Skip to main content
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1991 Apr;11(4):2004–2012. doi: 10.1128/mcb.11.4.2004

Point mutations upstream of the yeast ADH2 poly(A) site significantly reduce the efficiency of 3'-end formation.

L E Hyman 1, S H Seiler 1, J Whoriskey 1, C L Moore 1
PMCID: PMC359886  PMID: 2005893

Abstract

The sequences directing formation of mRNA 3' ends in Saccharomyces cerevisiae are not well defined. This is in contrast to the situation in higher eukaryotes in which the sequence AAUAAA is known to be crucial to proper 3'-end formation. The AAUAAA hexanucleotide is found upstream of the poly(A) site in some but not all yeast genes. One of these is the gene coding for alcohol dehydrogenase, ADH2. Deletion or a double point mutation of the AAUAAA has only a small effect on the efficiency of the reaction, and in contrast to the mammalian system, it is most likely not operating as a major processing signal in the yeast cell. However, we isolated point mutations which reveal that a region located approximately 80 nucleotides upstream of the poly(A) site plays a critical role in either transcription termination, polyadenylation, or both. These mutations represent the first point mutations in yeasts which significantly reduce the efficiency of 3'-end formation.

Full text

PDF
2012

Images in this article

Selected References

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

  1. Abe A., Hiraoka Y., Fukasawa T. Signal sequence for generation of mRNA 3' end in the Saccharomyces cerevisiae GAL7 gene. EMBO J. 1990 Nov;9(11):3691–3697. doi: 10.1002/j.1460-2075.1990.tb07581.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Adami G., Nevins J. R. Splice site selection dominates over poly(A) site choice in RNA production from complex adenovirus transcription units. EMBO J. 1988 Jul;7(7):2107–2116. doi: 10.1002/j.1460-2075.1988.tb03050.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Birchmeier C., Folk W., Birnstiel M. L. The terminal RNA stem-loop structure and 80 bp of spacer DNA are required for the formation of 3' termini of sea urchin H2A mRNA. Cell. 1983 Dec;35(2 Pt 1):433–440. doi: 10.1016/0092-8674(83)90176-9. [DOI] [PubMed] [Google Scholar]
  4. Brady H. A., Wold W. S. Competition between splicing and polyadenylation reactions determines which adenovirus region E3 mRNAs are synthesized. Mol Cell Biol. 1988 Aug;8(8):3291–3297. doi: 10.1128/mcb.8.8.3291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. 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]
  7. 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]
  8. Carswell S., Alwine J. C. Efficiency of utilization of the simian virus 40 late polyadenylation site: effects of upstream sequences. Mol Cell Biol. 1989 Oct;9(10):4248–4258. doi: 10.1128/mcb.9.10.4248. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. 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]
  11. Chu C. T., Parris D. S., Dixon R. A., Farber F. E., Schaffer P. A. Hydroxylamine mutagenesis of HSV DNA and DNA fragments: introduction of mutations into selected regions of the viral genome. Virology. 1979 Oct 15;98(1):168–181. doi: 10.1016/0042-6822(79)90535-x. [DOI] [PubMed] [Google Scholar]
  12. Couto J. R., Tamm J., Parker R., Guthrie C. A trans-acting suppressor restores splicing of a yeast intron with a branch point mutation. Genes Dev. 1987 Jul;1(5):445–455. doi: 10.1101/gad.1.5.445. [DOI] [PubMed] [Google Scholar]
  13. DeZazzo J. D., Imperiale M. J. Sequences upstream of AAUAAA influence poly(A) site selection in a complex transcription unit. Mol Cell Biol. 1989 Nov;9(11):4951–4961. doi: 10.1128/mcb.9.11.4951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Falck-Pedersen E., Logan J., Shenk T., Darnell J. E., Jr Transcription termination within the E1A gene of adenovirus induced by insertion of the mouse beta-major globin terminator element. Cell. 1985 Apr;40(4):897–905. doi: 10.1016/0092-8674(85)90349-6. [DOI] [PubMed] [Google Scholar]
  16. Gilmartin G. M., McDevitt M. A., Nevins J. R. Multiple factors are required for specific RNA cleavage at a poly(A) addition site. Genes Dev. 1988 May;2(5):578–587. doi: 10.1101/gad.2.5.578. [DOI] [PubMed] [Google Scholar]
  17. Guarente L. UASs and enhancers: common mechanism of transcriptional activation in yeast and mammals. Cell. 1988 Feb 12;52(3):303–305. doi: 10.1016/s0092-8674(88)80020-5. [DOI] [PubMed] [Google Scholar]
  18. Haggren W., Kolodrubetz D. The Saccharomyces cerevisiae ACP2 gene encodes an essential HMG1-like protein. Mol Cell Biol. 1988 Mar;8(3):1282–1289. doi: 10.1128/mcb.8.3.1282. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Halfter H., Gallwitz D. Impairment of yeast pre-mRNA splicing by potential secondary structure-forming sequences near the conserved branchpoint sequence. Nucleic Acids Res. 1988 Nov 25;16(22):10413–10423. doi: 10.1093/nar/16.22.10413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hart R. P., McDevitt M. A., Ali H., Nevins J. R. Definition of essential sequences and functional equivalence of elements downstream of the adenovirus E2A and the early simian virus 40 polyadenylation sites. Mol Cell Biol. 1985 Nov;5(11):2975–2983. doi: 10.1128/mcb.5.11.2975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Henikoff S., Cohen E. H. Sequences responsible for transcription termination on a gene segment in Saccharomyces cerevisiae. Mol Cell Biol. 1984 Aug;4(8):1515–1520. doi: 10.1128/mcb.4.8.1515. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hunt A. G., MacDonald M. H. Deletion analysis of the polyadenylation signal of a pea ribulose-1,5-bisphosphate carboxylase small-subunit gene. Plant Mol Biol. 1989 Aug;13(2):125–138. doi: 10.1007/BF00016132. [DOI] [PubMed] [Google Scholar]
  23. Joshi C. P. Putative polyadenylation signals in nuclear genes of higher plants: a compilation and analysis. Nucleic Acids Res. 1987 Dec 10;15(23):9627–9640. doi: 10.1093/nar/15.23.9627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Klinz F. J., Gallwitz D. Size and position of intervening sequences are critical for the splicing efficiency of pre-mRNA in the yeast Saccharomyces cerevisiae. Nucleic Acids Res. 1985 Jun 11;13(11):3791–3804. doi: 10.1093/nar/13.11.3791. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Levitt N., Briggs D., Gil A., Proudfoot N. J. Definition of an efficient synthetic poly(A) site. Genes Dev. 1989 Jul;3(7):1019–1025. doi: 10.1101/gad.3.7.1019. [DOI] [PubMed] [Google Scholar]
  26. Liu Y. X., Dieckmann C. L. Overproduction of yeast viruslike particles by strains deficient in a mitochondrial nuclease. Mol Cell Biol. 1989 Aug;9(8):3323–3331. doi: 10.1128/mcb.9.8.3323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Manley J. L. Polyadenylation of mRNA precursors. Biochim Biophys Acta. 1988 May 6;950(1):1–12. doi: 10.1016/0167-4781(88)90067-x. [DOI] [PubMed] [Google Scholar]
  28. McDevitt M. A., Gilmartin G. M., Reeves W. H., Nevins J. R. Multiple factors are required for poly(A) addition to a mRNA 3' end. Genes Dev. 1988 May;2(5):588–597. doi: 10.1101/gad.2.5.588. [DOI] [PubMed] [Google Scholar]
  29. Mogen B. D., MacDonald M. H., Graybosch R., Hunt A. G. Upstream sequences other than AAUAAA are required for efficient messenger RNA 3'-end formation in plants. Plant Cell. 1990 Dec;2(12):1261–1272. doi: 10.1105/tpc.2.12.1261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Osborne B. I., Guarente L. Mutational analysis of a yeast transcriptional terminator. Proc Natl Acad Sci U S A. 1989 Jun;86(11):4097–4101. doi: 10.1073/pnas.86.11.4097. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Pikielny C. W., Teem J. L., Rosbash M. Evidence for the biochemical role of an internal sequence in yeast nuclear mRNA introns: implications for U1 RNA and metazoan mRNA splicing. Cell. 1983 Sep;34(2):395–403. doi: 10.1016/0092-8674(83)90373-2. [DOI] [PubMed] [Google Scholar]
  32. Platt T. Transcription termination and the regulation of gene expression. Annu Rev Biochem. 1986;55:339–372. doi: 10.1146/annurev.bi.55.070186.002011. [DOI] [PubMed] [Google Scholar]
  33. Proudfoot N. J. How RNA polymerase II terminates transcription in higher eukaryotes. Trends Biochem Sci. 1989 Mar;14(3):105–110. doi: 10.1016/0968-0004(89)90132-1. [DOI] [PubMed] [Google Scholar]
  34. Ruohola H., Baker S. M., Parker R., Platt T. Orientation-dependent function of a short CYC1 DNA fragment in directing mRNA 3' end formation in yeast. Proc Natl Acad Sci U S A. 1988 Jul;85(14):5041–5045. doi: 10.1073/pnas.85.14.5041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Russell D. W., Smith M., Williamson V. M., Young E. T. Nucleotide sequence of the yeast alcohol dehydrogenase II gene. J Biol Chem. 1983 Feb 25;258(4):2674–2682. [PubMed] [Google Scholar]
  36. Russnak R., Ganem D. Sequences 5' to the polyadenylation signal mediate differential poly(A) site use in hepatitis B viruses. Genes Dev. 1990 May;4(5):764–776. doi: 10.1101/gad.4.5.764. [DOI] [PubMed] [Google Scholar]
  37. Russo P., Sherman F. Transcription terminates near the poly(A) site in the CYC1 gene of the yeast Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1989 Nov;86(21):8348–8352. doi: 10.1073/pnas.86.21.8348. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Snyder M., Sapolsky R. J., Davis R. W. Transcription interferes with elements important for chromosome maintenance in Saccharomyces cerevisiae. Mol Cell Biol. 1988 May;8(5):2184–2194. doi: 10.1128/mcb.8.5.2184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sutton A., Broach J. R. Signals for transcription initiation and termination in the Saccharomyces cerevisiae plasmid 2 micron circle. Mol Cell Biol. 1985 Oct;5(10):2770–2780. doi: 10.1128/mcb.5.10.2770. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Takagaki Y., Ryner L. C., Manley J. L. Separation and characterization of a poly(A) polymerase and a cleavage/specificity factor required for pre-mRNA polyadenylation. Cell. 1988 Mar 11;52(5):731–742. doi: 10.1016/0092-8674(88)90411-4. [DOI] [PubMed] [Google Scholar]
  41. Teem J. L., Rosbash M. Expression of a beta-galactosidase gene containing the ribosomal protein 51 intron is sensitive to the rna2 mutation of yeast. Proc Natl Acad Sci U S A. 1983 Jul;80(14):4403–4407. doi: 10.1073/pnas.80.14.4403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Whitelaw E., Proudfoot N. Alpha-thalassaemia caused by a poly(A) site mutation reveals that transcriptional termination is linked to 3' end processing in the human alpha 2 globin gene. EMBO J. 1986 Nov;5(11):2915–2922. doi: 10.1002/j.1460-2075.1986.tb04587.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Wickens M., Stephenson P. Role of the conserved AAUAAA sequence: four AAUAAA point mutants prevent messenger RNA 3' end formation. Science. 1984 Nov 30;226(4678):1045–1051. doi: 10.1126/science.6208611. [DOI] [PubMed] [Google Scholar]
  44. Woolford J. L., Jr Nuclear pre-mRNA splicing in yeast. Yeast. 1989 Nov-Dec;5(6):439–457. doi: 10.1002/yea.320050604. [DOI] [PubMed] [Google Scholar]
  45. Yarger J. G., Armilei G., Gorman M. C. Transcription terminator-like element within a Saccharomyces cerevisiae promoter region. Mol Cell Biol. 1986 Apr;6(4):1095–1101. doi: 10.1128/mcb.6.4.1095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Yu K., Elder R. T. Some of the signals for 3'-end formation in transcription of the Saccharomyces cerevisiae Ty-D15 element are immediately downstream of the initiation site. Mol Cell Biol. 1989 Jun;9(6):2431–2444. doi: 10.1128/mcb.9.6.2431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Zaret K. S., Sherman F. DNA sequence required for efficient transcription termination in yeast. Cell. 1982 Mar;28(3):563–573. doi: 10.1016/0092-8674(82)90211-2. [DOI] [PubMed] [Google Scholar]
  48. Zuker M., Stiegler P. Optimal computer folding of large RNA sequences using thermodynamics and auxiliary information. Nucleic Acids Res. 1981 Jan 10;9(1):133–148. doi: 10.1093/nar/9.1.133. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES