Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1995 Mar;15(3):1689–1697. doi: 10.1128/mcb.15.3.1689

REF2 encodes an RNA-binding protein directly involved in yeast mRNA 3'-end formation.

R Russnak 1, K W Nehrke 1, T Platt 1
PMCID: PMC230393  PMID: 7862160

Abstract

The Saccharomyces cerevisiae mutant ref2-1 (REF = RNA end formation) was originally identified by a genetic strategy predicted to detect decreases in the use of a CYC1 poly(A) site interposed within the intron of an ACT1-HIS4 fusion reporter gene. Direct RNA analysis now proves this effect and also demonstrates the trans action of the REF2 gene product on cryptic poly(A) sites located within the coding region of a plasmid-borne ACT1-lacZ gene. Despite impaired growth of ref2 strains, possibly because of a general defect in the efficiency of mRNA 3'-end processing, the steady-state characteristics of a variety of normal cellular mRNAs remain unaffected. Sequencing of the complementing gene predicts the Ref2p product to be a novel, basic protein of 429 amino acids (M(r), 48,000) with a high-level lysine/serine content and some unusual features. Analysis in vitro, with a number of defined RNA substrates, confirms that efficient use of weak poly(A) sites requires Ref2p: endonucleolytic cleavage is carried out accurately but at significantly lower rates in extracts prepared from delta ref2 cells. The addition of purified, epitope-tagged Ref2p (Ref2pF) reestablishes wild-type levels of activity in these extracts, demonstrating direct involvement of this protein in the cleavage step of 3' mRNA processing. Together with the RNA-binding characteristics of Ref2pF in vitro, our results support an important contributing role for the REF2 locus in 3'-end processing. As the first gene genetically identified to participate in mRNA 3'-end maturation prior to the final polyadenylation step, REF2 provides an ideal starting point for identifying related genes in this event.

Full Text

The Full Text of this article is available as a PDF (381.5 KB).

Selected References

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

  1. Ahmed Y. F., Gilmartin G. M., Hanly S. M., Nevins J. R., Greene W. C. The HTLV-I Rex response element mediates a novel form of mRNA polyadenylation. Cell. 1991 Feb 22;64(4):727–737. doi: 10.1016/0092-8674(91)90502-p. [DOI] [PubMed] [Google Scholar]
  2. Bardwell V. J., Wickens M., Bienroth S., Keller W., Sproat B. S., Lamond A. I. Site-directed ribose methylation identifies 2'-OH groups in polyadenylation substrates critical for AAUAAA recognition and poly(A) addition. Cell. 1991 Apr 5;65(1):125–133. doi: 10.1016/0092-8674(91)90414-t. [DOI] [PubMed] [Google Scholar]
  3. Boelens W. C., Jansen E. J., van Venrooij W. J., Stripecke R., Mattaj I. W., Gunderson S. I. The human U1 snRNP-specific U1A protein inhibits polyadenylation of its own pre-mRNA. Cell. 1993 Mar 26;72(6):881–892. doi: 10.1016/0092-8674(93)90577-d. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. 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]
  7. Chiang C. M., Roeder R. G. Expression and purification of general transcription factors by FLAG epitope-tagging and peptide elution. Pept Res. 1993 Mar-Apr;6(2):62–64. [PubMed] [Google Scholar]
  8. Chien C. T., Bartel P. L., Sternglanz R., Fields S. The two-hybrid system: a method to identify and clone genes for proteins that interact with a protein of interest. Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9578–9582. doi: 10.1073/pnas.88.21.9578. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Edwalds-Gilbert G., Prescott J., Falck-Pedersen E. 3' RNA processing efficiency plays a primary role in generating termination-competent RNA polymerase II elongation complexes. Mol Cell Biol. 1993 Jun;13(6):3472–3480. doi: 10.1128/mcb.13.6.3472. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Elder R. T., Loh E. Y., Davis R. W. RNA from the yeast transposable element Ty1 has both ends in the direct repeats, a structure similar to retrovirus RNA. Proc Natl Acad Sci U S A. 1983 May;80(9):2432–2436. doi: 10.1073/pnas.80.9.2432. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gietz R. D., Sugino A. New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene. 1988 Dec 30;74(2):527–534. doi: 10.1016/0378-1119(88)90185-0. [DOI] [PubMed] [Google Scholar]
  12. Gilmartin G. M., Fleming E. S., Oetjen J. Activation of HIV-1 pre-mRNA 3' processing in vitro requires both an upstream element and TAR. EMBO J. 1992 Dec;11(12):4419–4428. doi: 10.1002/j.1460-2075.1992.tb05542.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. Gunderson S. I., Beyer K., Martin G., Keller W., Boelens W. C., Mattaj L. W. The human U1A snRNP protein regulates polyadenylation via a direct interaction with poly(A) polymerase. Cell. 1994 Feb 11;76(3):531–541. doi: 10.1016/0092-8674(94)90116-3. [DOI] [PubMed] [Google Scholar]
  16. Hereford L. M., Osley M. A., Ludwig T. R., 2nd, McLaughlin C. S. Cell-cycle regulation of yeast histone mRNA. Cell. 1981 May;24(2):367–375. doi: 10.1016/0092-8674(81)90326-3. [DOI] [PubMed] [Google Scholar]
  17. Hou W., Russnak R., Platt T. Poly(A) site selection in the yeast Ty retroelement requires an upstream region and sequence-specific titratable factor(s) in vitro. EMBO J. 1994 Jan 15;13(2):446–452. doi: 10.1002/j.1460-2075.1994.tb06279.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Irniger S., Braus G. H. Saturation mutagenesis of a polyadenylation signal reveals a hexanucleotide element essential for mRNA 3' end formation in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1994 Jan 4;91(1):257–261. doi: 10.1073/pnas.91.1.257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Irniger S., Egli C. M., Braus G. H. Different classes of polyadenylation sites in the yeast Saccharomyces cerevisiae. Mol Cell Biol. 1991 Jun;11(6):3060–3069. doi: 10.1128/mcb.11.6.3060. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Larson G. P., Itakura K., Ito H., Rossi J. J. Saccharomyces cerevisiae actin--Escherichia coli lacZ gene fusions: synthetic-oligonucleotide-mediated deletion of the 309 base pair intervening sequence in the actin gene. Gene. 1983 Apr;22(1):31–39. doi: 10.1016/0378-1119(83)90061-6. [DOI] [PubMed] [Google Scholar]
  23. Liao X. C., Tang J., Rosbash M. An enhancer screen identifies a gene that encodes the yeast U1 snRNP A protein: implications for snRNP protein function in pre-mRNA splicing. Genes Dev. 1993 Mar;7(3):419–428. doi: 10.1101/gad.7.3.419. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Lutz C. S., Alwine J. C. Direct interaction of the U1 snRNP-A protein with the upstream efficiency element of the SV40 late polyadenylation signal. Genes Dev. 1994 Mar 1;8(5):576–586. doi: 10.1101/gad.8.5.576. [DOI] [PubMed] [Google Scholar]
  26. Mayer S. A., Dieckmann C. L. The yeast CBP1 gene produces two differentially regulated transcripts by alternative 3'-end formation. Mol Cell Biol. 1989 Oct;9(10):4161–4169. doi: 10.1128/mcb.9.10.4161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Parker R., Guthrie C. A point mutation in the conserved hexanucleotide at a yeast 5' splice junction uncouples recognition, cleavage, and ligation. Cell. 1985 May;41(1):107–118. doi: 10.1016/0092-8674(85)90065-0. [DOI] [PubMed] [Google Scholar]
  28. Passmore S., Maine G. T., Elble R., Christ C., Tye B. K. Saccharomyces cerevisiae protein involved in plasmid maintenance is necessary for mating of MAT alpha cells. J Mol Biol. 1988 Dec 5;204(3):593–606. doi: 10.1016/0022-2836(88)90358-0. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Philippsen P., Stotz A., Scherf C. DNA of Saccharomyces cerevisiae. Methods Enzymol. 1991;194:169–182. doi: 10.1016/0076-6879(91)94014-4. [DOI] [PubMed] [Google Scholar]
  31. Qian Z. W., Wilusz J. An RNA-binding protein specifically interacts with a functionally important domain of the downstream element of the simian virus 40 late polyadenylation signal. Mol Cell Biol. 1991 Oct;11(10):5312–5320. doi: 10.1128/mcb.11.10.5312. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Raabe T., Bollum F. J., Manley J. L. Primary structure and expression of bovine poly(A) polymerase. Nature. 1991 Sep 19;353(6341):229–234. doi: 10.1038/353229a0. [DOI] [PubMed] [Google Scholar]
  33. Robinson M. K., van Zyl W. H., Phizicky E. M., Broach J. R. TPD1 of Saccharomyces cerevisiae encodes a protein phosphatase 2C-like activity implicated in tRNA splicing and cell separation. Mol Cell Biol. 1994 Jun;14(6):3634–3645. doi: 10.1128/mcb.14.6.3634. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Rose M. D., Novick P., Thomas J. H., Botstein D., Fink G. R. A Saccharomyces cerevisiae genomic plasmid bank based on a centromere-containing shuttle vector. Gene. 1987;60(2-3):237–243. doi: 10.1016/0378-1119(87)90232-0. [DOI] [PubMed] [Google Scholar]
  35. 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]
  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., Li W. Z., Guo Z., Sherman F. Signals that produce 3' termini in CYC1 mRNA of the yeast Saccharomyces cerevisiae. Mol Cell Biol. 1993 Dec;13(12):7836–7849. doi: 10.1128/mcb.13.12.7836. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Russo P., Li W. Z., Hampsey D. M., Zaret K. S., Sherman F. Distinct cis-acting signals enhance 3' endpoint formation of CYC1 mRNA in the yeast Saccharomyces cerevisiae. EMBO J. 1991 Mar;10(3):563–571. doi: 10.1002/j.1460-2075.1991.tb07983.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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]
  40. Sachs A. B., Davis R. W. The poly(A) binding protein is required for poly(A) shortening and 60S ribosomal subunit-dependent translation initiation. Cell. 1989 Sep 8;58(5):857–867. doi: 10.1016/0092-8674(89)90938-0. [DOI] [PubMed] [Google Scholar]
  41. 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]
  42. Steinmetz E. J., Brennan C. A., Platt T. A short intervening structure can block rho factor helicase action at a distance. J Biol Chem. 1990 Oct 25;265(30):18408–18413. [PubMed] [Google Scholar]
  43. Takagaki Y., MacDonald C. C., Shenk T., Manley J. L. The human 64-kDa polyadenylylation factor contains a ribonucleoprotein-type RNA binding domain and unusual auxiliary motifs. Proc Natl Acad Sci U S A. 1992 Feb 15;89(4):1403–1407. doi: 10.1073/pnas.89.4.1403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Takagaki Y., Manley J. L. A human polyadenylation factor is a G protein beta-subunit homologue. J Biol Chem. 1992 Nov 25;267(33):23471–23474. [PubMed] [Google Scholar]
  45. 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]
  46. Wahle E., Martin G., Schiltz E., Keller W. Isolation and expression of cDNA clones encoding mammalian poly(A) polymerase. EMBO J. 1991 Dec;10(13):4251–4257. doi: 10.1002/j.1460-2075.1991.tb05003.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. 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