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. 1991 Jul 25;19(14):3953–3958. doi: 10.1093/nar/19.14.3953

Alterations of RNase H sensitivity of the 3' splice site region during the in vitro splicing reaction.

H Sawa 1, Y Shimura 1
PMCID: PMC328488  PMID: 1650457

Abstract

We have developed a splicing assay system with an immobilized pre-mRNA to study the mechanism of the splicing reaction after spliceosome assembly. Using this system, we have found that the second step of the splicing reaction could be dissected into two stages. After the 5' splice site reaction, at least two factors interact with the pre-formed spliceosome containing intermediate molecules in an ATP-independent manner to convert the spliceosome into a form competent for the 3' splice site reaction. Then, the 3' splice site reaction occurs on this spliceosome, if ATP is supplied to the reaction mixture. We have also investigated the dynamic state of the 3' splice site region in the spliceosomes during the splicing reaction by probing with RNase H sensitivity. Prior to the 5' splice site reaction, the 3' splice site region was protected from RNase H attack. The region became sensitive immediately after the 5' splice site reaction, and subsequently became resistant again as the spliceosome competent for the 3' splice site reaction was formed. These results suggest that the interaction of the 3' splice site region with some spliceosome components changes significantly during the splicing reaction.

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Selected References

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

  1. Abmayr S. M., Reed R., Maniatis T. Identification of a functional mammalian spliceosome containing unspliced pre-mRNA. Proc Natl Acad Sci U S A. 1988 Oct;85(19):7216–7220. doi: 10.1073/pnas.85.19.7216. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bindereif A., Green M. R. An ordered pathway of snRNP binding during mammalian pre-mRNA splicing complex assembly. EMBO J. 1987 Aug;6(8):2415–2424. doi: 10.1002/j.1460-2075.1987.tb02520.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brody E., Abelson J. The "spliceosome": yeast pre-messenger RNA associates with a 40S complex in a splicing-dependent reaction. Science. 1985 May 24;228(4702):963–967. doi: 10.1126/science.3890181. [DOI] [PubMed] [Google Scholar]
  4. Cheng S. C., Abelson J. Spliceosome assembly in yeast. Genes Dev. 1987 Nov;1(9):1014–1027. doi: 10.1101/gad.1.9.1014. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. 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]
  7. 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]
  8. Furneaux H. M., Perkins K. K., Freyer G. A., Arenas J., Hurwitz J. Isolation and characterization of two fractions from HeLa cells required for mRNA splicing in vitro. Proc Natl Acad Sci U S A. 1985 Jul;82(13):4351–4355. doi: 10.1073/pnas.82.13.4351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. García-Blanco M. A., Jamison S. F., Sharp P. A. Identification and purification of a 62,000-dalton protein that binds specifically to the polypyrimidine tract of introns. Genes Dev. 1989 Dec;3(12A):1874–1886. doi: 10.1101/gad.3.12a.1874. [DOI] [PubMed] [Google Scholar]
  10. Gerke V., Steitz J. A. A protein associated with small nuclear ribonucleoprotein particles recognizes the 3' splice site of premessenger RNA. Cell. 1986 Dec 26;47(6):973–984. doi: 10.1016/0092-8674(86)90812-3. [DOI] [PubMed] [Google Scholar]
  11. Grabowski P. J., Seiler S. R., Sharp P. A. A multicomponent complex is involved in the splicing of messenger RNA precursors. Cell. 1985 Aug;42(1):345–353. doi: 10.1016/s0092-8674(85)80130-6. [DOI] [PubMed] [Google Scholar]
  12. Grabowski P. J., Sharp P. A. Affinity chromatography of splicing complexes: U2, U5, and U4 + U6 small nuclear ribonucleoprotein particles in the spliceosome. Science. 1986 Sep 19;233(4770):1294–1299. doi: 10.1126/science.3638792. [DOI] [PubMed] [Google Scholar]
  13. Green M. R. Pre-mRNA splicing. Annu Rev Genet. 1986;20:671–708. doi: 10.1146/annurev.ge.20.120186.003323. [DOI] [PubMed] [Google Scholar]
  14. Guthrie C., Patterson B. Spliceosomal snRNAs. Annu Rev Genet. 1988;22:387–419. doi: 10.1146/annurev.ge.22.120188.002131. [DOI] [PubMed] [Google Scholar]
  15. Konarska M. M., Sharp P. A. Interactions between small nuclear ribonucleoprotein particles in formation of spliceosomes. Cell. 1987 Jun 19;49(6):763–774. doi: 10.1016/0092-8674(87)90614-3. [DOI] [PubMed] [Google Scholar]
  16. Krainer A. R., Maniatis T. Multiple factors including the small nuclear ribonucleoproteins U1 and U2 are necessary for pre-mRNA splicing in vitro. Cell. 1985 Oct;42(3):725–736. doi: 10.1016/0092-8674(85)90269-7. [DOI] [PubMed] [Google Scholar]
  17. Lamond A. I., Konarska M. M., Grabowski P. J., Sharp P. A. Spliceosome assembly involves the binding and release of U4 small nuclear ribonucleoprotein. Proc Natl Acad Sci U S A. 1988 Jan;85(2):411–415. doi: 10.1073/pnas.85.2.411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lamond A. I., Konarska M. M., Sharp P. A. A mutational analysis of spliceosome assembly: evidence for splice site collaboration during spliceosome formation. Genes Dev. 1987 Aug;1(6):532–543. doi: 10.1101/gad.1.6.532. [DOI] [PubMed] [Google Scholar]
  19. Lin R. J., Lustig A. J., Abelson J. Splicing of yeast nuclear pre-mRNA in vitro requires a functional 40S spliceosome and several extrinsic factors. Genes Dev. 1987 Mar;1(1):7–18. doi: 10.1101/gad.1.1.7. [DOI] [PubMed] [Google Scholar]
  20. Padgett R. A., Grabowski P. J., Konarska M. M., Seiler S., Sharp P. A. Splicing of messenger RNA precursors. Annu Rev Biochem. 1986;55:1119–1150. doi: 10.1146/annurev.bi.55.070186.005351. [DOI] [PubMed] [Google Scholar]
  21. Perkins K. K., Furneaux H. M., Hurwitz J. RNA splicing products formed with isolated fractions from HeLa cells are associated with fast-sedimenting complexes. Proc Natl Acad Sci U S A. 1986 Feb;83(4):887–891. doi: 10.1073/pnas.83.4.887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Pikielny C. W., Rymond B. C., Rosbash M. Electrophoresis of ribonucleoproteins reveals an ordered assembly pathway of yeast splicing complexes. 1986 Nov 27-Dec 3Nature. 324(6095):341–345. doi: 10.1038/324341a0. [DOI] [PubMed] [Google Scholar]
  23. Reed R., Griffith J., Maniatis T. Purification and visualization of native spliceosomes. Cell. 1988 Jun 17;53(6):949–961. doi: 10.1016/s0092-8674(88)90489-8. [DOI] [PubMed] [Google Scholar]
  24. Reed R. The organization of 3' splice-site sequences in mammalian introns. Genes Dev. 1989 Dec;3(12B):2113–2123. doi: 10.1101/gad.3.12b.2113. [DOI] [PubMed] [Google Scholar]
  25. Ruby S. W., Abelson J. An early hierarchic role of U1 small nuclear ribonucleoprotein in spliceosome assembly. Science. 1988 Nov 18;242(4881):1028–1035. doi: 10.1126/science.2973660. [DOI] [PubMed] [Google Scholar]
  26. Ruskin B., Green M. R. Specific and stable intron-factor interactions are established early during in vitro pre-mRNA splicing. Cell. 1985 Nov;43(1):131–142. doi: 10.1016/0092-8674(85)90018-2. [DOI] [PubMed] [Google Scholar]
  27. Ruskin B., Zamore P. D., Green M. R. A factor, U2AF, is required for U2 snRNP binding and splicing complex assembly. Cell. 1988 Jan 29;52(2):207–219. doi: 10.1016/0092-8674(88)90509-0. [DOI] [PubMed] [Google Scholar]
  28. Sawa H., Ohno M., Sakamoto H., Shimura Y. Requirement of ATP in the second step of the pre-mRNA splicing reaction. Nucleic Acids Res. 1988 Apr 25;16(8):3157–3164. doi: 10.1093/nar/16.8.3157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Schwer B., Guthrie C. PRP16 is an RNA-dependent ATPase that interacts transiently with the spliceosome. Nature. 1991 Feb 7;349(6309):494–499. doi: 10.1038/349494a0. [DOI] [PubMed] [Google Scholar]
  30. Swanson M. S., Dreyfuss G. RNA binding specificity of hnRNP proteins: a subset bind to the 3' end of introns. EMBO J. 1988 Nov;7(11):3519–3529. doi: 10.1002/j.1460-2075.1988.tb03228.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Tazi J., Alibert C., Temsamani J., Reveillaud I., Cathala G., Brunel C., Jeanteur P. A protein that specifically recognizes the 3' splice site of mammalian pre-mRNA introns is associated with a small nuclear ribonucleoprotein. Cell. 1986 Dec 5;47(5):755–766. doi: 10.1016/0092-8674(86)90518-0. [DOI] [PubMed] [Google Scholar]
  32. Vijayraghavan U., Abelson J. PRP18, a protein required for the second reaction in pre-mRNA splicing. Mol Cell Biol. 1990 Jan;10(1):324–332. doi: 10.1128/mcb.10.1.324. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Watakabe A., Inoue K., Sakamoto H., Shimura Y. A secondary structure at the 3' splice site affects the in vitro splicing reaction of mouse immunoglobulin mu chain pre-mRNAs. Nucleic Acids Res. 1989 Oct 25;17(20):8159–8169. doi: 10.1093/nar/17.20.8159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Zillmann M., Zapp M. L., Berget S. M. Gel electrophoretic isolation of splicing complexes containing U1 small nuclear ribonucleoprotein particles. Mol Cell Biol. 1988 Feb;8(2):814–821. doi: 10.1128/mcb.8.2.814. [DOI] [PMC free article] [PubMed] [Google Scholar]

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