Messenger RNA half-life measurements in mammalian cells.

The recognition of the importance of mRNA turnover in regulating eukaryotic gene expression has mandated the development of reliable, rigorous, and "user-friendly" methods to accurately measure changes in mRNA stability in mammalian cells. Frequently, mRNA stability is studied indirectly by analyzing the steady-state level of mRNA in the cytoplasm; in this case, changes in mRNA abundance are assumed to reflect only mRNA degradation, an assumption that is not always correct. Although direct measurements of mRNA decay rate can be performed with kinetic labeling techniques and transcriptional inhibitors, these techniques often introduce significant changes in cell physiology. Furthermore, many critical mechanistic issues as to deadenylation kinetics, decay intermediates, and precursor-product relationships cannot be readily addressed by these methods. In light of these concerns, we have previously reported transcriptional pulsing methods based on the c-fos serum-inducible promoter and the tetracycline-regulated (Tet-off) promoter systems to better explain mechanisms of mRNA turnover in mammalian cells. In this chapter, we describe and discuss in detail different protocols that use these two transcriptional pulsing methods. The information described here also provides guidelines to help develop optimal protocols for studying mammalian mRNA turnover in different cell types under a wide range of physiologic conditions.

[1]  A. Shyu,et al.  Rapid Deadenylation Triggered by a Nonsense Codon Precedes Decay of the RNA Body in a Mammalian Cytoplasmic Nonsense-Mediated Decay Pathway , 2003, Molecular and Cellular Biology.

[2]  Tsung-Cheng Chang,et al.  Versatile applications of transcriptional pulsing to study mRNA turnover in mammalian cells. , 2007, RNA.

[3]  M. Gossen,et al.  Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[4]  T. Yen,et al.  Autoregulated instability of β-tubulin mRNAs by recognition of the nascent amino terminus of βtubulin , 1988, Nature.

[5]  C. Y. Chen,et al.  AU-rich elements: characterization and importance in mRNA degradation. , 1995, Trends in biochemical sciences.

[6]  C. Speth,et al.  Expression of basement membrane proteins: evidence for complex post-transcriptional control mechanisms. , 1993, Experimental cell research.

[7]  R Parker,et al.  Deadenylation of the unstable mRNA encoded by the yeast MFA2 gene leads to decapping followed by 5'-->3' digestion of the transcript. , 1994, Genes & development.

[8]  S. Peltz,et al.  The cap-to-tail guide to mRNA turnover , 2001, Nature Reviews Molecular Cell Biology.

[9]  M. Greenberg,et al.  Two distinct destabilizing elements in the c-fos message trigger deadenylation as a first step in rapid mRNA decay. , 1991, Genes & development.

[10]  C. Y. Chen,et al.  Unraveling a cytoplasmic role for hnRNP D in the in vivo mRNA destabilization directed by the AU-rich element. , 1999, Genes & development.

[11]  J. Belasco,et al.  18 – Experimental Approaches to the Study of mRNA Decay , 1993 .

[12]  R D Klausner,et al.  Iron-responsive elements: regulatory RNA sequences that control mRNA levels and translation. , 1988, Science.

[13]  Elmar Wahle,et al.  Messenger RNA Turnover in Eukaryotes: Pathways and Enzymes , 2004, Critical reviews in biochemistry and molecular biology.

[14]  Richard Treisman,et al.  Transient accumulation of c-fos RNA following serum stimulation requires a conserved 5′ element and c-fos 3′ sequences , 1985, Cell.

[15]  C. Y. Chen,et al.  Selective degradation of early-response-gene mRNAs: functional analyses of sequence features of the AU-rich elements , 1994, Molecular and cellular biology.

[16]  C. Y. Chen,et al.  mRNA decay mediated by two distinct AU-rich elements from c-fos and granulocyte-macrophage colony-stimulating factor transcripts: different deadenylation kinetics and uncoupling from translation , 1995, Molecular and cellular biology.

[17]  N. Sonenberg,et al.  A Mechanism for Translationally Coupled mRNA Turnover Interaction between the Poly(A) Tail and a c-fos RNA Coding Determinant via a Protein Complex , 2000, Cell.

[18]  Michael E. Greenberg,et al.  Stimulation of 3T3 cells induces transcription of the c-fos proto-oncogene , 1984, Nature.

[19]  C. Thompson,et al.  Regulation of lymphokine messenger RNA stability by a surface-mediated T cell activation pathway. , 1989, Science.

[20]  P. Anderson,et al.  Posttranscriptional mechanisms regulating the inflammatory response. , 2006, Advances in immunology.

[21]  J. Belasco,et al.  Control of messenger RNA stability. , 1993 .

[22]  M. Greenberg,et al.  The c-fos transcript is targeted for rapid decay by two distinct mRNA degradation pathways. , 1989, Genes & development.

[23]  Ligang Wu,et al.  MicroRNAs direct rapid deadenylation of mRNA. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[24]  Haiwei Song,et al.  The enzymes and control of eukaryotic mRNA turnover , 2004, Nature Structural &Molecular Biology.

[25]  Klaus Resch,et al.  The p38 MAP kinase pathway signals for cytokine‐induced mRNA stabilization via MAP kinase‐activated protein kinase 2 and an AU‐rich region‐targeted mechanism , 1999, The EMBO journal.

[26]  W. Marzluff,et al.  The stem-loop structure at the 3' end of histone mRNA is necessary and sufficient for regulation of histone mRNA stability , 1987, Molecular and cellular biology.

[27]  S. Morrison,et al.  A comparison of apparent mRNA half-life using kinetic labeling techniques vs decay following administration of transcriptional inhibitors. , 1991, Analytical biochemistry.

[28]  Tsung-Cheng Chang,et al.  Concerted action of poly(A) nucleases and decapping enzyme in mammalian mRNA turnover , 2005, Nature Structural &Molecular Biology.

[29]  Barbara Neupert,et al.  A specific mRNA binding factor regulates the iron-dependent stability of cytoplasmic transferrin receptor mRNA , 1989, Cell.

[30]  Tsuey-Ming Chen,et al.  Interplay of two functionally and structurally distinct domains of the c-fos AU-rich element specifies its mRNA-destabilizing function , 1994, Molecular and cellular biology.

[31]  Hermann Bujard,et al.  Control of gene activity in higher eukaryotic cells by prokaryotic regulatory elements , 1993 .

[32]  J. Belasco,et al.  Mechanisms of mRNA decay in bacteria: a perspective. , 1988, Gene.

[33]  J. Ross,et al.  mRNA stability in mammalian cells. , 1995, Microbiological reviews.

[34]  A. Shyu,et al.  A broader role for AU-rich element-mediated mRNA turnover revealed by a new transcriptional pulse strategy. , 1998, Nucleic acids research.

[35]  Markus Posch,et al.  Effect of Transcription Inhibitors on the Iron-dependent Degradation of Transferrin Receptor mRNA * , 1995, The Journal of Biological Chemistry.

[36]  C. Y. Chen,et al.  Transcriptional pulsing approaches for analysis of mRNA turnover in mammalian cells. , 1999, Methods.