Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1995 Apr;15(4):2145–2156. doi: 10.1128/mcb.15.4.2145

Turnover mechanisms of the stable yeast PGK1 mRNA.

D Muhlrad 1, C J Decker 1, R Parker 1
PMCID: PMC230442  PMID: 7891709

Abstract

The first step in the decay of several yeast mRNAs is the shortening of the poly(A) tail, which for the MFA2 transcript triggers decapping and 5'-to-3' degradation. To understand the basis for differences in mRNA decay rates, it is important to determine if deadenylation-dependent decapping is specific to the unstable MFA2 transcript or is a general mechanism of mRNA degradation. To this end, we analyzed the turnover of the stable PGK1 mRNA by monitoring the decay of a pulse of newly synthesized transcripts while using two strategies to trap decay intermediates. First, we used strains deleted for the XRN1 gene, which encodes a major 5'-to-3' exonuclease in Saccharomyces cerevisiae. In xrn1 delta cells, PGK1 transcripts lacking the 5' cap structure and a few nucleotides at the 5' end were detected after deadenylation. Second, we inserted into the PGK1 5' untranslated region strong RNA secondary structures, which can slow exonucleolytic digestion and thereby trap decay intermediates. These secondary structures led to the accumulation of PGK1 mRNA fragments, following deadenylation, trimmed from the 5' end to the site of the secondary structure. The insertion of strong secondary structures into the 5' untranslated region also inhibited translation of the mRNA and greatly stimulated the decay of the PGK1 transcripts, suggesting that translation of the PGK1 mRNA is required for its normally slow rate of decay. These results suggest that one mechanism of degradation of the PGK1 transcript is deadenylation followed by decapping and subsequent 5'-to-3' exonucleolytic degradation. In addition, by blocking the 5'-to-3' degradation process, we observed PGK1 mRNA fragments that are consistent with a 3'-to-5' pathway of mRNA turnover that is slightly slower than the decapping/5'-to-3' decay pathway. These observations indicate that there are multiple mechanisms by which an individual transcript can be degraded following deadenylation.

Full Text

The Full Text of this article is available as a PDF (684.0 KB).

Selected References

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

  1. Aharon T., Schneider R. J. Selective destabilization of short-lived mRNAs with the granulocyte-macrophage colony-stimulating factor AU-rich 3' noncoding region is mediated by a cotranslational mechanism. Mol Cell Biol. 1993 Mar;13(3):1971–1980. doi: 10.1128/mcb.13.3.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Amberg D. C., Goldstein A. L., Cole C. N. Isolation and characterization of RAT1: an essential gene of Saccharomyces cerevisiae required for the efficient nucleocytoplasmic trafficking of mRNA. Genes Dev. 1992 Jul;6(7):1173–1189. doi: 10.1101/gad.6.7.1173. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Brewer G., Ross J. Poly(A) shortening and degradation of the 3' A+U-rich sequences of human c-myc mRNA in a cell-free system. Mol Cell Biol. 1988 Apr;8(4):1697–1708. doi: 10.1128/mcb.8.4.1697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Christensen A. K., Kahn L. E., Bourne C. M. Circular polysomes predominate on the rough endoplasmic reticulum of somatotropes and mammotropes in the rat anterior pituitary. Am J Anat. 1987 Jan;178(1):1–10. doi: 10.1002/aja.1001780102. [DOI] [PubMed] [Google Scholar]
  7. Cigan A. M., Pabich E. K., Donahue T. F. Mutational analysis of the HIS4 translational initiator region in Saccharomyces cerevisiae. Mol Cell Biol. 1988 Jul;8(7):2964–2975. doi: 10.1128/mcb.8.7.2964. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cleveland D. W. Gene regulation through messenger RNA stability. Curr Opin Cell Biol. 1989 Dec;1(6):1148–1153. doi: 10.1016/s0955-0674(89)80065-1. [DOI] [PubMed] [Google Scholar]
  9. Coutts M., Brawerman G. A 5' exoribonuclease from cytoplasmic extracts of mouse sarcoma 180 ascites cells. Biochim Biophys Acta. 1993 Apr 29;1173(1):57–62. doi: 10.1016/0167-4781(93)90242-6. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. 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]
  13. 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]
  14. Hsu C. L., Stevens A. Yeast cells lacking 5'-->3' exoribonuclease 1 contain mRNA species that are poly(A) deficient and partially lack the 5' cap structure. Mol Cell Biol. 1993 Aug;13(8):4826–4835. doi: 10.1128/mcb.13.8.4826. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Kenna M., Stevens A., McCammon M., Douglas M. G. An essential yeast gene with homology to the exonuclease-encoding XRN1/KEM1 gene also encodes a protein with exoribonuclease activity. Mol Cell Biol. 1993 Jan;13(1):341–350. doi: 10.1128/mcb.13.1.341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kozak M. Structural features in eukaryotic mRNAs that modulate the initiation of translation. J Biol Chem. 1991 Oct 25;266(30):19867–19870. [PubMed] [Google Scholar]
  18. Laird-Offringa I. A., de Wit C. L., Elfferich P., van der Eb A. J. Poly(A) tail shortening is the translation-dependent step in c-myc mRNA degradation. Mol Cell Biol. 1990 Dec;10(12):6132–6140. doi: 10.1128/mcb.10.12.6132. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Larimer F. W., Stevens A. Disruption of the gene XRN1, coding for a 5'----3' exoribonuclease, restricts yeast cell growth. Gene. 1990 Oct 30;95(1):85–90. doi: 10.1016/0378-1119(90)90417-p. [DOI] [PubMed] [Google Scholar]
  20. Mason J. O., Williams G. T., Neuberger M. S. The half-life of immunoglobulin mRNA increases during B-cell differentiation: a possible role for targeting to membrane-bound polysomes. Genes Dev. 1988 Aug;2(8):1003–1011. doi: 10.1101/gad.2.8.1003. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. Muhlrad D., Parker R. Premature translational termination triggers mRNA decapping. Nature. 1994 Aug 18;370(6490):578–581. doi: 10.1038/370578a0. [DOI] [PubMed] [Google Scholar]
  24. Munns T. W., Liszewski M. K., Tellam J. T., Sims H. F., Rhoads R. E. Antibody-nucleic acid complexes. Immunospecific retention of globin messenger ribonucleic acid with antibodies specific for 7-methylguanosine. Biochemistry. 1982 Jun 8;21(12):2922–2928. doi: 10.1021/bi00541a018. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Peltz S. W., Brewer G., Bernstein P., Hart P. A., Ross J. Regulation of mRNA turnover in eukaryotic cells. Crit Rev Eukaryot Gene Expr. 1991;1(2):99–126. [PubMed] [Google Scholar]
  27. Peltz S. W., Brown A. H., Jacobson A. mRNA destabilization triggered by premature translational termination depends on at least three cis-acting sequence elements and one trans-acting factor. Genes Dev. 1993 Sep;7(9):1737–1754. doi: 10.1101/gad.7.9.1737. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Savant-Bhonsale S., Cleveland D. W. Evidence for instability of mRNAs containing AUUUA motifs mediated through translation-dependent assembly of a > 20S degradation complex. Genes Dev. 1992 Oct;6(10):1927–1939. doi: 10.1101/gad.6.10.1927. [DOI] [PubMed] [Google Scholar]
  30. Shyu A. B., Belasco J. G., Greenberg M. E. Two distinct destabilizing elements in the c-fos message trigger deadenylation as a first step in rapid mRNA decay. Genes Dev. 1991 Feb;5(2):221–231. doi: 10.1101/gad.5.2.221. [DOI] [PubMed] [Google Scholar]
  31. Swartwout S. G., Kinniburgh A. J. c-myc RNA degradation in growing and differentiating cells: possible alternate pathways. Mol Cell Biol. 1989 Jan;9(1):288–295. doi: 10.1128/mcb.9.1.288. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Vega Laso M. R., Zhu D., Sagliocco F., Brown A. J., Tuite M. F., McCarthy J. E. Inhibition of translational initiation in the yeast Saccharomyces cerevisiae as a function of the stability and position of hairpin structures in the mRNA leader. J Biol Chem. 1993 Mar 25;268(9):6453–6462. [PubMed] [Google Scholar]
  33. Vreken P., Raué H. A. The rate-limiting step in yeast PGK1 mRNA degradation is an endonucleolytic cleavage in the 3'-terminal part of the coding region. Mol Cell Biol. 1992 Jul;12(7):2986–2996. doi: 10.1128/mcb.12.7.2986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Williamson J. R., Raghuraman M. K., Cech T. R. Monovalent cation-induced structure of telomeric DNA: the G-quartet model. Cell. 1989 Dec 1;59(5):871–880. doi: 10.1016/0092-8674(89)90610-7. [DOI] [PubMed] [Google Scholar]
  35. Wilson T., Treisman R. Removal of poly(A) and consequent degradation of c-fos mRNA facilitated by 3' AU-rich sequences. Nature. 1988 Nov 24;336(6197):396–399. doi: 10.1038/336396a0. [DOI] [PubMed] [Google Scholar]
  36. Wisdom R., Lee W. The protein-coding region of c-myc mRNA contains a sequence that specifies rapid mRNA turnover and induction by protein synthesis inhibitors. Genes Dev. 1991 Feb;5(2):232–243. doi: 10.1101/gad.5.2.232. [DOI] [PubMed] [Google Scholar]
  37. Wisdom R., Lee W. Translation of c-myc mRNA is required for its post-transcriptional regulation during myogenesis. J Biol Chem. 1990 Nov 5;265(31):19015–19021. [PubMed] [Google Scholar]
  38. Yang H., Moss M. L., Lund E., Dahlberg J. E. Nuclear processing of the 3'-terminal nucleotides of pre-U1 RNA in Xenopus laevis oocytes. Mol Cell Biol. 1992 Apr;12(4):1553–1560. doi: 10.1128/mcb.12.4.1553. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Zambetti G., Schmidt W., Stein G., Stein J. Subcellular localization of histone messenger RNAs on cytoskeleton-associated free polysomes in HeLa S3 cells. J Cell Physiol. 1985 Nov;125(2):345–353. doi: 10.1002/jcp.1041250225. [DOI] [PubMed] [Google Scholar]
  40. Zimmerman S. B., Cohen G. H., Davies D. R. X-ray fiber diffraction and model-building study of polyguanylic acid and polyinosinic acid. J Mol Biol. 1975 Feb 25;92(2):181–192. doi: 10.1016/0022-2836(75)90222-3. [DOI] [PubMed] [Google Scholar]

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

RESOURCES