Abstract
We previously reported the isolation of PRP28, a gene in Saccharomyces cerevisiae whose activity is required for the first step of nuclear mRNA splicing in vivo. Sequence analysis revealed that PRP28 is included in the 'DEAD-box' gene family, members of which are thought to function as ATP-dependent RNA helicases. Genetic interactions led us to suggest that PRP28 is functionally associated with the U4/U5/U6 snRNP. We have now purified the PRP28 protein from S. cerevisiae and demonstrated that it is required for the first step of splicing in vitro. Interestingly, PRP28 is not a stably associated snRNP protein. Strand displacement assays indicate that PRP28 does not exhibit RNA helicase activity, suggesting that an additional factor or factors may be required for its activation.
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- Abramson R. D., Dever T. E., Lawson T. G., Ray B. K., Thach R. E., Merrick W. C. The ATP-dependent interaction of eukaryotic initiation factors with mRNA. J Biol Chem. 1987 Mar 15;262(8):3826–3832. [PubMed] [Google Scholar]
- Abramson R. D., Dever T. E., Merrick W. C. Biochemical evidence supporting a mechanism for cap-independent and internal initiation of eukaryotic mRNA. J Biol Chem. 1988 May 5;263(13):6016–6019. [PubMed] [Google Scholar]
- 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]
- Blencowe B. J., Sproat B. S., Ryder U., Barabino S., Lamond A. I. Antisense probing of the human U4/U6 snRNP with biotinylated 2'-OMe RNA oligonucleotides. Cell. 1989 Nov 3;59(3):531–539. doi: 10.1016/0092-8674(89)90036-6. [DOI] [PubMed] [Google Scholar]
- Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
- Bringmann P., Appel B., Rinke J., Reuter R., Theissen H., Lührmann R. Evidence for the existence of snRNAs U4 and U6 in a single ribonucleoprotein complex and for their association by intermolecular base pairing. EMBO J. 1984 Jun;3(6):1357–1363. doi: 10.1002/j.1460-2075.1984.tb01977.x. [DOI] [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. doi: 10.1126/science.3890181. [DOI] [PubMed] [Google Scholar]
- Brow D. A., Guthrie C. Spliceosomal RNA U6 is remarkably conserved from yeast to mammals. Nature. 1988 Jul 21;334(6179):213–218. doi: 10.1038/334213a0. [DOI] [PubMed] [Google Scholar]
- Burgess S., Couto J. R., Guthrie C. A putative ATP binding protein influences the fidelity of branchpoint recognition in yeast splicing. Cell. 1990 Mar 9;60(5):705–717. doi: 10.1016/0092-8674(90)90086-t. [DOI] [PubMed] [Google Scholar]
- Cech T. R. The generality of self-splicing RNA: relationship to nuclear mRNA splicing. Cell. 1986 Jan 31;44(2):207–210. doi: 10.1016/0092-8674(86)90751-8. [DOI] [PubMed] [Google Scholar]
- Chen J. H., Lin R. J. 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. doi: 10.1093/nar/18.21.6447. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cheng S. C., Abelson J. Fractionation and characterization of a yeast mRNA splicing extract. Proc Natl Acad Sci U S A. 1986 Apr;83(8):2387–2391. doi: 10.1073/pnas.83.8.2387. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Claude A., Arenas J., Hurwitz J. The isolation and characterization of an RNA helicase from nuclear extracts of HeLa cells. J Biol Chem. 1991 Jun 5;266(16):10358–10367. [PubMed] [Google Scholar]
- 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]
- Dalbadie-McFarland G., Abelson J. PRP5: a helicase-like protein required for mRNA splicing in yeast. Proc Natl Acad Sci U S A. 1990 Jun;87(11):4236–4240. doi: 10.1073/pnas.87.11.4236. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dreusicke D., Karplus P. A., Schulz G. E. Refined structure of porcine cytosolic adenylate kinase at 2.1 A resolution. J Mol Biol. 1988 Jan 20;199(2):359–371. doi: 10.1016/0022-2836(88)90319-1. [DOI] [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. doi: 10.1016/s0092-8674(85)80131-8. [DOI] [PubMed] [Google Scholar]
- Grabowski P. J., Padgett R. A., Sharp P. A. Messenger RNA splicing in vitro: an excised intervening sequence and a potential intermediate. Cell. 1984 Jun;37(2):415–427. doi: 10.1016/0092-8674(84)90372-6. [DOI] [PubMed] [Google Scholar]
- 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]
- Grifo J. A., Abramson R. D., Satler C. A., Merrick W. C. RNA-stimulated ATPase activity of eukaryotic initiation factors. J Biol Chem. 1984 Jul 10;259(13):8648–8654. [PubMed] [Google Scholar]
- 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]
- Hashimoto C., Steitz J. A. U4 and U6 RNAs coexist in a single small nuclear ribonucleoprotein particle. Nucleic Acids Res. 1984 Apr 11;12(7):3283–3293. doi: 10.1093/nar/12.7.3283. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hay B., Jan L. Y., Jan Y. N. A protein component of Drosophila polar granules is encoded by vasa and has extensive sequence similarity to ATP-dependent helicases. Cell. 1988 Nov 18;55(4):577–587. doi: 10.1016/0092-8674(88)90216-4. [DOI] [PubMed] [Google Scholar]
- Hirling H., Scheffner M., Restle T., Stahl H. RNA helicase activity associated with the human p68 protein. Nature. 1989 Jun 15;339(6225):562–564. doi: 10.1038/339562a0. [DOI] [PubMed] [Google Scholar]
- Iggo R. D., Jamieson D. J., MacNeill S. A., Southgate J., McPheat J., Lane D. P. p68 RNA helicase: identification of a nucleolar form and cloning of related genes containing a conserved intron in yeasts. Mol Cell Biol. 1991 Mar;11(3):1326–1333. doi: 10.1128/mcb.11.3.1326. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Iggo R., Picksley S., Southgate J., McPheat J., Lane D. P. Identification of a putative RNA helicase in E.coli. Nucleic Acids Res. 1990 Sep 25;18(18):5413–5417. doi: 10.1093/nar/18.18.5413. [DOI] [PMC free article] [PubMed] [Google Scholar]
- King D. S., Beggs J. D. Interactions of PRP2 protein with pre-mRNA splicing complexes in Saccharomyces cerevisiae. Nucleic Acids Res. 1990 Nov 25;18(22):6559–6564. doi: 10.1093/nar/18.22.6559. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- Lasko P. F., Ashburner M. The product of the Drosophila gene vasa is very similar to eukaryotic initiation factor-4A. Nature. 1988 Oct 13;335(6191):611–617. doi: 10.1038/335611a0. [DOI] [PubMed] [Google Scholar]
- Lee C. G., Hurwitz J. A new RNA helicase isolated from HeLa cells that catalytically translocates in the 3' to 5' direction. J Biol Chem. 1992 Mar 5;267(7):4398–4407. [PubMed] [Google Scholar]
- Legrain P., Seraphin B., Rosbash M. Early commitment of yeast pre-mRNA to the spliceosome pathway. Mol Cell Biol. 1988 Sep;8(9):3755–3760. doi: 10.1128/mcb.8.9.3755. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Leroy P., Alzari P., Sassoon D., Wolgemuth D., Fellous M. The protein encoded by a murine male germ cell-specific transcript is a putative ATP-dependent RNA helicase. Cell. 1989 May 19;57(4):549–559. doi: 10.1016/0092-8674(89)90125-6. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- Lin R. J., Newman A. J., Cheng S. C., Abelson J. Yeast mRNA splicing in vitro. J Biol Chem. 1985 Nov 25;260(27):14780–14792. [PubMed] [Google Scholar]
- Linder P., Lasko P. F., Ashburner M., Leroy P., Nielsen P. J., Nishi K., Schnier J., Slonimski P. P. Birth of the D-E-A-D box. Nature. 1989 Jan 12;337(6203):121–122. doi: 10.1038/337121a0. [DOI] [PubMed] [Google Scholar]
- Linder P., Slonimski P. P. An essential yeast protein, encoded by duplicated genes TIF1 and TIF2 and homologous to the mammalian translation initiation factor eIF-4A, can suppress a mitochondrial missense mutation. Proc Natl Acad Sci U S A. 1989 Apr;86(7):2286–2290. doi: 10.1073/pnas.86.7.2286. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Matson S. W., Kaiser-Rogers K. A. DNA helicases. Annu Rev Biochem. 1990;59:289–329. doi: 10.1146/annurev.bi.59.070190.001445. [DOI] [PubMed] [Google Scholar]
- Michel F., Umesono K., Ozeki H. Comparative and functional anatomy of group II catalytic introns--a review. Gene. 1989 Oct 15;82(1):5–30. doi: 10.1016/0378-1119(89)90026-7. [DOI] [PubMed] [Google Scholar]
- Nielsen P. J., McMaster G. K., Trachsel H. Cloning of eukaryotic protein synthesis initiation factor genes: isolation and characterization of cDNA clones encoding factor eIF-4A. Nucleic Acids Res. 1985 Oct 11;13(19):6867–6880. doi: 10.1093/nar/13.19.6867. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nishi K., Morel-Deville F., Hershey J. W., Leighton T., Schnier J. An eIF-4A-like protein is a suppressor of an Escherichia coli mutant defective in 50S ribosomal subunit assembly. Nature. 1988 Dec 1;336(6198):496–498. doi: 10.1038/336496a0. [DOI] [PubMed] [Google Scholar]
- 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]
- Padgett R. A., Konarska M. M., Grabowski P. J., Hardy S. F., Sharp P. A. Lariat RNA's as intermediates and products in the splicing of messenger RNA precursors. Science. 1984 Aug 31;225(4665):898–903. doi: 10.1126/science.6206566. [DOI] [PubMed] [Google Scholar]
- Pai E. F., Kabsch W., Krengel U., Holmes K. C., John J., Wittinghofer A. Structure of the guanine-nucleotide-binding domain of the Ha-ras oncogene product p21 in the triphosphate conformation. Nature. 1989 Sep 21;341(6239):209–214. doi: 10.1038/341209a0. [DOI] [PubMed] [Google Scholar]
- Pause A., Méthot N., Sonenberg N. The HRIGRXXR region of the DEAD box RNA helicase eukaryotic translation initiation factor 4A is required for RNA binding and ATP hydrolysis. Mol Cell Biol. 1993 Nov;13(11):6789–6798. doi: 10.1128/mcb.13.11.6789. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Peebles C. L., Perlman P. S., Mecklenburg K. L., Petrillo M. L., Tabor J. H., Jarrell K. A., Cheng H. L. A self-splicing RNA excises an intron lariat. Cell. 1986 Jan 31;44(2):213–223. doi: 10.1016/0092-8674(86)90755-5. [DOI] [PubMed] [Google Scholar]
- 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]
- Ray B. K., Lawson T. G., Kramer J. C., Cladaras M. H., Grifo J. A., Abramson R. D., Merrick W. C., Thach R. E. ATP-dependent unwinding of messenger RNA structure by eukaryotic initiation factors. J Biol Chem. 1985 Jun 25;260(12):7651–7658. [PubMed] [Google Scholar]
- Rozen F., Edery I., Meerovitch K., Dever T. E., Merrick W. C., Sonenberg N. Bidirectional RNA helicase activity of eucaryotic translation initiation factors 4A and 4F. Mol Cell Biol. 1990 Mar;10(3):1134–1144. doi: 10.1128/mcb.10.3.1134. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Ruby S. W., Abelson J. Pre-mRNA splicing in yeast. Trends Genet. 1991 Mar;7(3):79–85. doi: 10.1016/0168-9525(91)90276-V. [DOI] [PubMed] [Google Scholar]
- Sachs A. B., Davis R. W. Translation initiation and ribosomal biogenesis: involvement of a putative rRNA helicase and RPL46. Science. 1990 Mar 2;247(4946):1077–1079. doi: 10.1126/science.2408148. [DOI] [PubMed] [Google Scholar]
- Sarkar G., Edery I., Sonenberg N. Photoaffinity labeling of the cap-binding protein complex with ATP/dATP. Differential labeling of free eukaryotic initiation factor 4A and the eukaryotic initiation factor 4A component of the cap-binding protein complex with [alpha-32P]ATP/dATP. J Biol Chem. 1985 Nov 5;260(25):13831–13837. [PubMed] [Google Scholar]
- 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]
- Schmelzer C., Schweyen R. J. Self-splicing of group II introns in vitro: mapping of the branch point and mutational inhibition of lariat formation. Cell. 1986 Aug 15;46(4):557–565. doi: 10.1016/0092-8674(86)90881-0. [DOI] [PubMed] [Google Scholar]
- Schmid S. R., Linder P. D-E-A-D protein family of putative RNA helicases. Mol Microbiol. 1992 Feb;6(3):283–291. doi: 10.1111/j.1365-2958.1992.tb01470.x. [DOI] [PubMed] [Google Scholar]
- 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. doi: 10.1016/0092-8674(89)90296-1. [DOI] [PubMed] [Google Scholar]
- Shannon K. W., Guthrie C. Suppressors of a U4 snRNA mutation define a novel U6 snRNP protein with RNA-binding motifs. Genes Dev. 1991 May;5(5):773–785. doi: 10.1101/gad.5.5.773. [DOI] [PubMed] [Google Scholar]
- 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]
- Siliciano P. G., Brow D. A., Roiha H., Guthrie C. An essential snRNA from S. cerevisiae has properties predicted for U4, including interaction with a U6-like snRNA. Cell. 1987 Aug 14;50(4):585–592. doi: 10.1016/0092-8674(87)90031-6. [DOI] [PubMed] [Google Scholar]
- Strauss E. J., Guthrie C. A cold-sensitive mRNA splicing mutant is a member of the RNA helicase gene family. Genes Dev. 1991 Apr;5(4):629–641. doi: 10.1101/gad.5.4.629. [DOI] [PubMed] [Google Scholar]
- Thompson S. T., Cass K. H., Stellwagen E. Blue dextran-sepharose: an affinity column for the dinucleotide fold in proteins. Proc Natl Acad Sci U S A. 1975 Feb;72(2):669–672. doi: 10.1073/pnas.72.2.669. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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. doi: 10.1002/j.1460-2075.1986.tb04412.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wassarman D. A., Steitz J. A. RNA splicing. Alive with DEAD proteins. Nature. 1991 Feb 7;349(6309):463–464. doi: 10.1038/349463a0. [DOI] [PubMed] [Google Scholar]
- Wray W., Boulikas T., Wray V. P., Hancock R. Silver staining of proteins in polyacrylamide gels. Anal Biochem. 1981 Nov 15;118(1):197–203. doi: 10.1016/0003-2697(81)90179-2. [DOI] [PubMed] [Google Scholar]
- Yean S. L., Lin R. J. 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. doi: 10.1128/mcb.11.11.5571. [DOI] [PMC free article] [PubMed] [Google Scholar]
- de Vos A. M., Tong L., Milburn M. V., Matias P. M., Jancarik J., Noguchi S., Nishimura S., Miura K., Ohtsuka E., Kim S. H. Three-dimensional structure of an oncogene protein: catalytic domain of human c-H-ras p21. Science. 1988 Feb 19;239(4842):888–893. doi: 10.1126/science.2448879. [DOI] [PubMed] [Google Scholar]
- van der Veen R., Arnberg A. C., van der Horst G., Bonen L., Tabak H. F., Grivell L. A. Excised group II introns in yeast mitochondria are lariats and can be formed by self-splicing in vitro. Cell. 1986 Jan 31;44(2):225–234. doi: 10.1016/0092-8674(86)90756-7. [DOI] [PubMed] [Google Scholar]