Microarray analysis of newly synthesized RNA in cells and animals

Current methods to analyze gene expression measure steady-state levels of mRNA. To specifically analyze mRNA transcription, we have developed a technique that can be applied in vivo in intact cells and animals. Our method makes use of the cellular pyrimidine salvage pathway and is based on affinity-chromatographic isolation of thiolated mRNA. When combined with data on mRNA steady-state levels, this method is able to assess the relative contributions of mRNA synthesis and degradation/stabilization. It overcomes limitations associated with currently available methods such as mechanistic intervention that disrupts cellular physiology, or the inability to apply the techniques in vivo. Our method was first tested in serum response of cultured fibroblast cells and then applied to the study of renal ischemia reperfusion injury, demonstrating its applicability for whole organs in vivo. Combined with data on mRNA steady-state levels, this method provided a detailed analysis of regulatory mechanisms of mRNA expression and the relative contributions of RNA synthesis and turnover within distinct pathways, and identification of genes expressed at low abundance at the transcriptional level.

[1]  J. Mattick,et al.  RNA : Networks & Imaging , 2006 .

[2]  M. Gorospe,et al.  Egad, More Forms of Gene Regulation: The gadd45a Story , 2006, Cell cycle.

[3]  J. Boothroyd,et al.  Biosynthetic labeling of RNA with uracil phosphoribosyltransferase allows cell-specific microarray analysis of mRNA synthesis and decay , 2005, Nature Biotechnology.

[4]  B. Molitoris,et al.  Evolving basic concepts in ischemic injury , 2004 .

[5]  J. Pérez-Ortín,et al.  Genomic run-on evaluates transcription rates for all yeast genes and identifies gene regulatory mechanisms. , 2004, Molecular cell.

[6]  Matthew Hurles,et al.  Gene Duplication: The Genomic Trade in Spare Parts , 2004, PLoS biology.

[7]  J. LaMarre,et al.  AU-rich elements and the control of gene expression through regulated mRNA stability. , 2004 .

[8]  M. Hergenhahn,et al.  High-accuracy amplification of nanogram total RNA amounts for gene profiling. , 2004, Genomics.

[9]  L. Bénard.  Inhibition of 5' to 3' mRNA degradation under stress conditions in Saccharomyces cerevisiae: from GCN4 to MET16. , 2004, RNA.

[10]  Russell D. Wolfinger,et al.  Comparison of Li-Wong and loglinear mixed models for the statistical analysis of oligonucleotide arrays , 2004, Bioinform..

[11]  L. Lai,et al.  Pathogenesis of renal ischemia/reperfusion injury: lessons from knockout mice. , 2003, Life sciences.

[12]  K. Matsushima,et al.  J Am Soc Nephrol 14: 2503–2515, 2003 CCR2 Signaling Contributes to Ischemia-Reperfusion Injury , 2022 .

[13]  M. Daly,et al.  PGC-1α-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes , 2003, Nature Genetics.

[14]  R. Kucherlapati,et al.  Differential gene expression following early renal ischemia/reperfusion. , 2003, Kidney international.

[15]  A. Khodursky,et al.  Life after transcription--revisiting the fate of messenger RNA. , 2003, Trends in genetics : TIG.

[16]  T. Sasaguri,et al.  Transcriptional and Posttranscriptional Regulation of Cyclooxygenase-2 Expression by Fluid Shear Stress in Vascular Endothelial Cells , 2002, Arteriosclerosis, thrombosis, and vascular biology.

[17]  M. Gorospe,et al.  Global analysis of stress-regulated mRNA turnover by using cDNA arrays , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[18]  R. Bendayan,et al.  Drug transporters in the central nervous system: brain barriers and brain parenchyma considerations. , 2001, Pharmacological reviews.

[19]  J. Guhaniyogi,et al.  Regulation of mRNA stability in mammalian cells. , 2001, Gene.

[20]  B. McEwen,et al.  Neurotrophic and neuroprotective actions of estrogens and their therapeutic implications. , 2001, Annual review of pharmacology and toxicology.

[21]  G. Hager.  Methods issue on nuclear structure and chromatin. , 1999, Methods.

[22]  J. Lis,et al.  Nuclear run-on assays: assessing transcription by measuring density of engaged RNA polymerases. , 1999, Methods in enzymology.

[23]  D. Botstein,et al.  The transcriptional program in the response of human fibroblasts to serum. , 1999, Science.

[24]  N. Çíftçíoglu,et al.  Interaction of nanobacteria with cultured mammalian cells , 1998 .

[25]  A. Favre,et al.  4‐THIOURIDINE INCORPORATION INTO THE RNA OF MONKEY KIDNEY CELLS (CV‐1) TRIGGERS NEAR‐UV LIGHT LONG‐TERM INHIBITION OF DNA, RNA AND PROTEIN SYNTHESIS , 1993, Photochemistry and photobiology.

[26]  D. Zaharevitz,et al.  Contribution of de-novo and salvage synthesis to the uracil nucleotide pool in mouse tissues and tumors in vivo. , 1992, European journal of biochemistry.

[27]  R. Zager.  Adenine nucleotide changes in kidney, liver, and small intestine during different forms of ischemic injury. , 1991, Circulation research.

[28]  R. Schlegel,et al.  Selective isolation of newly synthesized mammalian mRNA after in vivo labeling with 4-thiouridine or 6-thioguanosine. , 1988, Analytical biochemistry.

[29]  R. Handschumacher,et al.  Tissue uridine pools: evidence in vivo of a concentrative mechanism for uridine uptake. , 1986, Cancer research.

[30]  R. Spector.  Uridine Transport and Metabolism in the Central Nervous System , 1985, Journal of neurochemistry.

[31]  J. Moyer,et al.  Salvage of circulating pyrimidine nucleosides by tissues of the mouse. , 1985, The Journal of biological chemistry.

[32]  R. Handschumacher,et al.  Novel single-pass exchange of circulating uridine in rat liver. , 1981, Science.

[33]  M. Ono,et al.  Separation of newly-synthesized RNA by organomercurial agarose affinity chromatography. , 1977, Journal of biochemistry.

[34]  H. Pakkenberg,et al.  Relative uptake of tritiated uridine in various brain areas in the mouse , 1976, Journal of neurochemistry.

[35]  H. Pakkenberg,et al.  Kinetics of 3 H-5-uridine incorporation in brain cells of the mouse. , 1972, Experimental neurology.

[36]  Canellakis Es.  Pyrimidine metabolism. III. The interaction of the catabolic and anabolic pathways of uracil metabolism. , 1957 .