10.1038/384379a0. [DOI] [PubMed] [Google Scholar]
  44. 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. 1998 May 15;17(10):2926–2937. doi: 10.1093/emboj/17.10.2926. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Wang Y., Wagner J. D., Guthrie C. The DEAH-box splicing factor Prp16 unwinds RNA duplexes in vitro. Curr Biol. 1998 Apr 9;8(8):441–451. doi: 10.1016/s0960-9822(98)70178-2. [DOI] [PubMed] [Google Scholar]
  46. Will C. L., Lührmann R. Protein functions in pre-mRNA splicing. Curr Opin Cell Biol. 1997 Jun;9(3):320–328. doi: 10.1016/s0955-0674(97)80003-8. [DOI] [PubMed] [Google Scholar]
  47. Xu D., Nouraini S., Field D., Tang S. J., Friesen J. D. An RNA-dependent ATPase associated with U2/U6 snRNAs in pre-mRNA splicing. Nature. 1996 Jun 20;381(6584):709–713. doi: 10.1038/381709a0. [DOI] [PubMed] [Google Scholar]
  48. Yao N., Hesson T., Cable M., Hong Z., Kwong A. D., Le H. V., Weber P. C. Structure of the hepatitis C virus RNA helicase domain. Nat Struct Biol. 1997 Jun;4(6):463–467. doi: 10.1038/nsb0697-463. [DOI] [PubMed] [Google Scholar]

Articles from RNA are provided here courtesy of The RNA Society

RESOURCES