Skip to main content
PMC home page
RNA logoLink to RNA
. 1998 Aug;4(8):915–927. doi: 10.1017/s1355838298980591

A role for U2/U6 helix Ib in 5' splice site selection.

B G Luukkonen 1, B Séraphin 1
PMCID: PMC1369669  PMID: 9701283

Abstract

Selection of pre-mRNA splice sites is a highly accurate process involving many trans-acting factors. Recently, we described a role for U6 snRNA position G52 in selection of the first intron nucleotide (+1G). Because some U2 alleles suppress U6-G52 mutations, we investigated whether the corresponding U2 snRNA region also influenced 5' splice site selection. Our results demonstrate that U2 snRNAs mutated at position U23, but not adjacent nucleotides, specifically affect 5' splice site cleavage. Furthermore, all U2 position U23 mutations are synthetic lethal with the thermosensitive U6-G52U allele. Interestingly, the U2-U23C substitution has an unprecedented hyperaccurate splicing phenotype in which cleavage of introns with a +1G substitution is reduced, whereas the strain grows with wild-type kinetics. U2 position U23 forms the first base pair with U6 position A59 in U2/U6 helix Ib. Restoration of the helical structure suppresses 5' splice site cleavage defects, showing an important role for the helix Ib structure in 5' splice site selection. U2/U6 helix Ib and helix II have recently been described as being functionally redundant. This report demonstrates a unique role for helix Ib in 5' splice site selection that is not shared with helix II.

Full Text

The Full Text of this article is available as a PDF (683.3 KB).

Selected References

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

  1. Abovich N., Rosbash M. Cross-intron bridging interactions in the yeast commitment complex are conserved in mammals. Cell. 1997 May 2;89(3):403–412. doi: 10.1016/s0092-8674(00)80221-4. [DOI] [PubMed] [Google Scholar]
  2. Aebi M., Hornig H., Weissmann C. 5' cleavage site in eukaryotic pre-mRNA splicing is determined by the overall 5' splice region, not by the conserved 5' GU. Cell. 1987 Jul 17;50(2):237–246. doi: 10.1016/0092-8674(87)90219-4. [DOI] [PubMed] [Google Scholar]
  3. Arning S., Grüter P., Bilbe G., Krämer A. Mammalian splicing factor SF1 is encoded by variant cDNAs and binds to RNA. RNA. 1996 Aug;2(8):794–810. [PMC free article] [PubMed] [Google Scholar]
  4. Berglund J. A., Chua K., Abovich N., Reed R., Rosbash M. The splicing factor BBP interacts specifically with the pre-mRNA branchpoint sequence UACUAAC. Cell. 1997 May 30;89(5):781–787. doi: 10.1016/s0092-8674(00)80261-5. [DOI] [PubMed] [Google Scholar]
  5. Boulanger S. C., Belcher S. M., Schmidt U., Dib-Hajj S. D., Schmidt T., Perlman P. S. Studies of point mutants define three essential paired nucleotides in the domain 5 substructure of a group II intron. Mol Cell Biol. 1995 Aug;15(8):4479–4488. doi: 10.1128/mcb.15.8.4479. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Brow D. A., Guthrie C. Splicing a spliceosomal RNA. Nature. 1989 Jan 5;337(6202):14–15. doi: 10.1038/337014a0. [DOI] [PubMed] [Google Scholar]
  8. Cech T. R. The generality of self-splicing RNA: relationship to nuclear mRNA splicing. Cell. 1986 Jan 31;44(2):207–210. doi: 10.1016/0092-8674(86)90751-8. [DOI] [PubMed] [Google Scholar]
  9. Chanfreau G., Jacquier A. Catalytic site components common to both splicing steps of a group II intron. Science. 1994 Nov 25;266(5189):1383–1387. doi: 10.1126/science.7973729. [DOI] [PubMed] [Google Scholar]
  10. Chanfreau G., Legrain P., Dujon B., Jacquier A. Interaction between the first and last nucleotides of pre-mRNA introns is a determinant of 3' splice site selection in S. cerevisiae. Nucleic Acids Res. 1994 Jun 11;22(11):1981–1987. doi: 10.1093/nar/22.11.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. Deirdre A., Scadden J., Smith C. W. Interactions between the terminal bases of mammalian introns are retained in inosine-containing pre-mRNAs. EMBO J. 1995 Jul 3;14(13):3236–3246. doi: 10.1002/j.1460-2075.1995.tb07326.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fabrizio P., Abelson J. Thiophosphates in yeast U6 snRNA specifically affect pre-mRNA splicing in vitro. Nucleic Acids Res. 1992 Jul 25;20(14):3659–3664. doi: 10.1093/nar/20.14.3659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. Field D. J., Friesen J. D. Functionally redundant interactions between U2 and U6 spliceosomal snRNAs. Genes Dev. 1996 Feb 15;10(4):489–501. doi: 10.1101/gad.10.4.489. [DOI] [PubMed] [Google Scholar]
  18. Gorini L. Ribosomal discrimination of tRNAs. Nat New Biol. 1971 Dec 29;234(52):261–264. doi: 10.1038/newbio234261a0. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. 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]
  22. Jacquier A., Jacquesson-Breuleux N. Splice site selection and role of the lariat in a group II intron. J Mol Biol. 1991 Jun 5;219(3):415–428. doi: 10.1016/0022-2836(91)90183-7. [DOI] [PubMed] [Google Scholar]
  23. Jacquier A., Rodriguez J. R., Rosbash M. A quantitative analysis of the effects of 5' junction and TACTAAC box mutants and mutant combinations on yeast mRNA splicing. Cell. 1985 Dec;43(2 Pt 1):423–430. doi: 10.1016/0092-8674(85)90172-2. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Kim C. H., Abelson J. Site-specific crosslinks of yeast U6 snRNA to the pre-mRNA near the 5' splice site. RNA. 1996 Oct;2(10):995–1010. [PMC free article] [PubMed] [Google Scholar]
  26. Konforti B. B., Koziolkiewicz M. J., Konarska M. M. Disruption of base pairing between the 5' splice site and the 5' end of U1 snRNA is required for spliceosome assembly. Cell. 1993 Dec 3;75(5):863–873. doi: 10.1016/0092-8674(93)90531-t. [DOI] [PubMed] [Google Scholar]
  27. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lesser C. F., Guthrie C. Mutations in U6 snRNA that alter splice site specificity: implications for the active site. Science. 1993 Dec 24;262(5142):1982–1988. doi: 10.1126/science.8266093. [DOI] [PubMed] [Google Scholar]
  29. Luukkonen B. G., Séraphin B. Genetic interaction between U6 snRNA and the first intron nucleotide in Saccharomyces cerevisiae. RNA. 1998 Feb;4(2):167–180. [PMC free article] [PubMed] [Google Scholar]
  30. 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. 1997 Feb 17;16(4):779–792. doi: 10.1093/emboj/16.4.779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lygerou Z., Conesa C., Lesage P., Swanson R. N., Ruet A., Carlson M., Sentenac A., Séraphin B. The yeast BDF1 gene encodes a transcription factor involved in the expression of a broad class of genes including snRNAs. Nucleic Acids Res. 1994 Dec 11;22(24):5332–5340. doi: 10.1093/nar/22.24.5332. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. Madhani H. D., Guthrie C. A novel base-pairing interaction between U2 and U6 snRNAs suggests a mechanism for the catalytic activation of the spliceosome. Cell. 1992 Nov 27;71(5):803–817. doi: 10.1016/0092-8674(92)90556-r. [DOI] [PubMed] [Google Scholar]
  34. Madhani H. D., Guthrie C. Dynamic RNA-RNA interactions in the spliceosome. Annu Rev Genet. 1994;28:1–26. doi: 10.1146/annurev.ge.28.120194.000245. [DOI] [PubMed] [Google Scholar]
  35. Madhani H. D., Guthrie C. Randomization-selection analysis of snRNAs in vivo: evidence for a tertiary interaction in the spliceosome. Genes Dev. 1994 May 1;8(9):1071–1086. doi: 10.1101/gad.8.9.1071. [DOI] [PubMed] [Google Scholar]
  36. McPheeters D. S. Interactions of the yeast U6 RNA with the pre-mRNA branch site. RNA. 1996 Nov;2(11):1110–1123. [PMC free article] [PubMed] [Google Scholar]
  37. Michel F., Ferat J. L. Structure and activities of group II introns. Annu Rev Biochem. 1995;64:435–461. doi: 10.1146/annurev.bi.64.070195.002251. [DOI] [PubMed] [Google Scholar]
  38. Moore M. J., Sharp P. A. Evidence for two active sites in the spliceosome provided by stereochemistry of pre-mRNA splicing. Nature. 1993 Sep 23;365(6444):364–368. doi: 10.1038/365364a0. [DOI] [PubMed] [Google Scholar]
  39. Newman A. J., Norman C. U5 snRNA interacts with exon sequences at 5' and 3' splice sites. Cell. 1992 Feb 21;68(4):743–754. doi: 10.1016/0092-8674(92)90149-7. [DOI] [PubMed] [Google Scholar]
  40. Newman A. J., Teigelkamp S., Beggs J. D. snRNA interactions at 5' and 3' splice sites monitored by photoactivated crosslinking in yeast spliceosomes. RNA. 1995 Nov;1(9):968–980. [PMC free article] [PubMed] [Google Scholar]
  41. 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]
  42. 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]
  43. 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. 1993 Feb 18;361(6413):660–662. doi: 10.1038/361660a0. [DOI] [PubMed] [Google Scholar]
  44. Parker R., Siliciano P. G., Guthrie C. Recognition of the TACTAAC box during mRNA splicing in yeast involves base pairing to the U2-like snRNA. Cell. 1987 Apr 24;49(2):229–239. doi: 10.1016/0092-8674(87)90564-2. [DOI] [PubMed] [Google Scholar]
  45. Pascolo E., Séraphin B. The branchpoint residue is recognized during commitment complex formation before being bulged out of the U2 snRNA-pre-mRNA duplex. Mol Cell Biol. 1997 Jul;17(7):3469–3476. doi: 10.1128/mcb.17.7.3469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Pikielny C. W., Rosbash M. mRNA splicing efficiency in yeast and the contribution of nonconserved sequences. Cell. 1985 May;41(1):119–126. doi: 10.1016/0092-8674(85)90066-2. [DOI] [PubMed] [Google Scholar]
  47. Query C. C., Moore M. J., Sharp P. A. Branch nucleophile selection in pre-mRNA splicing: evidence for the bulged duplex model. Genes Dev. 1994 Mar 1;8(5):587–597. doi: 10.1101/gad.8.5.587. [DOI] [PubMed] [Google Scholar]
  48. 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]
  49. Siliciano P. G., Guthrie C. 5' splice site selection in yeast: genetic alterations in base-pairing with U1 reveal additional requirements. Genes Dev. 1988 Oct;2(10):1258–1267. doi: 10.1101/gad.2.10.1258. [DOI] [PubMed] [Google Scholar]
  50. 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]
  51. 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]
  52. Sontheimer E. J., Sun S., Piccirilli J. A. Metal ion catalysis during splicing of premessenger RNA. Nature. 1997 Aug 21;388(6644):801–805. doi: 10.1038/42068. [DOI] [PubMed] [Google Scholar]
  53. Steitz T. A., Steitz J. A. A general two-metal-ion mechanism for catalytic RNA. Proc Natl Acad Sci U S A. 1993 Jul 15;90(14):6498–6502. doi: 10.1073/pnas.90.14.6498. [DOI] [PMC free article] [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. Séraphin B., Kretzner L., Rosbash M. A U1 snRNA:pre-mRNA base pairing interaction is required early in yeast spliceosome assembly but does not uniquely define the 5' cleavage site. EMBO J. 1988 Aug;7(8):2533–2538. doi: 10.1002/j.1460-2075.1988.tb03101.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Séraphin B., Rosbash M. Exon mutations uncouple 5' splice site selection from U1 snRNA pairing. Cell. 1990 Nov 2;63(3):619–629. doi: 10.1016/0092-8674(90)90457-p. [DOI] [PubMed] [Google Scholar]
  57. Tani T., Ohshima Y. The gene for the U6 small nuclear RNA in fission yeast has an intron. Nature. 1989 Jan 5;337(6202):87–90. doi: 10.1038/337087a0. [DOI] [PubMed] [Google Scholar]
  58. Teem J. L., Rosbash M. Expression of a beta-galactosidase gene containing the ribosomal protein 51 intron is sensitive to the rna2 mutation of yeast. Proc Natl Acad Sci U S A. 1983 Jul;80(14):4403–4407. doi: 10.1073/pnas.80.14.4403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Weiner A. M. mRNA splicing and autocatalytic introns: distant cousins or the products of chemical determinism? Cell. 1993 Jan 29;72(2):161–164. doi: 10.1016/0092-8674(93)90654-9. [DOI] [PubMed] [Google Scholar]
  60. 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]
  61. 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]
  62. Wu J., Manley J. L. Mammalian pre-mRNA branch site selection by U2 snRNP involves base pairing. Genes Dev. 1989 Oct;3(10):1553–1561. doi: 10.1101/gad.3.10.1553. [DOI] [PubMed] [Google Scholar]
  63. 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]
  64. Xu D., Field D. J., Tang S. J., Moris A., Bobechko B. P., Friesen J. D. Synthetic lethality of yeast slt mutations with U2 small nuclear RNA mutations suggests functional interactions between U2 and U5 snRNPs that are important for both steps of pre-mRNA splicing. Mol Cell Biol. 1998 Apr;18(4):2055–2066. doi: 10.1128/mcb.18.4.2055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Yan D., Ares M., Jr Invariant U2 RNA sequences bordering the branchpoint recognition region are essential for interaction with yeast SF3a and SF3b subunits. Mol Cell Biol. 1996 Mar;16(3):818–828. doi: 10.1128/mcb.16.3.818. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Yu Y. T., Maroney P. A., Darzynkiwicz E., Nilsen T. W. U6 snRNA function in nuclear pre-mRNA splicing: a phosphorothioate interference analysis of the U6 phosphate backbone. RNA. 1995 Mar;1(1):46–54. [PMC free article] [PubMed] [Google Scholar]
  67. Zhuang Y., Weiner A. M. A compensatory base change in U1 snRNA suppresses a 5' splice site mutation. Cell. 1986 Sep 12;46(6):827–835. doi: 10.1016/0092-8674(86)90064-4. [DOI] [PubMed] [Google Scholar]
  68. Zhuang Y., Weiner A. M. A compensatory base change in human U2 snRNA can suppress a branch site mutation. Genes Dev. 1989 Oct;3(10):1545–1552. doi: 10.1101/gad.3.10.1545. [DOI] [PubMed] [Google Scholar]

Articles from RNA are provided here courtesy of The RNA Society

RESOURCES