Skip to main content
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1995 Dec;15(12):6979–6986. doi: 10.1128/mcb.15.12.6979

Effects of mutations in the Saccharomyces cerevisiae RNA14, RNA15, and PAP1 genes on polyadenylation in vivo.

E Mandart 1, R Parker 1
PMCID: PMC230953  PMID: 8524265

Abstract

The RNA14 and RNA15 gene products have been implicated in a variety of cellular processes. Mutations in these genes lead to faster decay of some mRNAs and yield extracts that are deficient in cleavage and polyadenylation in vitro. These results suggest that the RNA14 and RNA15 gene products may be involved in both adenylation and deadenylation in vivo. To explore the roles of these gene products in vivo, we examined the site of adenylation and the rate of deadenylation for individual mRNAs in rna14 and rna15 mutant strains. We observed that the rates of deadenylation are not affected by lesions in either the RNA14 or the RNA15 gene. This result suggests that the proteins encoded by these genes are not involved in regulation of the deadenylation rate. In contrast, we observed that the site of adenylation for the ACT1 transcript can be altered in these mutants. Interestingly, we also observed that mutation of the poly(A) polymerase gene altered the site of ACT1 polyadenylation. These observations suggest that the RNA14, RNA15, and PAP1 proteins are involved in poly(A) site choice. This alteration in poly(A) site choice in the rna14 mutant can be corrected by the ssm4 suppressor, indicating that this suppression acts at the level of polyadenylation and not by slowing mRNA degradation.

Full Text

The Full Text of this article is available as a PDF (1,013.7 KB).

Selected References

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

  1. Beelman C. A., Parker R. Degradation of mRNA in eukaryotes. Cell. 1995 Apr 21;81(2):179–183. doi: 10.1016/0092-8674(95)90326-7. [DOI] [PubMed] [Google Scholar]
  2. Beelman C. A., Parker R. Differential effects of translational inhibition in cis and in trans on the decay of the unstable yeast MFA2 mRNA. J Biol Chem. 1994 Apr 1;269(13):9687–9692. [PubMed] [Google Scholar]
  3. Bienroth S., Keller W., Wahle E. Assembly of a processive messenger RNA polyadenylation complex. EMBO J. 1993 Feb;12(2):585–594. doi: 10.1002/j.1460-2075.1993.tb05690.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bonneaud N., Minvielle-Sebastia L., Cullin C., Lacroute F. Cellular localization of RNA14p and RNA15p, two yeast proteins involved in mRNA stability. J Cell Sci. 1994 Apr;107(Pt 4):913–921. doi: 10.1242/jcs.107.4.913. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Caponigro G., Muhlrad D., Parker R. A small segment of the MAT alpha 1 transcript promotes mRNA decay in Saccharomyces cerevisiae: a stimulatory role for rare codons. Mol Cell Biol. 1993 Sep;13(9):5141–5148. doi: 10.1128/mcb.13.9.5141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Decker C. J., Parker R. A turnover pathway for both stable and unstable mRNAs in yeast: evidence for a requirement for deadenylation. Genes Dev. 1993 Aug;7(8):1632–1643. doi: 10.1101/gad.7.8.1632. [DOI] [PubMed] [Google Scholar]
  9. Decker C. J., Parker R. Mechanisms of mRNA degradation in eukaryotes. Trends Biochem Sci. 1994 Aug;19(8):336–340. doi: 10.1016/0968-0004(94)90073-6. [DOI] [PubMed] [Google Scholar]
  10. Felici F., Cesareni G., Hughes J. M. The most abundant small cytoplasmic RNA of Saccharomyces cerevisiae has an important function required for normal cell growth. Mol Cell Biol. 1989 Aug;9(8):3260–3268. doi: 10.1128/mcb.9.8.3260. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Gallwitz D., Perrin F., Seidel R. The actin gene in yeast Saccharomyces cerevisiae: 5' and 3' end mapping, flanking and putative regulatory sequences. Nucleic Acids Res. 1981 Dec 11;9(23):6339–6350. doi: 10.1093/nar/9.23.6339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gietz D., St Jean A., Woods R. A., Schiestl R. H. Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res. 1992 Mar 25;20(6):1425–1425. doi: 10.1093/nar/20.6.1425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Heaton B., Decker C., Muhlrad D., Donahue J., Jacobson A., Parker R. Analysis of chimeric mRNAs derived from the STE3 mRNA identifies multiple regions within yeast mRNAs that modulate mRNA decay. Nucleic Acids Res. 1992 Oct 25;20(20):5365–5373. doi: 10.1093/nar/20.20.5365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Heidmann S., Obermaier B., Vogel K., Domdey H. Identification of pre-mRNA polyadenylation sites in Saccharomyces cerevisiae. Mol Cell Biol. 1992 Sep;12(9):4215–4229. doi: 10.1128/mcb.12.9.4215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hyman L. E., Moore C. L. Termination and pausing of RNA polymerase II downstream of yeast polyadenylation sites. Mol Cell Biol. 1993 Sep;13(9):5159–5167. doi: 10.1128/mcb.13.9.5159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Keller W., Bienroth S., Lang K. M., Christofori G. Cleavage and polyadenylation factor CPF specifically interacts with the pre-mRNA 3' processing signal AAUAAA. EMBO J. 1991 Dec;10(13):4241–4249. doi: 10.1002/j.1460-2075.1991.tb05002.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Kessler M. M., Zhelkovsky A. M., Skvorak A., Moore C. L. Monoclonal antibodies to yeast poly(A) polymerase (PAP) provide evidence for association of PAP with cleavage factor I. Biochemistry. 1995 Feb 7;34(5):1750–1759. doi: 10.1021/bi00005a032. [DOI] [PubMed] [Google Scholar]
  21. Lingner J., Kellermann J., Keller W. Cloning and expression of the essential gene for poly(A) polymerase from S. cerevisiae. Nature. 1991 Dec 12;354(6353):496–498. doi: 10.1038/354496a0. [DOI] [PubMed] [Google Scholar]
  22. Lingner J., Radtke I., Wahle E., Keller W. Purification and characterization of poly(A) polymerase from Saccharomyces cerevisiae. J Biol Chem. 1991 May 15;266(14):8741–8746. [PubMed] [Google Scholar]
  23. MacDonald C. C., Wilusz J., Shenk T. The 64-kilodalton subunit of the CstF polyadenylation factor binds to pre-mRNAs downstream of the cleavage site and influences cleavage site location. Mol Cell Biol. 1994 Oct;14(10):6647–6654. doi: 10.1128/mcb.14.10.6647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Mandart E., Dufour M. E., Lacroute F. Inactivation of SSM4, a new Saccharomyces cerevisiae gene, suppresses mRNA instability due to rna14 mutations. Mol Gen Genet. 1994 Nov 1;245(3):323–333. doi: 10.1007/BF00290112. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. 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]
  27. 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]
  28. Mitchelson A., Simonelig M., Williams C., O'Hare K. Homology with Saccharomyces cerevisiae RNA14 suggests that phenotypic suppression in Drosophila melanogaster by suppressor of forked occurs at the level of RNA stability. Genes Dev. 1993 Feb;7(2):241–249. doi: 10.1101/gad.7.2.241. [DOI] [PubMed] [Google Scholar]
  29. Muhlrad D., Decker C. J., Parker R. Deadenylation of the unstable mRNA encoded by the yeast MFA2 gene leads to decapping followed by 5'-->3' digestion of the transcript. Genes Dev. 1994 Apr 1;8(7):855–866. doi: 10.1101/gad.8.7.855. [DOI] [PubMed] [Google Scholar]
  30. Muhlrad D., Decker C. J., Parker R. Turnover mechanisms of the stable yeast PGK1 mRNA. Mol Cell Biol. 1995 Apr;15(4):2145–2156. doi: 10.1128/mcb.15.4.2145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Muhlrad D., Parker R. Mutations affecting stability and deadenylation of the yeast MFA2 transcript. Genes Dev. 1992 Nov;6(11):2100–2111. doi: 10.1101/gad.6.11.2100. [DOI] [PubMed] [Google Scholar]
  32. Munroe D., Jacobson A. mRNA poly(A) tail, a 3' enhancer of translational initiation. Mol Cell Biol. 1990 Jul;10(7):3441–3455. doi: 10.1128/mcb.10.7.3441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Nonet M., Scafe C., Sexton J., Young R. Eucaryotic RNA polymerase conditional mutant that rapidly ceases mRNA synthesis. Mol Cell Biol. 1987 May;7(5):1602–1611. doi: 10.1128/mcb.7.5.1602. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Piñol-Roma S., Dreyfuss G. Shuttling of pre-mRNA binding proteins between nucleus and cytoplasm. Nature. 1992 Feb 20;355(6362):730–732. doi: 10.1038/355730a0. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. 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]
  37. Russnak R., Nehrke K. W., Platt T. REF2 encodes an RNA-binding protein directly involved in yeast mRNA 3'-end formation. Mol Cell Biol. 1995 Mar;15(3):1689–1697. doi: 10.1128/mcb.15.3.1689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sachs A., Wahle E. Poly(A) tail metabolism and function in eucaryotes. J Biol Chem. 1993 Nov 5;268(31):22955–22958. [PubMed] [Google Scholar]
  39. 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]
  40. Wahle E., Keller W. The biochemistry of 3'-end cleavage and polyadenylation of messenger RNA precursors. Annu Rev Biochem. 1992;61:419–440. doi: 10.1146/annurev.bi.61.070192.002223. [DOI] [PubMed] [Google Scholar]
  41. Weiss E. A., Gilmartin G. M., Nevins J. R. Poly(A) site efficiency reflects the stability of complex formation involving the downstream element. EMBO J. 1991 Jan;10(1):215–219. doi: 10.1002/j.1460-2075.1991.tb07938.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES