Abstract
Cleavage and polyadenylation factor I (CF I) is one of four factors required in vitro for yeast pre-mRNA 3'-end processing. Two protein components of this factor, encoded by genes RNA14 and RNA15, have already been identified. We describe here another gene, PCF11 (for protein 1 of CF I), that genetically interacts with RNA14 and RNA15 and which presumably codes for a third protein component of CF I. This gene was isolated in a two-hybrid screening designed to identify proteins interacting with Rna14 and Rna15. PCF11 is an essential gene encoding for a protein of 626 amino acids having an apparent molecular mass of 70 kDa. Thermosensitive mutations in PCF11 are synergistically lethal with thermosensitive alleles of RNA14 and RNA15. The Pcf11-2 thermosensitive strain shows a shortening of the poly(A) tails and a strong decrease in the steady-state level of actin transcripts after a shift to the nonpermissive temperature as do the thermosensitive alleles of RNA14 and RNA15. Extracts from the pcf11-1 and pcf11-2 thermosensitive strains and the wild-type strain, when Pcf11 is neutralized by specific antibodies, are deficient in cleavage and polyadenylation. Moreover, fractions obtained by anion-exchange chromatography of extracts from the wild-type strain contain both Pcf11 and Rna15 in the same fractions, as shown by immunoblotting with a Pcf11-specific antibody.
Full Text
The Full Text of this article is available as a PDF (2.9 MB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Amrani N., Dufour M. E., Bonneaud N., Lacroute F. Mutations in STS1 suppress the defect in 3' mRNA processing caused by the rna15-2 mutation in Saccharomyces cerevisiae. Mol Gen Genet. 1996 Oct 16;252(5):552–562. doi: 10.1007/BF02172401. [DOI] [PubMed] [Google Scholar]
- Aström J., Aström A., Virtanen A. In vitro deadenylation of mammalian mRNA by a HeLa cell 3' exonuclease. EMBO J. 1991 Oct;10(10):3067–3071. doi: 10.1002/j.1460-2075.1991.tb07858.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Aström J., Aström A., Virtanen A. Properties of a HeLa cell 3' exonuclease specific for degrading poly(A) tails of mammalian mRNA. J Biol Chem. 1992 Sep 5;267(25):18154–18159. [PubMed] [Google Scholar]
- Baudin A., Ozier-Kalogeropoulos O., Denouel A., Lacroute F., Cullin C. A simple and efficient method for direct gene deletion in Saccharomyces cerevisiae. Nucleic Acids Res. 1993 Jul 11;21(14):3329–3330. doi: 10.1093/nar/21.14.3329. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bernstein P., Peltz S. W., Ross J. The poly(A)-poly(A)-binding protein complex is a major determinant of mRNA stability in vitro. Mol Cell Biol. 1989 Feb;9(2):659–670. doi: 10.1128/mcb.9.2.659. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bloch J. C., Perrin F., Lacroute F. Yeast temperature-sensitive mutants specifically impaired in processing of poly(A)-containing RNAs. Mol Gen Genet. 1978 Oct 4;165(2):123–127. doi: 10.1007/BF00269900. [DOI] [PubMed] [Google Scholar]
- Boeck R., Tarun S., Jr, Rieger M., Deardorff J. A., Müller-Auer S., Sachs A. B. The yeast Pan2 protein is required for poly(A)-binding protein-stimulated poly(A)-nuclease activity. J Biol Chem. 1996 Jan 5;271(1):432–438. doi: 10.1074/jbc.271.1.432. [DOI] [PubMed] [Google Scholar]
- Boeke J. D., LaCroute F., Fink G. R. A positive selection for mutants lacking orotidine-5'-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol Gen Genet. 1984;197(2):345–346. doi: 10.1007/BF00330984. [DOI] [PubMed] [Google Scholar]
- Butler J. S., Platt T. RNA processing generates the mature 3' end of yeast CYC1 messenger RNA in vitro. Science. 1988 Dec 2;242(4883):1270–1274. doi: 10.1126/science.2848317. [DOI] [PubMed] [Google Scholar]
- Butler J. S., Sadhale P. P., Platt T. RNA processing in vitro produces mature 3' ends of a variety of Saccharomyces cerevisiae mRNAs. Mol Cell Biol. 1990 Jun;10(6):2599–2605. doi: 10.1128/mcb.10.6.2599. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chen J., Moore C. Separation of factors required for cleavage and polyadenylation of yeast pre-mRNA. Mol Cell Biol. 1992 Aug;12(8):3470–3481. doi: 10.1128/mcb.12.8.3470. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Christofori G., Keller W. 3' cleavage and polyadenylation of mRNA precursors in vitro requires a poly(A) polymerase, a cleavage factor, and a snRNP. Cell. 1988 Sep 9;54(6):875–889. doi: 10.1016/s0092-8674(88)91263-9. [DOI] [PubMed] [Google Scholar]
- Christofori G., Keller W. Poly(A) polymerase purified from HeLa cell nuclear extract is required for both cleavage and polyadenylation of pre-mRNA in vitro. Mol Cell Biol. 1989 Jan;9(1):193–203. doi: 10.1128/mcb.9.1.193. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cullin C., Minvielle-Sebastia L. Multipurpose vectors designed for the fast generation of N- or C-terminal epitope-tagged proteins. Yeast. 1994 Jan;10(1):105–112. doi: 10.1002/yea.320100110. [DOI] [PubMed] [Google Scholar]
- Curtis D., Lehmann R., Zamore P. D. Translational regulation in development. Cell. 1995 Apr 21;81(2):171–178. doi: 10.1016/0092-8674(95)90325-9. [DOI] [PubMed] [Google Scholar]
- Fields S., Sternglanz R. The two-hybrid system: an assay for protein-protein interactions. Trends Genet. 1994 Aug;10(8):286–292. doi: 10.1016/0168-9525(90)90012-u. [DOI] [PubMed] [Google Scholar]
- Gallie D. R. The cap and poly(A) tail function synergistically to regulate mRNA translational efficiency. Genes Dev. 1991 Nov;5(11):2108–2116. doi: 10.1101/gad.5.11.2108. [DOI] [PubMed] [Google Scholar]
- Gietz R. D., Schiestl R. H., Willems A. R., Woods R. A. Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure. Yeast. 1995 Apr 15;11(4):355–360. doi: 10.1002/yea.320110408. [DOI] [PubMed] [Google Scholar]
- Gilmartin G. M., Nevins J. R. Molecular analyses of two poly(A) site-processing factors that determine the recognition and efficiency of cleavage of the pre-mRNA. Mol Cell Biol. 1991 May;11(5):2432–2438. doi: 10.1128/mcb.11.5.2432. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jackson R. J., Standart N. Do the poly(A) tail and 3' untranslated region control mRNA translation? Cell. 1990 Jul 13;62(1):15–24. doi: 10.1016/0092-8674(90)90235-7. [DOI] [PubMed] [Google Scholar]
- Keller W. No end yet to messenger RNA 3' processing! Cell. 1995 Jun 16;81(6):829–832. doi: 10.1016/0092-8674(95)90001-2. [DOI] [PubMed] [Google Scholar]
- Kuras L., Thomas D. Functional analysis of Met4, a yeast transcriptional activator responsive to S-adenosylmethionine. Mol Cell Biol. 1995 Jan;15(1):208–216. doi: 10.1128/mcb.15.1.208. [DOI] [PMC free article] [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]
- Manley J. L., Proudfoot N. J. RNA 3' ends: formation and function--meeting review. Genes Dev. 1994 Feb 1;8(3):259–264. doi: 10.1101/gad.8.3.259. [DOI] [PubMed] [Google Scholar]
- Maquat L. E. Nuclear mRNA export. Curr Opin Cell Biol. 1991 Dec;3(6):1004–1012. doi: 10.1016/0955-0674(91)90121-e. [DOI] [PubMed] [Google Scholar]
- Minvielle-Sebastia L., Preker P. J., Keller W. RNA14 and RNA15 proteins as components of a yeast pre-mRNA 3'-end processing factor. Science. 1994 Dec 9;266(5191):1702–1705. doi: 10.1126/science.7992054. [DOI] [PubMed] [Google Scholar]
- Minvielle-Sebastia L., Winsor B., Bonneaud N., Lacroute F. Mutations in the yeast RNA14 and RNA15 genes result in an abnormal mRNA decay rate; sequence analysis reveals an RNA-binding domain in the RNA15 protein. Mol Cell Biol. 1991 Jun;11(6):3075–3087. doi: 10.1128/mcb.11.6.3075. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mortimer R. K., Hawthorne D. C. Genetic mapping in Saccharomyces. Genetics. 1966 Jan;53(1):165–173. doi: 10.1093/genetics/53.1.165. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Muhlrad D., Hunter R., Parker R. A rapid method for localized mutagenesis of yeast genes. Yeast. 1992 Feb;8(2):79–82. doi: 10.1002/yea.320080202. [DOI] [PubMed] [Google Scholar]
- Patel D., Butler J. S. Conditional defect in mRNA 3' end processing caused by a mutation in the gene for poly(A) polymerase. Mol Cell Biol. 1992 Jul;12(7):3297–3304. doi: 10.1128/mcb.12.7.3297. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Preker P. J., Lingner J., Minvielle-Sebastia L., Keller W. The FIP1 gene encodes a component of a yeast pre-mRNA polyadenylation factor that directly interacts with poly(A) polymerase. Cell. 1995 May 5;81(3):379–389. doi: 10.1016/0092-8674(95)90391-7. [DOI] [PubMed] [Google Scholar]
- Proweller A., Butler S. Efficient translation of poly(A)-deficient mRNAs in Saccharomyces cerevisiae. Genes Dev. 1994 Nov 1;8(21):2629–2640. doi: 10.1101/gad.8.21.2629. [DOI] [PubMed] [Google Scholar]
- Sachs A. B., Deardorff J. A. Translation initiation requires the PAB-dependent poly(A) ribonuclease in yeast. Cell. 1992 Sep 18;70(6):961–973. doi: 10.1016/0092-8674(92)90246-9. [DOI] [PubMed] [Google Scholar]
- Sachs A. B. Messenger RNA degradation in eukaryotes. Cell. 1993 Aug 13;74(3):413–421. doi: 10.1016/0092-8674(93)80043-e. [DOI] [PubMed] [Google Scholar]
- Sachs A. The role of poly(A) in the translation and stability of mRNA. Curr Opin Cell Biol. 1990 Dec;2(6):1092–1098. doi: 10.1016/0955-0674(90)90161-7. [DOI] [PubMed] [Google Scholar]
- Takagaki Y., Manley J. L. A polyadenylation factor subunit is the human homologue of the Drosophila suppressor of forked protein. Nature. 1994 Dec 1;372(6505):471–474. doi: 10.1038/372471a0. [DOI] [PubMed] [Google Scholar]
- Takagaki Y., Manley J. L., MacDonald C. C., Wilusz J., Shenk T. A multisubunit factor, CstF, is required for polyadenylation of mammalian pre-mRNAs. Genes Dev. 1990 Dec;4(12A):2112–2120. doi: 10.1101/gad.4.12a.2112. [DOI] [PubMed] [Google Scholar]
- Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wahle E., Keller W. The biochemistry of polyadenylation. Trends Biochem Sci. 1996 Jul;21(7):247–250. [PubMed] [Google Scholar]
- Wickens M. How the messenger got its tail: addition of poly(A) in the nucleus. Trends Biochem Sci. 1990 Jul;15(7):277–281. doi: 10.1016/0968-0004(90)90054-f. [DOI] [PubMed] [Google Scholar]
- Winston F., Dollard C., Ricupero-Hovasse S. L. Construction of a set of convenient Saccharomyces cerevisiae strains that are isogenic to S288C. Yeast. 1995 Jan;11(1):53–55. doi: 10.1002/yea.320110107. [DOI] [PubMed] [Google Scholar]
- Zarkower D., Stephenson P., Sheets M., Wickens M. The AAUAAA sequence is required both for cleavage and for polyadenylation of simian virus 40 pre-mRNA in vitro. Mol Cell Biol. 1986 Jul;6(7):2317–2323. doi: 10.1128/mcb.6.7.2317. [DOI] [PMC free article] [PubMed] [Google Scholar]