Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1995 Dec 5;92(25):11461–11464. doi: 10.1073/pnas.92.25.11461

In vitro trans-splicing in Saccharomyces cerevisiae.

A Ghetti 1, J N Abelson 1
PMCID: PMC40421  PMID: 8524783

Abstract

The interactions established at the 5'-splice site during spliceosome assembly are likely to be important for both precise recognition of the upstream intron boundary and for positioning this site in the active center of the spliceosome. Definition of the RNA-RNA and the RNA-protein interactions at the 5' splice site would be facilitated by the use of a small substrate amenable to modification during chemical synthesis. We describe a trans-splicing reaction performed in Saccharomyces cerevisiae extracts in which the 5' splice site and the 3' splice site are on separate molecules. The RNA contributing the 5' splice site is only 20 nucleotides long and was synthesized chemically. The trans-splicing reaction is accurate and has the same sequence, ATP, and Mg2+ requirements as cis-splicing. We also report how deoxy substitutions around the 5'-splice site affect trans-splicing efficiency.

Full text

PDF

Images in this article

11462Fig. 1
on p.11462
11463Fig. 2
on p.11463
11463Fig. 3
on p.11463

Selected References

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

  1. Bevilacqua P. C., Turner D. H. Comparison of binding of mixed ribose-deoxyribose analogues of CUCU to a ribozyme and to GGAGAA by equilibrium dialysis: evidence for ribozyme specific interactions with 2' OH groups. Biochemistry. 1991 Nov 5;30(44):10632–10640. doi: 10.1021/bi00108a005. [DOI] [PubMed] [Google Scholar]
  2. Bruzik J. P., Maniatis T. Enhancer-dependent interaction between 5' and 3' splice sites in trans. Proc Natl Acad Sci U S A. 1995 Jul 18;92(15):7056–7059. doi: 10.1073/pnas.92.15.7056. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bruzik J. P., Maniatis T. Spliced leader RNAs from lower eukaryotes are trans-spliced in mammalian cells. Nature. 1992 Dec 17;360(6405):692–695. doi: 10.1038/360692a0. [DOI] [PubMed] [Google Scholar]
  4. Chastain M., Tinoco I., Jr A base-triple structural domain in RNA. Biochemistry. 1992 Dec 29;31(51):12733–12741. doi: 10.1021/bi00166a004. [DOI] [PubMed] [Google Scholar]
  5. Chiara M. D., Reed R. A two-step mechanism for 5' and 3' splice-site pairing. Nature. 1995 Jun 8;375(6531):510–513. doi: 10.1038/375510a0. [DOI] [PubMed] [Google Scholar]
  6. Eul J., Graessmann M., Graessmann A. Experimental evidence for RNA trans-splicing in mammalian cells. EMBO J. 1995 Jul 3;14(13):3226–3235. doi: 10.1002/j.1460-2075.1995.tb07325.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hall K. B., Konarska M. M. The 5' splice site consensus RNA oligonucleotide induces assembly of U2/U4/U5/U6 small nuclear ribonucleoprotein complexes. Proc Natl Acad Sci U S A. 1992 Nov 15;89(22):10969–10973. doi: 10.1073/pnas.89.22.10969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Herschlag D., Cech T. R. DNA cleavage catalysed by the ribozyme from Tetrahymena. Nature. 1990 Mar 29;344(6265):405–409. doi: 10.1038/344405a0. [DOI] [PubMed] [Google Scholar]
  9. Horowitz D. S., Krainer A. R. Mechanisms for selecting 5' splice sites in mammalian pre-mRNA splicing. Trends Genet. 1994 Mar;10(3):100–106. doi: 10.1016/0168-9525(94)90233-x. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Konarska M. M., Padgett R. A., Sharp P. A. Trans splicing of mRNA precursors in vitro. Cell. 1985 Aug;42(1):165–171. doi: 10.1016/s0092-8674(85)80112-4. [DOI] [PubMed] [Google Scholar]
  12. Konforti B. B., Konarska M. M. U4/U5/U6 snRNP recognizes the 5' splice site in the absence of U2 snRNP. Genes Dev. 1994 Aug 15;8(16):1962–1973. doi: 10.1101/gad.8.16.1962. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. 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]
  16. Mei H. Y., Kaaret T. W., Bruice T. C. A computational approach to the mechanism of self-cleavage of hammerhead RNA. Proc Natl Acad Sci U S A. 1989 Dec;86(24):9727–9731. doi: 10.1073/pnas.86.24.9727. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. Nilsen T. W. RNA-RNA interactions in the spliceosome: unraveling the ties that bind. Cell. 1994 Jul 15;78(1):1–4. doi: 10.1016/0092-8674(94)90563-0. [DOI] [PubMed] [Google Scholar]
  20. Nilsen T. W. Trans-splicing of nematode premessenger RNA. Annu Rev Microbiol. 1993;47:413–440. doi: 10.1146/annurev.mi.47.100193.002213. [DOI] [PubMed] [Google Scholar]
  21. Pyle A. M., Cech T. R. Ribozyme recognition of RNA by tertiary interactions with specific ribose 2'-OH groups. Nature. 1991 Apr 18;350(6319):628–631. doi: 10.1038/350628a0. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Sawa H., Shimura Y. Association of U6 snRNA with the 5'-splice site region of pre-mRNA in the spliceosome. Genes Dev. 1992 Feb;6(2):244–254. doi: 10.1101/gad.6.2.244. [DOI] [PubMed] [Google Scholar]
  24. Sharp P. A. "Five easy pieces". Science. 1991 Nov 1;254(5032):663–663. doi: 10.1126/science.1948046. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Solnick D. Trans splicing of mRNA precursors. Cell. 1985 Aug;42(1):157–164. doi: 10.1016/s0092-8674(85)80111-2. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. 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]
  29. Ullu E., Matthews K. R., Tschudi C. Temporal order of RNA-processing reactions in trypanosomes: rapid trans splicing precedes polyadenylation of newly synthesized tubulin transcripts. Mol Cell Biol. 1993 Jan;13(1):720–725. doi: 10.1128/mcb.13.1.720. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Wassarman D. A., Steitz J. A. Interactions of small nuclear RNA's with precursor messenger RNA during in vitro splicing. Science. 1992 Sep 25;257(5078):1918–1925. doi: 10.1126/science.1411506. [DOI] [PubMed] [Google Scholar]
  31. 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]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES