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Mol Cell Biol. Dec 1996; 16(12): 6810–6819.
PMCID: PMC231684

Spliceosome activation by PRP2 ATPase prior to the first transesterification reaction of pre-mRNA splicing.

Abstract

In addition to small nuclear RNAs and spliceosomal proteins, ATP hydrolysis is needed for nuclear pre-mRNA splicing. A number of RNA-dependent ATPases which are involved in several distinct ATP-dependent steps in splicing have been identified in Saccharomyces cerevisiae and mammals. These so-called DEAD/H ATPases contain conserved RNA helicase motifs, although RNA unwinding activity has not been demonstrated in purified proteins. Here we report the role of one such DEAH protein, PRP2 of S. cerevisiae, in spliceosome activation. PRP2 bound to a precatalytic spliceosome prior to the first step of splicing. By blocking the activity of a novel splicing factor(s), HP, which was involved in a post-PRP2 step, we found that PRP2 hydrolyzed ATP to cause a change in the spliceosome without the occurrence of splicing. The change was quite dramatic and could account for the previously reported differences between the precatalytic, pre-mRNA-containing spliceosome and the "active," intermediate-containing spliceosome. The post-PRP2-ATP spliceosome was further isolated and could carry out the subsequent reaction apparently in the absence of PRP2 and ATP. We hypothesize that PRP2 functions as a molecular motor, similar to some DExH ATPases in transcription, in the activation of the precatalytic spliceosome for the transesterification reaction.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Alberts B, Miake-Lye R. Unscrambling the puzzle of biological machines: the importance of the details. Cell. 1992 Feb 7;68(3):415–420. [PubMed]
  • 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. [PMC free article] [PubMed]
  • Beggs JD. Yeast protein splicing factors involved in nuclear pre-mRNA splicing. Mol Biol Rep. 1993 Aug;18(2):99–103. [PubMed]
  • 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]
  • Buratowski S. The basics of basal transcription by RNA polymerase II. Cell. 1994 Apr 8;77(1):1–3. [PubMed]
  • Buratowski S, Hahn S, Guarente L, Sharp PA. Five intermediate complexes in transcription initiation by RNA polymerase II. Cell. 1989 Feb 24;56(4):549–561. [PubMed]
  • Burgess SM, Guthrie C. A mechanism to enhance mRNA splicing fidelity: the RNA-dependent ATPase Prp16 governs usage of a discard pathway for aberrant lariat intermediates. Cell. 1993 Jul 2;73(7):1377–1391. [PubMed]
  • Chen JH, Lin RJ. The yeast PRP2 protein, a putative RNA-dependent ATPase, shares extensive sequence homology with two other pre-mRNA splicing factors. Nucleic Acids Res. 1990 Nov 11;18(21):6447–6447. [PMC free article] [PubMed]
  • Cheng SC, Abelson J. Spliceosome assembly in yeast. Genes Dev. 1987 Nov;1(9):1014–1027. [PubMed]
  • Clark MW, Goelz S, Abelson J. Electron microscopic identification of the yeast spliceosome. EMBO J. 1988 Dec 1;7(12):3829–3836. [PMC free article] [PubMed]
  • Côté J, Quinn J, Workman JL, Peterson CL. Stimulation of GAL4 derivative binding to nucleosomal DNA by the yeast SWI/SNF complex. Science. 1994 Jul 1;265(5168):53–60. [PubMed]
  • Drapkin R, Sancar A, Reinberg D. Where transcription meets repair. Cell. 1994 Apr 8;77(1):9–12. [PubMed]
  • Fuller-Pace FV. RNA helicases: modulators of RNA structure. Trends Cell Biol. 1994 Aug;4(8):271–274. [PubMed]
  • Goodrich JA, Tjian R. Transcription factors IIE and IIH and ATP hydrolysis direct promoter clearance by RNA polymerase II. Cell. 1994 Apr 8;77(1):145–156. [PubMed]
  • Guan KL, Dixon JE. Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase. Anal Biochem. 1991 Feb 1;192(2):262–267. [PubMed]
  • Guthrie C. Messenger RNA splicing in yeast: clues to why the spliceosome is a ribonucleoprotein. Science. 1991 Jul 12;253(5016):157–163. [PubMed]
  • Herschlag D. RNA chaperones and the RNA folding problem. J Biol Chem. 1995 Sep 8;270(36):20871–20874. [PubMed]
  • 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]
  • Jones MH, Frank DN, 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. [PMC free article] [PubMed]
  • Kim SH, Lin RJ. Pre-mRNA splicing within an assembled yeast spliceosome requires an RNA-dependent ATPase and ATP hydrolysis. Proc Natl Acad Sci U S A. 1993 Feb 1;90(3):888–892. [PMC free article] [PubMed]
  • 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]
  • King DS, Beggs JD. Interactions of PRP2 protein with pre-mRNA splicing complexes in Saccharomyces cerevisiae. Nucleic Acids Res. 1990 Nov 25;18(22):6559–6564. [PMC free article] [PubMed]
  • Konarska MM, Sharp PA. Interactions between small nuclear ribonucleoprotein particles in formation of spliceosomes. Cell. 1987 Jun 19;49(6):763–774. [PubMed]
  • Laggerbauer B, Lauber J, Lührmann R. Identification of an RNA-dependent ATPase activity in mammalian U5 snRNPs. Nucleic Acids Res. 1996 Mar 1;24(5):868–875. [PMC free article] [PubMed]
  • Lamond AI, Konarska MM, Grabowski PJ, Sharp PA. 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. [PMC free article] [PubMed]
  • 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]
  • Lin RJ, Newman AJ, Cheng SC, Abelson J. Yeast mRNA splicing in vitro. J Biol Chem. 1985 Nov 25;260(27):14780–14792. [PubMed]
  • Lustig AJ, Lin RJ, Abelson J. The yeast RNA gene products are essential for mRNA splicing in vitro. Cell. 1986 Dec 26;47(6):953–963. [PubMed]
  • Madhani HD, Guthrie C. Dynamic RNA-RNA interactions in the spliceosome. Annu Rev Genet. 1994;28:1–26. [PubMed]
  • Nilsen TW. RNA-RNA interactions in the spliceosome: unraveling the ties that bind. Cell. 1994 Jul 15;78(1):1–4. [PubMed]
  • Ohno M, Shimura Y. A human RNA helicase-like protein, HRH1, facilitates nuclear export of spliced mRNA by releasing the RNA from the spliceosome. Genes Dev. 1996 Apr 15;10(8):997–1007. [PubMed]
  • Ono Y, Ohno M, Shimura Y. Identification of a putative RNA helicase (HRH1), a human homolog of yeast Prp22. Mol Cell Biol. 1994 Nov;14(11):7611–7620. [PMC free article] [PubMed]
  • Peterson CL, Tamkun JW. The SWI-SNF complex: a chromatin remodeling machine? Trends Biochem Sci. 1995 Apr;20(4):143–146. [PubMed]
  • Pikielny CW, Rymond BC, Rosbash M. Electrophoresis of ribonucleoproteins reveals an ordered assembly pathway of yeast splicing complexes. Nature. 324(6095):341–345. [PubMed]
  • 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]
  • Rosbash M, Harris PK, Woolford JL, Jr, Teem JL. The effect of temperature-sensitive RNA mutants on the transcription products from cloned ribosomal protein genes of yeast. Cell. 1981 Jun;24(3):679–686. [PubMed]
  • Roy J, Kim K, Maddock JR, Anthony JG, Woolford JL., Jr The final stages of spliceosome maturation require Spp2p that can interact with the DEAH box protein Prp2p and promote step 1 of splicing. RNA. 1995 Jun;1(4):375–390. [PMC free article] [PubMed]
  • Ruby SW, Abelson J. Pre-mRNA splicing in yeast. Trends Genet. 1991 Mar;7(3):79–85. [PubMed]
  • Schmid SR, Linder P. D-E-A-D protein family of putative RNA helicases. Mol Microbiol. 1992 Feb;6(3):283–291. [PubMed]
  • 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]
  • 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]
  • Seraphin B, Rosbash M. Identification of functional U1 snRNA-pre-mRNA complexes committed to spliceosome assembly and splicing. Cell. 1989 Oct 20;59(2):349–358. [PubMed]
  • Sharp PA. Split genes and RNA splicing. Cell. 1994 Jun 17;77(6):805–815. [PubMed]
  • Smith DB, Johnson KS. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988 Jul 15;67(1):31–40. [PubMed]
  • Teigelkamp S, McGarvey M, Plumpton M, Beggs JD. The splicing factor PRP2, a putative RNA helicase, interacts directly with pre-mRNA. EMBO J. 1994 Feb 15;13(4):888–897. [PMC free article] [PubMed]
  • Umen JG, Guthrie C. The second catalytic step of pre-mRNA splicing. RNA. 1995 Nov;1(9):869–885. [PMC free article] [PubMed]
  • Vijayraghavan U, Parker R, Tamm J, Iimura Y, Rossi J, Abelson J, Guthrie C. Mutations in conserved intron sequences affect multiple steps in the yeast splicing pathway, particularly assembly of the spliceosome. EMBO J. 1986 Jul;5(7):1683–1695. [PMC free article] [PubMed]
  • Wassarman DA, Steitz JA. RNA splicing. Alive with DEAD proteins. Nature. 1991 Feb 7;349(6309):463–464. [PubMed]
  • Yean SL, Lin RJ. U4 small nuclear RNA dissociates from a yeast spliceosome and does not participate in the subsequent splicing reaction. Mol Cell Biol. 1991 Nov;11(11):5571–5577. [PMC free article] [PubMed]
  • Yean SL, Lin RJ. Analysis of small nuclear RNAs in a precatalytic spliceosome. Gene Expr. 1996;5(6):301–313. [PubMed]

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