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Messenger RNA Turnover in Cell-Free Extracts from Higher Eukaryotes

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Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 118))

Abstract

Two groups of observations support the notion that mRNA turnover influences gene expression in virtually all cells (13). (1) The steady-state levels of many mRNAs are determined more by their half-lives than by their gene transcription rates. In other words, mRNA levels often fluctuate without any measurable change in transcription. (2) The half-lives of some mRNAs change when the environment of the cell, its replication cycle status, or its stage of differentiation changes. The sequences and structures that determine the half-lives of many mRNAs have been mapped in great detail. In contrast, little is known about the RNases that specifically degrade mRNAs (mRNases) and the regulatory factors that influence mRNA stability. How many mRNases are expressed in each cell? Do different cells express different mRNases with different specificities? How do these enzymes function? Are they endonucleases or exonucleases, and where do they first begin to attack the mRNA molecule? What are the major pathways of mRNA decay, and what sorts of degradation intermediates are generated as mRNAs are destroyed? How does translation affect mRNA turnover? What trans-acting factors regulate mRNA turnover? How do they function—by binding to the mRNA molecules they affect, by up- or down-regulating mRNase activity, or by indirect mechanisms?

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References

  1. Belasco, J. G. (1993) mRNA degradation in prokaryotic cells: an overview, in Control of Messenger RNA Stability (Belasco, J. and Brawerman, G., eds.), Academic Press, New York, pp. 3–12.

    Google Scholar 

  2. Caponigro, G. and Parker, R. (1996) Mechanisms and control of mRNA turnover in Saccharomyces cerevisiae. Microbiol. Rev. 60, 233–249.

    PubMed  CAS  Google Scholar 

  3. Ross, J. (1995) mRNA stability in mammalian cells. Microbiol. Rev. 59, 423–450.

    PubMed  CAS  Google Scholar 

  4. Hajnsdorf, E., Carpousis, A. J., and Régnier, P. (1994) Nucleolytic inactivation and degradation of RNase III processed pnp message encoding polynucleotide phosphorylase of Escherichia coli. J. Mol. Biol. 239, 439–454.

    Article  PubMed  CAS  Google Scholar 

  5. Beelman, C. A., Stevens, A., Caponigro, G., LaGrandeur, T. E., Hatfield, L., Fortner, D. M., and Parker, R. (1996) An essential component of the decapping enzyme required for normal rates of mRNA turnover. Nature 382, 642–646.

    Article  PubMed  CAS  Google Scholar 

  6. Ross, J. and Kobs, G. (1986) H4 histone mRNA decay in cell-free extracts initiates at or near the 3′ terminus and proceeds 3′ to 5′. J. Mol. Biol. 188, 579–593.

    Article  PubMed  CAS  Google Scholar 

  7. Bernstein, P. L., Herrick, D. J., Prokipcak, R. D., and Ross, J. (1992) Control of c-myc mRNA half-life in vitro by a protein capable of binding to a coding region stability determinant. Genes Devel. 6, 642–654.

    Article  PubMed  CAS  Google Scholar 

  8. Caruccio, N. and Ross, J. (1994) Purification and characterization of a ribosome-associated 3′ to 5′ exoribonuclease from human cells. J. Biol. Chem. 269, 31,814–31,821.

    PubMed  CAS  Google Scholar 

  9. Zelus, B. D., Stewart, R. S., and Ross, J. (1996) The virion host shutoff protein of Herpes simplex virus type 1: messenger ribonucleolytic activity in vitro. J. Virol. 70, 2411–2419.

    PubMed  CAS  Google Scholar 

  10. Amara, F. M., Chen, F. Y., and Wright, J. A. (1994) Phorbol ester modulation of a novel cytoplasmic protein binding activity at the 3′-untranslated region of mammalian ribonucleotide reductase R2 mRNA and role in message stability. J. Biol. Chem. 269, 6709–6715.

    PubMed  CAS  Google Scholar 

  11. Dompenciel, R. E., Garnepudi, V. R., and Schoenberg, D. R. (1995) Purification and characterization of an estrogen-regulated Xenopus liver polysomal nuclease involved in the selective destabilization of albumin mRNA. J. Biol. Chem. 270, 6108–6118.

    Article  PubMed  CAS  Google Scholar 

  12. Krikorian, C. R. and Read, G. S. (1990) In vitro mRNA degradation system to study the virion host shutoff function of herpes simplex virus. J. Virol. 65, 112–122.

    Google Scholar 

  13. Brewer, G (1991) An A + U-rich element RNA-binding factor regulates c-myc mRNA stability in vitro. Mol. Cell. Biol. 11, 2460–2466.

    PubMed  CAS  Google Scholar 

  14. Amara, F. M., Chen, F. Y., and Wright, J. A. (1993) A novel transforming growth factor-β1 responsive cytoplasmic trans-acting factor binds selectively to the 3′-untranslated region of mammalian ribonucleotide reductase R2 mRNA: role in message stability. Nucleic Acids Res. 21, 4803–4809.

    Article  PubMed  CAS  Google Scholar 

  15. Brown, B. D., Zipkin, I. D., and Harland, R. M. (1993) Sequence-specific endonucleolytic cleavage and protection of mRNA in Xenopus and Drosophila. Genes Devel. 7, 1620–1631.

    Article  PubMed  CAS  Google Scholar 

  16. Wreschner, D. H. and Rechavi, G (1988) Differential mRNA stability to reticulocyte ribonucleases correlates with 3′ non-coding (U)nA sequences. Eur. J. Biochem. 172, 333–340.

    Article  PubMed  CAS  Google Scholar 

  17. Hepler, J. E., Van Wyk, J. J., and Lund, P. K. (1990) Different half-lives of insulin-like growth factor I mRNAs that differ in length of 3′-untranslated sequence. Endocrinology 127, 1550–1552.

    Article  PubMed  CAS  Google Scholar 

  18. Gorospe, M. and Baglioni, C. (1994) Degradation of unstable interleukin-1α mRNA in a rabbit reticulocyte cell-free system. Localization of an instability determinant to a cluster of AUUUA motifs. J. Biol. Chem. 269, 11845–11851.

    PubMed  CAS  Google Scholar 

  19. Maquat, L. E. (1995) When cells stop making sense: effects of nonsense codons on RNA metabolism in vertebrate cells. RNA 1, 453–465.

    PubMed  CAS  Google Scholar 

  20. Molla, M., Paul, A. V., and Wimmer E. (1991) Cell-free, de novo synthesis of poliovirus. Science 254, 1647–1651.

    Article  PubMed  CAS  Google Scholar 

  21. Osley, M. A. (1991) The regulation of histone synthesis in the cell cycle. Annu. Rev. Biochem. 60, 827–861.

    Article  PubMed  CAS  Google Scholar 

  22. Zambetti, G, Stein, J., and Stein, G. (1990) Role of messenger RNA subcellular localization in the posttranscriptional regulation of human histone gene expression. J. Cell. Physiol. 144, 175–182.

    Article  PubMed  CAS  Google Scholar 

  23. Blume, J. E. and Shapiro, D. J. (1989) Ribosome loading, but not protein synthesis, is required for estrogen stabilization of Xenopus laevis vitellogenin mRNA. Nucleic Acids Res. 17, 9003–9014.

    Article  PubMed  CAS  Google Scholar 

  24. Hargrove, J. L., Hulsey, M. G., and Beale, E. G. (1991) The kinetics of mammalian gene expression. BioEssays 13, 667–674.

    Article  PubMed  CAS  Google Scholar 

  25. Brewer, G and Ross, J. (1989) Regulation of c-myc mRNA stability in vitro by a labile destabilizer with an essential nucleic acid component. Mol. Cell. Biol. 9, 1996–2006.

    PubMed  CAS  Google Scholar 

  26. Ross, J., Kobs, G Brewer, G., and Peltz, S. W. (1987) Properties of the exonuclease activity that degrades H4 histone mRNA. J. Biol. Chem. 262, 9374–9381.

    PubMed  CAS  Google Scholar 

  27. Peltz, S. W., Brewer, G, Kobs, G, and Ross, J. (1987) Substrate specificity of the exonuclease activity that degrades H4 histone mRNA. J. Biol. Chem. 262, 9382–9388.

    PubMed  Google Scholar 

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© 1999 Humana Press Inc.

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Ross, J. (1999). Messenger RNA Turnover in Cell-Free Extracts from Higher Eukaryotes. In: Haynes, S.R. (eds) RNA-Protein Interaction Protocols. Methods in Molecular Biology™, vol 118. Humana Press. https://doi.org/10.1385/1-59259-676-2:459

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  • DOI: https://doi.org/10.1385/1-59259-676-2:459

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-568-3

  • Online ISBN: 978-1-59259-676-8

  • eBook Packages: Springer Protocols

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