SLU7 and a novel activity, SSF1, act during the PRP16-dependent step of yeast pre-mRNA splicing.

A Ansari; B Schwer

doi:10.1002/j.1460-2075.1995.tb00071.x

EMBO J. 1995 Aug 15; 14(16): 4001–4009.

doi: 10.1002/j.1460-2075.1995.tb00071.x

PMCID: PMC394478

PMID: 7664739

SLU7 and a novel activity, SSF1, act during the PRP16-dependent step of yeast pre-mRNA splicing.

A Ansari and B Schwer

Author information Copyright and License information PMC Disclaimer

Abstract

Understanding the mechanism of pre-mRNA splicing requires the characterization of all components involved. In the present study, we used the genetically and biochemically defined yeast PRP16 protein as a point of departure for the identification of additional factors required for the second catalytic step in vitro. We isolated by glycerol gradient sedimentation spliceosomes that were formed in yeast extracts depleted of PRP16. This procedure separated the spliceosomal complexes containing lariat intermediate and exon 1 from free proteins present in the whole-cell yeast extract. We then supplemented these spliceosomes with purified proteins or yeast extract fractions as a functional assay for second-step splicing factors. We show that SLU7 protein and a novel activity that we named SSF1 (second-step factor 1) were required in concert with PRP16 to promote progression through the second catalytic step of splicing. Taking advantage of a differential ATP requirement for PRP16 and SLU7 function, we show that SLU7 can act after PRP16 in the splicing pathway.

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Selected References

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Bindereif A, Green MR. An ordered pathway of snRNP binding during mammalian pre-mRNA splicing complex assembly. EMBO J. 1987 Aug;6(8):2415–2424. [PMC free article] [PubMed] [Google Scholar]
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. [PubMed] [Google Scholar]
Brown JD, Beggs JD. Roles of PRP8 protein in the assembly of splicing complexes. EMBO J. 1992 Oct;11(10):3721–3729. [PMC free article] [PubMed] [Google Scholar]
Cheng SC, Abelson J. Spliceosome assembly in yeast. Genes Dev. 1987 Nov;1(9):1014–1027. [PubMed] [Google Scholar]
Frank D, Guthrie C. An essential splicing factor, SLU7, mediates 3' splice site choice in yeast. Genes Dev. 1992 Nov;6(11):2112–2124. [PubMed] [Google Scholar]
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. [PMC free article] [PubMed] [Google Scholar]
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. [PubMed] [Google Scholar]
Gozani O, Patton JG, Reed R. A novel set of spliceosome-associated proteins and the essential splicing factor PSF bind stably to pre-mRNA prior to catalytic step II of the splicing reaction. EMBO J. 1994 Jul 15;13(14):3356–3367. [PMC free article] [PubMed] [Google Scholar]
Grabowski PJ, Seiler SR, Sharp PA. A multicomponent complex is involved in the splicing of messenger RNA precursors. Cell. 1985 Aug;42(1):345–353. [PubMed] [Google Scholar]
Guthrie C. Messenger RNA splicing in yeast: clues to why the spliceosome is a ribonucleoprotein. Science. 1991 Jul 12;253(5016):157–163. [PubMed] [Google Scholar]
Guthrie C, Patterson B. Spliceosomal snRNAs. Annu Rev Genet. 1988;22:387–419. [PubMed] [Google Scholar]
Horowitz DS, Abelson J. Stages in the second reaction of pre-mRNA splicing: the final step is ATP independent. Genes Dev. 1993 Feb;7(2):320–329. [PubMed] [Google Scholar]
Horowitz DS, Abelson J. A U5 small nuclear ribonucleoprotein particle protein involved only in the second step of pre-mRNA splicing in Saccharomyces cerevisiae. Mol Cell Biol. 1993 May;13(5):2959–2970. [PMC free article] [PubMed] [Google Scholar]
Jones EW. Tackling the protease problem in Saccharomyces cerevisiae. Methods Enzymol. 1991;194:428–453. [PubMed] [Google Scholar]
Kim SH, Smith J, Claude A, Lin RJ. The purified yeast pre-mRNA splicing factor PRP2 is an RNA-dependent NTPase. EMBO J. 1992 Jun;11(6):2319–2326. [PMC free article] [PubMed] [Google Scholar]
Krainer AR, 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. [PubMed] [Google Scholar]
Krämer A. Mammalian protein factors involved in nuclear pre-mRNA splicing. Mol Biol Rep. 1993 Aug;18(2):93–98. [PubMed] [Google Scholar]
Lamm GM, Lamond AI. Non-snRNP protein splicing factors. Biochim Biophys Acta. 1993 Jun 25;1173(3):247–265. [PubMed] [Google Scholar]
Lin RJ, Newman AJ, Cheng SC, Abelson J. Yeast mRNA splicing in vitro. J Biol Chem. 1985 Nov 25;260(27):14780–14792. [PubMed] [Google Scholar]
Lin RJ, Lustig AJ, 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. [PubMed] [Google Scholar]
Madhani HD, Guthrie C. Dynamic RNA-RNA interactions in the spliceosome. Annu Rev Genet. 1994;28:1–26. [PubMed] [Google Scholar]
Perkins KK, Furneaux HM, 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. [PMC free article] [PubMed] [Google Scholar]
Plumpton M, McGarvey M, Beggs JD. A dominant negative mutation in the conserved RNA helicase motif 'SAT' causes splicing factor PRP2 to stall in spliceosomes. EMBO J. 1994 Feb 15;13(4):879–887. [PMC free article] [PubMed] [Google Scholar]
Ruby SW, Abelson J. Pre-mRNA splicing in yeast. Trends Genet. 1991 Mar;7(3):79–85. [PubMed] [Google Scholar]
Ruskin B, Green MR. An RNA processing activity that debranches RNA lariats. Science. 1985 Jul 12;229(4709):135–140. [PubMed] [Google Scholar]
Sawa H, Shimura Y. Alterations of RNase H sensitivity of the 3' splice site region during the in vitro splicing reaction. Nucleic Acids Res. 1991 Jul 25;19(14):3953–3958. [PMC free article] [PubMed] [Google Scholar]
Schwer B, Guthrie C. PRP16 is an RNA-dependent ATPase that interacts transiently with the spliceosome. Nature. 1991 Feb 7;349(6309):494–499. [PubMed] [Google Scholar]
Schwer B, Guthrie C. A dominant negative mutation in a spliceosomal ATPase affects ATP hydrolysis but not binding to the spliceosome. Mol Cell Biol. 1992 Aug;12(8):3540–3547. [PMC free article] [PubMed] [Google Scholar]
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. [PMC free article] [PubMed] [Google Scholar]
Studier FW, Moffatt BA. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. [PubMed] [Google Scholar]
Teigelkamp S, Whittaker E, Beggs JD. 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. [PMC free article] [PubMed] [Google Scholar]
Umen JG, Guthrie C. A novel role for a U5 snRNP protein in 3' splice site selection. Genes Dev. 1995 Apr 1;9(7):855–868. [PubMed] [Google Scholar]
Vijayraghavan U, Company M, Abelson J. Isolation and characterization of pre-mRNA splicing mutants of Saccharomyces cerevisiae. Genes Dev. 1989 Aug;3(8):1206–1216. [PubMed] [Google Scholar]

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