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. 1997 Jul 1;16(13):4092–4106. doi: 10.1093/emboj/16.13.4092

An evolutionarily conserved U5 snRNP-specific protein is a GTP-binding factor closely related to the ribosomal translocase EF-2.

P Fabrizio 1, B Laggerbauer 1, J Lauber 1, W S Lane 1, R Lührmann 1
PMCID: PMC1170032  PMID: 9233818

Abstract

The driving forces behind the many RNA conformational changes occurring in the spliceosome are not well understood. Here we characterize an evolutionarily conserved human U5 small nuclear ribonucleoprotein (snRNP) protein (U5-116kD) that is strikingly homologous to the ribosomal elongation factor EF-2 (ribosomal translocase). A 114 kDa protein (Snu114p) homologous to U5-116kD was identified in Saccharomyces cerevisiae and was shown to be essential for yeast cell viability. Genetic depletion of Snu114p results in accumulation of unspliced pre-mRNA, indicating that Snu114p is essential for splicing in vivo. Antibodies specific for U5-116kD inhibit pre-mRNA splicing in a HeLa nuclear extract in vitro. In HeLa cells, U5-116kD is located in the nucleus and colocalizes with snRNP-containing subnuclear structures referred to as speckles. The G domain of U5-116kD/Snu114p contains the consensus sequence elements G1-G5 important for binding and hydrolyzing GTP. Consistent with this, U5-116kD can be cross-linked specifically to GTP by UV irradiation of U5 snRNPs. Moreover, a single amino acid substitution in the G1 sequence motif of Snu114p, expected to abolish GTP-binding activity, is lethal, suggesting that GTP binding and probably GTP hydrolysis is important for the function of U5-116kD/Snu114p. This is to date the first evidence that a G domain-containing protein plays an essential role in the pre-mRNA splicing process.

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Selected References

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  1. AEvarsson A., Brazhnikov E., Garber M., Zheltonosova J., Chirgadze Y., al-Karadaghi S., Svensson L. A., Liljas A. Three-dimensional structure of the ribosomal translocase: elongation factor G from Thermus thermophilus. EMBO J. 1994 Aug 15;13(16):3669–3677. doi: 10.1002/j.1460-2075.1994.tb06676.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Abel K., Jurnak F. A complex profile of protein elongation: translating chemical energy into molecular movement. Structure. 1996 Mar 15;4(3):229–238. doi: 10.1016/S0969-2126(96)00027-5. [DOI] [PubMed] [Google Scholar]
  3. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  4. Bennett M., Michaud S., Kingston J., Reed R. Protein components specifically associated with prespliceosome and spliceosome complexes. Genes Dev. 1992 Oct;6(10):1986–2000. doi: 10.1101/gad.6.10.1986. [DOI] [PubMed] [Google Scholar]
  5. Berchtold H., Reshetnikova L., Reiser C. O., Schirmer N. K., Sprinzl M., Hilgenfeld R. Crystal structure of active elongation factor Tu reveals major domain rearrangements. Nature. 1993 Sep 9;365(6442):126–132. doi: 10.1038/365126a0. [DOI] [PubMed] [Google Scholar]
  6. Bochnig P., Reuter R., Bringmann P., Lührmann R. 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. 1987 Oct 15;168(2):461–467. doi: 10.1111/j.1432-1033.1987.tb13439.x. [DOI] [PubMed] [Google Scholar]
  7. Boeke J. D., Trueheart J., Natsoulis G., Fink G. R. 5-Fluoroorotic acid as a selective agent in yeast molecular genetics. Methods Enzymol. 1987;154:164–175. doi: 10.1016/0076-6879(87)54076-9. [DOI] [PubMed] [Google Scholar]
  8. Bourne H. R., Sanders D. A., McCormick F. The GTPase superfamily: a conserved switch for diverse cell functions. Nature. 1990 Nov 8;348(6297):125–132. doi: 10.1038/348125a0. [DOI] [PubMed] [Google Scholar]
  9. Bourne H. R., Sanders D. A., McCormick F. The GTPase superfamily: conserved structure and molecular mechanism. Nature. 1991 Jan 10;349(6305):117–127. doi: 10.1038/349117a0. [DOI] [PubMed] [Google Scholar]
  10. Bringmann P., Appel B., Rinke J., Reuter R., Theissen H., Lührmann R. Evidence for the existence of snRNAs U4 and U6 in a single ribonucleoprotein complex and for their association by intermolecular base pairing. EMBO J. 1984 Jun;3(6):1357–1363. doi: 10.1002/j.1460-2075.1984.tb01977.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Brow D. A., Guthrie C. Spliceosomal RNA U6 is remarkably conserved from yeast to mammals. Nature. 1988 Jul 21;334(6179):213–218. doi: 10.1038/334213a0. [DOI] [PubMed] [Google Scholar]
  12. Burgess S. M., Guthrie C. Beat the clock: paradigms for NTPases in the maintenance of biological fidelity. Trends Biochem Sci. 1993 Oct;18(10):381–384. doi: 10.1016/0968-0004(93)90094-4. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. 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]
  16. Cortes J. J., Sontheimer E. J., Seiwert S. D., Steitz J. A. Mutations in the conserved loop of human U5 snRNA generate use of novel cryptic 5' splice sites in vivo. EMBO J. 1993 Dec 15;12(13):5181–5189. doi: 10.1002/j.1460-2075.1993.tb06213.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Czworkowski J., Wang J., Steitz T. A., Moore P. B. The crystal structure of elongation factor G complexed with GDP, at 2.7 A resolution. EMBO J. 1994 Aug 15;13(16):3661–3668. doi: 10.1002/j.1460-2075.1994.tb06675.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Dalbadie-McFarland G., Abelson J. PRP5: a helicase-like protein required for mRNA splicing in yeast. Proc Natl Acad Sci U S A. 1990 Jun;87(11):4236–4240. doi: 10.1073/pnas.87.11.4236. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Datta B., Weiner A. M. Genetic evidence for base pairing between U2 and U6 snRNA in mammalian mRNA splicing. Nature. 1991 Aug 29;352(6338):821–824. doi: 10.1038/352821a0. [DOI] [PubMed] [Google Scholar]
  20. Dever T. E., Glynias M. J., Merrick W. C. GTP-binding domain: three consensus sequence elements with distinct spacing. Proc Natl Acad Sci U S A. 1987 Apr;84(7):1814–1818. doi: 10.1073/pnas.84.7.1814. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Dignam J. D., Lebovitz R. M., Roeder R. G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983 Mar 11;11(5):1475–1489. doi: 10.1093/nar/11.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Dujon B., Alexandraki D., André B., Ansorge W., Baladron V., Ballesta J. P., Banrevi A., Bolle P. A., Bolotin-Fukuhara M., Bossier P. Complete DNA sequence of yeast chromosome XI. Nature. 1994 Jun 2;369(6479):371–378. doi: 10.1038/369371a0. [DOI] [PubMed] [Google Scholar]
  23. Eperon I. C., Ireland D. C., Smith R. A., Mayeda A., Krainer A. R. Pathways for selection of 5' splice sites by U1 snRNPs and SF2/ASF. EMBO J. 1993 Sep;12(9):3607–3617. doi: 10.1002/j.1460-2075.1993.tb06034.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Fabrizio P., Abelson J. Two domains of yeast U6 small nuclear RNA required for both steps of nuclear precursor messenger RNA splicing. Science. 1990 Oct 19;250(4979):404–409. doi: 10.1126/science.2145630. [DOI] [PubMed] [Google Scholar]
  25. Fabrizio P., McPheeters D. S., Abelson J. In vitro assembly of yeast U6 snRNP: a functional assay. Genes Dev. 1989 Dec;3(12B):2137–2150. doi: 10.1101/gad.3.12b.2137. [DOI] [PubMed] [Google Scholar]
  26. Frendewey D., Keller W. Stepwise assembly of a pre-mRNA splicing complex requires U-snRNPs and specific intron sequences. Cell. 1985 Aug;42(1):355–367. doi: 10.1016/s0092-8674(85)80131-8. [DOI] [PubMed] [Google Scholar]
  27. Fu X. D., Maniatis T. The 35-kDa mammalian splicing factor SC35 mediates specific interactions between U1 and U2 small nuclear ribonucleoprotein particles at the 3' splice site. Proc Natl Acad Sci U S A. 1992 Mar 1;89(5):1725–1729. doi: 10.1073/pnas.89.5.1725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Gozani O., Patton J. G., Reed R. A novel set of spliceosome-associated proteins and the essential splicing factor PSF bind stably to pre-mRNA prior to catalytic step II of the splicing reaction. EMBO J. 1994 Jul 15;13(14):3356–3367. doi: 10.1002/j.1460-2075.1994.tb06638.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Hardy S. F., Grabowski P. J., Padgett R. A., Sharp P. A. Cofactor requirements of splicing of purified messenger RNA precursors. Nature. 1984 Mar 22;308(5957):375–377. doi: 10.1038/308375a0. [DOI] [PubMed] [Google Scholar]
  31. Hashimoto C., Steitz J. A. U4 and U6 RNAs coexist in a single small nuclear ribonucleoprotein particle. Nucleic Acids Res. 1984 Apr 11;12(7):3283–3293. doi: 10.1093/nar/12.7.3283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Hodges P. E., Beggs J. D. RNA splicing. U2 fulfils a commitment. Curr Biol. 1994 Mar 1;4(3):264–267. doi: 10.1016/s0960-9822(00)00061-0. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. Kandels-Lewis S., Séraphin B. Involvement of U6 snRNA in 5' splice site selection. Science. 1993 Dec 24;262(5142):2035–2039. doi: 10.1126/science.8266100. [DOI] [PubMed] [Google Scholar]
  35. Kaziro Y. The role of guanosine 5'-triphosphate in polypeptide chain elongation. Biochim Biophys Acta. 1978 Sep 21;505(1):95–127. doi: 10.1016/0304-4173(78)90009-5. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. Kjeldgaard M., Nissen P., Thirup S., Nyborg J. The crystal structure of elongation factor EF-Tu from Thermus aquaticus in the GTP conformation. Structure. 1993 Sep 15;1(1):35–50. doi: 10.1016/0969-2126(93)90007-4. [DOI] [PubMed] [Google Scholar]
  38. Krainer A. R., Maniatis T., Ruskin B., Green M. R. Normal and mutant human beta-globin pre-mRNAs are faithfully and efficiently spliced in vitro. Cell. 1984 Apr;36(4):993–1005. doi: 10.1016/0092-8674(84)90049-7. [DOI] [PubMed] [Google Scholar]
  39. Laggerbauer B., Lauber J., Lührmann R. Identification of an RNA-dependent ATPase activity in mammalian U5 snRNPs. Nucleic Acids Res. 1996 Mar 1;24(5):868–875. doi: 10.1093/nar/24.5.868. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Lamm G. M., Lamond A. I. Non-snRNP protein splicing factors. Biochim Biophys Acta. 1993 Jun 25;1173(3):247–265. doi: 10.1016/0167-4781(93)90122-t. [DOI] [PubMed] [Google Scholar]
  41. 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]
  42. Myslinski E., Ségault V., Branlant C. An intron in the genes for U3 small nucleolar RNAs of the yeast Saccharomyces cerevisiae. Science. 1990 Mar 9;247(4947):1213–1216. doi: 10.1126/science.1690452. [DOI] [PubMed] [Google Scholar]
  43. Newman A., Norman C. Mutations in yeast U5 snRNA alter the specificity of 5' splice-site cleavage. Cell. 1991 Apr 5;65(1):115–123. doi: 10.1016/0092-8674(91)90413-s. [DOI] [PubMed] [Google Scholar]
  44. 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]
  45. Reed R. Initial splice-site recognition and pairing during pre-mRNA splicing. Curr Opin Genet Dev. 1996 Apr;6(2):215–220. doi: 10.1016/s0959-437x(96)80053-0. [DOI] [PubMed] [Google Scholar]
  46. Rinke J., Appel B., Digweed M., Lührmann R. Localization of a base-paired interaction between small nuclear RNAs U4 and U6 in intact U4/U6 ribonucleoprotein particles by psoralen cross-linking. J Mol Biol. 1985 Oct 20;185(4):721–731. doi: 10.1016/0022-2836(85)90057-9. [DOI] [PubMed] [Google Scholar]
  47. Saraste M., Sibbald P. R., Wittinghofer A. The P-loop--a common motif in ATP- and GTP-binding proteins. Trends Biochem Sci. 1990 Nov;15(11):430–434. doi: 10.1016/0968-0004(90)90281-f. [DOI] [PubMed] [Google Scholar]
  48. Sawa H., Abelson J. Evidence for a base-pairing interaction between U6 small nuclear RNA and 5' splice site during the splicing reaction in yeast. Proc Natl Acad Sci U S A. 1992 Dec 1;89(23):11269–11273. doi: 10.1073/pnas.89.23.11269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Scherer S., Davis R. W. Replacement of chromosome segments with altered DNA sequences constructed in vitro. Proc Natl Acad Sci U S A. 1979 Oct;76(10):4951–4955. doi: 10.1073/pnas.76.10.4951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. 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]
  51. Sikorski R. S., Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989 May;122(1):19–27. doi: 10.1093/genetics/122.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Sontheimer E. J., Steitz J. A. The U5 and U6 small nuclear RNAs as active site components of the spliceosome. Science. 1993 Dec 24;262(5142):1989–1996. doi: 10.1126/science.8266094. [DOI] [PubMed] [Google Scholar]
  53. 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]
  54. Sun J. S., Manley J. L. A novel U2-U6 snRNA structure is necessary for mammalian mRNA splicing. Genes Dev. 1995 Apr 1;9(7):843–854. doi: 10.1101/gad.9.7.843. [DOI] [PubMed] [Google Scholar]
  55. Utans U., Behrens S. E., Lührmann R., Kole R., Krämer A. A splicing factor that is inactivated during in vivo heat shock is functionally equivalent to the [U4/U6.U5] triple snRNP-specific proteins. Genes Dev. 1992 Apr;6(4):631–641. doi: 10.1101/gad.6.4.631. [DOI] [PubMed] [Google Scholar]
  56. 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]
  57. 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]
  58. Wilson R., Ainscough R., Anderson K., Baynes C., Berks M., Bonfield J., Burton J., Connell M., Copsey T., Cooper J. 2.2 Mb of contiguous nucleotide sequence from chromosome III of C. elegans. Nature. 1994 Mar 3;368(6466):32–38. doi: 10.1038/368032a0. [DOI] [PubMed] [Google Scholar]
  59. Wu J. A., Manley J. L. Base pairing between U2 and U6 snRNAs is necessary for splicing of a mammalian pre-mRNA. Nature. 1991 Aug 29;352(6338):818–821. doi: 10.1038/352818a0. [DOI] [PubMed] [Google Scholar]
  60. Wyatt J. R., Sontheimer E. J., Steitz J. A. Site-specific cross-linking of mammalian U5 snRNP to the 5' splice site before the first step of pre-mRNA splicing. Genes Dev. 1992 Dec;6(12B):2542–2553. doi: 10.1101/gad.6.12b.2542. [DOI] [PubMed] [Google Scholar]
  61. 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]
  62. Zamore P. D., Patton J. G., Green M. R. Cloning and domain structure of the mammalian splicing factor U2AF. Nature. 1992 Feb 13;355(6361):609–614. doi: 10.1038/355609a0. [DOI] [PubMed] [Google Scholar]
  63. Zuo P., Manley J. L. The human splicing factor ASF/SF2 can specifically recognize pre-mRNA 5' splice sites. Proc Natl Acad Sci U S A. 1994 Apr 12;91(8):3363–3367. doi: 10.1073/pnas.91.8.3363. [DOI] [PMC free article] [PubMed] [Google Scholar]

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