Skip to main content
NCBI home page
RNA logoLink to RNA
. 2000 Aug;6(8):1120–1130. doi: 10.1017/s1355838200000133

Identification of a U2/U6 helix la mutant that influences 3' splice site selection during nuclear pre-mRNA splicing.

J S Chang 1, D S McPheeters 1
PMCID: PMC1369986  PMID: 10943891

Abstract

Base substitutions in U2/U6 helix I, a conserved base-pairing interaction between the U6 and U2 snRNAs, have previously been found to specifically block the second catalytic step of nuclear pre-mRNA splicing. To further assess the role of U2/U6 helix I in the second catalytic step, we have screened mutations in U2/U6 helix I to identify those that influence 3' splice site selection using a derivative of the yeast actin pre-mRNA. In these derivatives, the spacing between the branch site adenosine and 3' splice site has been reduced from 43 to 12 nt and this results in enhanced splicing of mutants in the conserved 3' terminal intron residue. In this context, mutation of the conserved 3' intron terminal G to a C also results in the partial activation of a nearby cryptic 3' splice site with U as the 3' terminal intron nucleotide. Using this highly sensitive mutant substrate, we have identified a mutation in the U6 snRNA (U57A) that significantly increases the selection of the cryptic 3' splice site over the normal 3' splice site and augments its utilization relative to that observed with the wild-type U2 or U6 snRNAs. In a previous study, we found that the same U6 mutation suppressed the effects of an A-to-G branch site mutation in an allele-specific fashion. The ability of U6-U57 mutants to influence the fidelity of both branch site and 3' splice site recognition suggests that this nucleotide may participate in the formation of the active site(s) of the spliceosome.

Full Text

The Full Text of this article is available as a PDF (272.3 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. Ansari A., Schwer B. SLU7 and a novel activity, SSF1, act during the PRP16-dependent step of yeast pre-mRNA splicing. EMBO J. 1995 Aug 15;14(16):4001–4009. doi: 10.1002/j.1460-2075.1995.tb00071.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. 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]
  5. Brys A., Schwer B. Requirement for SLU7 in yeast pre-mRNA splicing is dictated by the distance between the branchpoint and the 3' splice site. RNA. 1996 Jul;2(7):707–717. [PMC free article] [PubMed] [Google Scholar]
  6. Cech T. R. RNA splicing: three themes with variations. Cell. 1983 Oct;34(3):713–716. doi: 10.1016/0092-8674(83)90527-5. [DOI] [PubMed] [Google Scholar]
  7. Collins C. A., Guthrie C. Allele-specific genetic interactions between Prp8 and RNA active site residues suggest a function for Prp8 at the catalytic core of the spliceosome. Genes Dev. 1999 Aug 1;13(15):1970–1982. doi: 10.1101/gad.13.15.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. 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]
  11. 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]
  12. Fouser L. A., Friesen J. D. Mutations in a yeast intron demonstrate the importance of specific conserved nucleotides for the two stages of nuclear mRNA splicing. Cell. 1986 Apr 11;45(1):81–93. doi: 10.1016/0092-8674(86)90540-4. [DOI] [PubMed] [Google Scholar]
  13. Frank D., Guthrie C. An essential splicing factor, SLU7, mediates 3' splice site choice in yeast. Genes Dev. 1992 Nov;6(11):2112–2124. doi: 10.1101/gad.6.11.2112. [DOI] [PubMed] [Google Scholar]
  14. Frank D., Patterson B., Guthrie C. Synthetic lethal mutations suggest interactions between U5 small nuclear RNA and four proteins required for the second step of splicing. Mol Cell Biol. 1992 Nov;12(11):5197–5205. doi: 10.1128/mcb.12.11.5197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Green R., Noller H. F. Ribosomes and translation. Annu Rev Biochem. 1997;66:679–716. doi: 10.1146/annurev.biochem.66.1.679. [DOI] [PubMed] [Google Scholar]
  16. Jones M. H., Frank D. N., Guthrie C. Characterization and functional ordering of Slu7p and Prp17p during the second step of pre-mRNA splicing in yeast. Proc Natl Acad Sci U S A. 1995 Oct 10;92(21):9687–9691. doi: 10.1073/pnas.92.21.9687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Kivens W., Siliciano P. G. RNA sequences upstream of the 3' splice site repress splicing of mutantyeast ACT1 introns. RNA. 1996 May;2(5):492–505. [PMC free article] [PubMed] [Google Scholar]
  19. Lesser C. F., Guthrie C. Mutational analysis of pre-mRNA splicing in Saccharomyces cerevisiae using a sensitive new reporter gene, CUP1. Genetics. 1993 Apr;133(4):851–863. doi: 10.1093/genetics/133.4.851. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Luukkonen B. G., Séraphin B. A role for U2/U6 helix Ib in 5' splice site selection. RNA. 1998 Aug;4(8):915–927. doi: 10.1017/s1355838298980591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. 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]
  24. 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]
  25. 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]
  26. 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]
  27. McPheeters D. S., Abelson J. Mutational analysis of the yeast U2 snRNA suggests a structural similarity to the catalytic core of group I introns. Cell. 1992 Nov 27;71(5):819–831. doi: 10.1016/0092-8674(92)90557-s. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. McPheeters D. S., Schwer B., Muhlenkamp P. Interaction of the yeast DExH-box RNA helicase prp22p with the 3' splice site during the second step of nuclear pre-mRNA splicing. Nucleic Acids Res. 2000 Mar 15;28(6):1313–1321. doi: 10.1093/nar/28.6.1313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Merendino L., Guth S., Bilbao D., Martínez C., Valcárcel J. Inhibition of msl-2 splicing by Sex-lethal reveals interaction between U2AF35 and the 3' splice site AG. Nature. 1999 Dec 16;402(6763):838–841. doi: 10.1038/45602. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. 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]
  33. 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]
  34. Ng R., Abelson J. Isolation and sequence of the gene for actin in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3912–3916. doi: 10.1073/pnas.77.7.3912. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. 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]
  37. Ruis B. L., Kivens W. J., Siliciano P. G. The interaction between the first and last intron nucleotides in the second step of pre-mRNA splicing is independent of other conserved intron nucleotides. Nucleic Acids Res. 1994 Dec 11;22(24):5190–5195. doi: 10.1093/nar/22.24.5190. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Rymond B. C., Rosbash M. Cleavage of 5' splice site and lariat formation are independent of 3' splice site in yeast mRNA splicing. Nature. 1985 Oct 24;317(6039):735–737. doi: 10.1038/317735a0. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. Schwer B., Guthrie C. A conformational rearrangement in the spliceosome is dependent on PRP16 and ATP hydrolysis. EMBO J. 1992 Dec;11(13):5033–5039. doi: 10.1002/j.1460-2075.1992.tb05610.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. 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]
  42. Shuster E. O., Guthrie C. Human U2 snRNA can function in pre-mRNA splicing in yeast. Nature. 1990 May 17;345(6272):270–273. doi: 10.1038/345270a0. [DOI] [PubMed] [Google Scholar]
  43. Siatecka M., Reyes J. L., Konarska M. M. Functional interactions of Prp8 with both splice sites at the spliceosomal catalytic center. Genes Dev. 1999 Aug 1;13(15):1983–1993. doi: 10.1101/gad.13.15.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Spingola M., Grate L., Haussler D., Ares M., Jr Genome-wide bioinformatic and molecular analysis of introns in Saccharomyces cerevisiae. RNA. 1999 Feb;5(2):221–234. doi: 10.1017/s1355838299981682. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. 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]
  46. 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]
  47. Stotz A., Linder P. The ADE2 gene from Saccharomyces cerevisiae: sequence and new vectors. Gene. 1990 Oct 30;95(1):91–98. doi: 10.1016/0378-1119(90)90418-q. [DOI] [PubMed] [Google Scholar]
  48. Ségault V., Will C. L., Polycarpou-Schwarz M., Mattaj I. W., Branlant C., Lührmann R. Conserved loop I of U5 small nuclear RNA is dispensable for both catalytic steps of pre-mRNA splicing in HeLa nuclear extracts. Mol Cell Biol. 1999 Apr;19(4):2782–2790. doi: 10.1128/mcb.19.4.2782. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. 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]
  50. Umen J. G., Guthrie C. Prp16p, Slu7p, and Prp8p interact with the 3' splice site in two distinct stages during the second catalytic step of pre-mRNA splicing. RNA. 1995 Aug;1(6):584–597. [PMC free article] [PubMed] [Google Scholar]
  51. Umen J. G., Guthrie C. The second catalytic step of pre-mRNA splicing. RNA. 1995 Nov;1(9):869–885. [PMC free article] [PubMed] [Google Scholar]
  52. Wu S., Romfo C. M., Nilsen T. W., Green M. R. Functional recognition of the 3' splice site AG by the splicing factor U2AF35. Nature. 1999 Dec 16;402(6763):832–835. doi: 10.1038/45590. [DOI] [PubMed] [Google Scholar]
  53. 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]
  54. 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]
  55. Zhang X., Schwer B. Functional and physical interaction between the yeast splicing factors Slu7 and Prp18. Nucleic Acids Res. 1997 Jun 1;25(11):2146–2152. doi: 10.1093/nar/25.11.2146. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Zorio D. A., Blumenthal T. Both subunits of U2AF recognize the 3' splice site in Caenorhabditis elegans. Nature. 1999 Dec 16;402(6763):835–838. doi: 10.1038/45597. [DOI] [PubMed] [Google Scholar]

Articles from RNA are provided here courtesy of The RNA Society

RESOURCES