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. 1999 Nov;5(11):1470–1481. doi: 10.1017/s1355838299991355

Characterization of U6 snRNA-protein interactions.

V P Vidal 1, L Verdone 1, A E Mayes 1, J D Beggs 1
PMCID: PMC1369868  PMID: 10580475

Abstract

Through a combination of in vitro snRNP reconstitution, photocross-linking and immunoprecipitation techniques, we have investigated the interaction of proteins with the spliceosomal U6 snRNA in U6 snRNPs, U4/U6 di-snRNPs and U4/U6.U5 tri-snRNPs. Of the seven Lsm (Sm-like) proteins that associate specifically with this spliceosomal snRNA, three were shown to contact the RNA directly, and to maintain contact as the U6 RNA is incorporated into tri-snRNPs. In tri-snRNPs, the U5 snRNP protein Prp8 contacts position 54 of U6, which is in the conserved region that contributes to the formation of the catalytic core of the spliceosome. Other tri-snRNP-specific contacts were also detected, indicating the dynamic nature of protein interactions with this important snRNA. The uridine-rich extreme 3' end of U6 RNA was shown to be essential but not sufficient for the association of the Lsm proteins. Interestingly, the Lsm proteins associate efficiently with the 3' half of U6, which contains the 3' stem-loop and uridine-rich 3' end, suggesting that the Lsm and Sm proteins may recognize similar features in RNAs.

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Selected References

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  1. Achsel T., Ahrens K., Brahms H., Teigelkamp S., Lührmann R. The human U5-220kD protein (hPrp8) forms a stable RNA-free complex with several U5-specific proteins, including an RNA unwindase, a homologue of ribosomal elongation factor EF-2, and a novel WD-40 protein. Mol Cell Biol. 1998 Nov;18(11):6756–6766. doi: 10.1128/mcb.18.11.6756. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Beggs J. D., Teigelkamp S., Newman A. J. The role of PRP8 protein in nuclear pre-mRNA splicing in yeast. J Cell Sci Suppl. 1995;19:101–105. doi: 10.1242/jcs.1995.supplement_19.15. [DOI] [PubMed] [Google Scholar]
  3. Bordonné R., Tarassov I. The yeast SME1 gene encodes the homologue of the human E core protein. Gene. 1996 Oct 17;176(1-2):111–117. doi: 10.1016/0378-1119(96)00230-2. [DOI] [PubMed] [Google Scholar]
  4. Branlant C., Krol A., Ebel J. P., Lazar E., Haendler B., Jacob M. U2 RNA shares a structural domain with U1, U4, and U5 RNAs. EMBO J. 1982;1(10):1259–1265. doi: 10.1002/j.1460-2075.1982.tb00022.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Brow D. A., Vidaver R. M. An element in human U6 RNA destabilizes the U4/U6 spliceosomal RNA complex. RNA. 1995 Apr;1(2):122–131. [PMC free article] [PubMed] [Google Scholar]
  7. Brown J. D., Beggs J. D. Roles of PRP8 protein in the assembly of splicing complexes. EMBO J. 1992 Oct;11(10):3721–3729. doi: 10.1002/j.1460-2075.1992.tb05457.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cooper M., Johnston L. H., Beggs J. D. Identification and characterization of Uss1p (Sdb23p): a novel U6 snRNA-associated protein with significant similarity to core proteins of small nuclear ribonucleoproteins. EMBO J. 1995 May 1;14(9):2066–2075. doi: 10.1002/j.1460-2075.1995.tb07198.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dix I., Russell C. S., O'Keefe R. T., Newman A. J., Beggs J. D. Protein-RNA interactions in the U5 snRNP of Saccharomyces cerevisiae. RNA. 1998 Dec;4(12):1675–1686. doi: 10.1017/s1355838298412998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fabrizio P., Esser S., Kastner B., Lührmann R. Isolation of S. cerevisiae snRNPs: comparison of U1 and U4/U6.U5 to their human counterparts. Science. 1994 Apr 8;264(5156):261–265. doi: 10.1126/science.8146658. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Fisher D. E., Conner G. E., Reeves W. H., Wisniewolski R., Blobel G. Small nuclear ribonucleoprotein particle assembly in vivo: demonstration of a 6S RNA-free core precursor and posttranslational modification. Cell. 1985 Oct;42(3):751–758. doi: 10.1016/0092-8674(85)90271-5. [DOI] [PubMed] [Google Scholar]
  13. Fromont-Racine M., Rain J. C., Legrain P. Toward a functional analysis of the yeast genome through exhaustive two-hybrid screens. Nat Genet. 1997 Jul;16(3):277–282. doi: 10.1038/ng0797-277. [DOI] [PubMed] [Google Scholar]
  14. Ghetti A., Company M., Abelson J. Specificity of Prp24 binding to RNA: a role for Prp24 in the dynamic interaction of U4 and U6 snRNAs. RNA. 1995 Apr;1(2):132–145. [PMC free article] [PubMed] [Google Scholar]
  15. Gottschalk A., Tang J., Puig O., Salgado J., Neubauer G., Colot H. V., Mann M., Séraphin B., Rosbash M., Lührmann R. A comprehensive biochemical and genetic analysis of the yeast U1 snRNP reveals five novel proteins. RNA. 1998 Apr;4(4):374–393. [PMC free article] [PubMed] [Google Scholar]
  16. Heinrichs V., Hackl W., Lührmann R. Direct binding of small nuclear ribonucleoprotein G to the Sm site of small nuclear RNA. Ultraviolet light cross-linking of protein G to the AAU stretch within the Sm site (AAUUUGUGG) of U1 small nuclear ribonucleoprotein reconstituted in vitro. J Mol Biol. 1992 Sep 5;227(1):15–28. doi: 10.1016/0022-2836(92)90678-d. [DOI] [PubMed] [Google Scholar]
  17. Hermann H., Fabrizio P., Raker V. A., Foulaki K., Hornig H., Brahms H., Lührmann R. snRNP Sm proteins share two evolutionarily conserved sequence motifs which are involved in Sm protein-protein interactions. EMBO J. 1995 May 1;14(9):2076–2088. doi: 10.1002/j.1460-2075.1995.tb07199.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hodges P. E., Jackson S. P., Brown J. D., Beggs J. D. Extraordinary sequence conservation of the PRP8 splicing factor. Yeast. 1995 Apr 15;11(4):337–342. doi: 10.1002/yea.320110406. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Jandrositz A., Guthrie C. Evidence for a Prp24 binding site in U6 snRNA and in a putative intermediate in the annealing of U6 and U4 snRNAs. EMBO J. 1995 Feb 15;14(4):820–832. doi: 10.1002/j.1460-2075.1995.tb07060.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Jarmolowski A., Mattaj I. W. The determinants for Sm protein binding to Xenopus U1 and U5 snRNAs are complex and non-identical. EMBO J. 1993 Jan;12(1):223–232. doi: 10.1002/j.1460-2075.1993.tb05648.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kambach C., Walke S., Young R., Avis J. M., de la Fortelle E., Raker V. A., Lührmann R., Li J., Nagai K. Crystal structures of two Sm protein complexes and their implications for the assembly of the spliceosomal snRNPs. Cell. 1999 Feb 5;96(3):375–387. doi: 10.1016/s0092-8674(00)80550-4. [DOI] [PubMed] [Google Scholar]
  23. Kim C. H., Ryan D. E., Marciniec T., Abelson J. Site-specific deoxynucleotide substitutions in yeast U6 snRNA block splicing of pre-mRNA in vitro. EMBO J. 1997 Apr 15;16(8):2119–2129. doi: 10.1093/emboj/16.8.2119. [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]
  24. Krämer A. The structure and function of proteins involved in mammalian pre-mRNA splicing. Annu Rev Biochem. 1996;65:367–409. doi: 10.1146/annurev.bi.65.070196.002055. [DOI] [PubMed] [Google Scholar]
  25. Kuhn A. N., Li Z., Brow D. A. Splicing factor Prp8 governs U4/U6 RNA unwinding during activation of the spliceosome. Mol Cell. 1999 Jan;3(1):65–75. doi: 10.1016/s1097-2765(00)80175-6. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. 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]
  29. 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]
  30. Lossky M., Anderson G. J., Jackson S. P., Beggs J. Identification of a yeast snRNP protein and detection of snRNP-snRNP interactions. Cell. 1987 Dec 24;51(6):1019–1026. doi: 10.1016/0092-8674(87)90588-5. [DOI] [PubMed] [Google Scholar]
  31. Lührmann R., Kastner B., Bach M. Structure of spliceosomal snRNPs and their role in pre-mRNA splicing. Biochim Biophys Acta. 1990 Nov 30;1087(3):265–292. doi: 10.1016/0167-4781(90)90001-i. [DOI] [PubMed] [Google Scholar]
  32. MacMillan A. M., Query C. C., Allerson C. R., Chen S., Verdine G. L., Sharp P. A. Dynamic association of proteins with the pre-mRNA branch region. Genes Dev. 1994 Dec 15;8(24):3008–3020. doi: 10.1101/gad.8.24.3008. [DOI] [PubMed] [Google Scholar]
  33. Madhani H. D., Bordonné R., Guthrie C. Multiple roles for U6 snRNA in the splicing pathway. Genes Dev. 1990 Dec;4(12B):2264–2277. doi: 10.1101/gad.4.12b.2264. [DOI] [PubMed] [Google Scholar]
  34. Mayes A. E., Verdone L., Legrain P., Beggs J. D. Characterization of Sm-like proteins in yeast and their association with U6 snRNA. EMBO J. 1999 Aug 2;18(15):4321–4331. doi: 10.1093/emboj/18.15.4321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Moore M. J., Sharp P. A. Site-specific modification of pre-mRNA: the 2'-hydroxyl groups at the splice sites. Science. 1992 May 15;256(5059):992–997. doi: 10.1126/science.1589782. [DOI] [PubMed] [Google Scholar]
  36. Neubauer G., Gottschalk A., Fabrizio P., Séraphin B., Lührmann R., Mann M. Identification of the proteins of the yeast U1 small nuclear ribonucleoprotein complex by mass spectrometry. Proc Natl Acad Sci U S A. 1997 Jan 21;94(2):385–390. doi: 10.1073/pnas.94.2.385. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. 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]
  38. O'Keefe R. T., Newman A. J. Functional analysis of the U5 snRNA loop 1 in the second catalytic step of yeast pre-mRNA splicing. EMBO J. 1998 Jan 15;17(2):565–574. doi: 10.1093/emboj/17.2.565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. O'Keefe R. T., Norman C., Newman A. J. The invariant U5 snRNA loop 1 sequence is dispensable for the first catalytic step of pre-mRNA splicing in yeast. Cell. 1996 Aug 23;86(4):679–689. doi: 10.1016/s0092-8674(00)80140-3. [DOI] [PubMed] [Google Scholar]
  40. Pannone B. K., Xue D., Wolin S. L. A role for the yeast La protein in U6 snRNP assembly: evidence that the La protein is a molecular chaperone for RNA polymerase III transcripts. EMBO J. 1998 Dec 15;17(24):7442–7453. doi: 10.1093/emboj/17.24.7442. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Plessel G., Lührmann R., Kastner B. Electron microscopy of assembly intermediates of the snRNP core: morphological similarities between the RNA-free (E.F.G) protein heteromer and the intact snRNP core. J Mol Biol. 1997 Jan 17;265(2):87–94. doi: 10.1006/jmbi.1996.0713. [DOI] [PubMed] [Google Scholar]
  42. Raghunathan P. L., Guthrie C. A spliceosomal recycling factor that reanneals U4 and U6 small nuclear ribonucleoprotein particles. Science. 1998 Feb 6;279(5352):857–860. doi: 10.1126/science.279.5352.857. [DOI] [PubMed] [Google Scholar]
  43. 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]
  44. Raker V. A., Plessel G., Lührmann R. The snRNP core assembly pathway: identification of stable core protein heteromeric complexes and an snRNP subcore particle in vitro. EMBO J. 1996 May 1;15(9):2256–2269. [PMC free article] [PubMed] [Google Scholar]
  45. Reyes J. L., Kois P., Konforti B. B., Konarska M. M. The canonical GU dinucleotide at the 5' splice site is recognized by p220 of the U5 snRNP within the spliceosome. RNA. 1996 Mar;2(3):213–225. [PMC free article] [PubMed] [Google Scholar]
  46. Roy J., Zheng B., Rymond B. C., Woolford J. L., Jr Structurally related but functionally distinct yeast Sm D core small nuclear ribonucleoprotein particle proteins. Mol Cell Biol. 1995 Jan;15(1):445–455. doi: 10.1128/mcb.15.1.445. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Rymond B. C., Rokeach L. A., Hoch S. O. Human snRNP polypeptide D1 promotes pre-mRNA splicing in yeast and defines nonessential yeast Smd1p sequences. Nucleic Acids Res. 1993 Jul 25;21(15):3501–3505. doi: 10.1093/nar/21.15.3501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Shannon K. W., Guthrie C. Suppressors of a U4 snRNA mutation define a novel U6 snRNP protein with RNA-binding motifs. Genes Dev. 1991 May;5(5):773–785. doi: 10.1101/gad.5.5.773. [DOI] [PubMed] [Google Scholar]
  49. Stevens S. W., Abelson J. Purification of the yeast U4/U6.U5 small nuclear ribonucleoprotein particle and identification of its proteins. Proc Natl Acad Sci U S A. 1999 Jun 22;96(13):7226–7231. doi: 10.1073/pnas.96.13.7226. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Séraphin B. Sm and Sm-like proteins belong to a large family: identification of proteins of the U6 as well as the U1, U2, U4 and U5 snRNPs. EMBO J. 1995 May 1;14(9):2089–2098. doi: 10.1002/j.1460-2075.1995.tb07200.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Teigelkamp S., Newman A. J., Beggs J. D. Extensive interactions of PRP8 protein with the 5' and 3' splice sites during splicing suggest a role in stabilization of exon alignment by U5 snRNA. EMBO J. 1995 Jun 1;14(11):2602–2612. doi: 10.1002/j.1460-2075.1995.tb07258.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Teigelkamp S., Whittaker E., Beggs J. D. Interaction of the yeast splicing factor PRP8 with substrate RNA during both steps of splicing. Nucleic Acids Res. 1995 Feb 11;23(3):320–326. doi: 10.1093/nar/23.3.320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Umen J. G., Guthrie C. A novel role for a U5 snRNP protein in 3' splice site selection. Genes Dev. 1995 Apr 1;9(7):855–868. doi: 10.1101/gad.9.7.855. [DOI] [PubMed] [Google Scholar]
  54. 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]
  55. 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]
  56. 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]

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