High-resolution gene expression profiling for simultaneous kinetic parameter analysis of RNA synthesis and decay
Top Cited Papers
- 24 July 2008
- journal article
- research article
- Published by Cold Spring Harbor Laboratory in RNA
- Vol. 14 (9), 1959-1972
- https://doi.org/10.1261/rna.1136108
Abstract
RNA levels in a cell are determined by the relative rates of RNA synthesis and decay. State-of-the-art transcriptional analyses only employ total cellular RNA. Therefore, changes in RNA levels cannot be attributed to RNA synthesis or decay, and temporal resolution is poor. Recently, it was reported that newly transcribed RNA can be biosynthetically labeled for 1–2 h using thiolated nucleosides, purified from total cellular RNA and subjected to microarray analysis. However, in order to study signaling events at molecular level, analysis of changes occurring within minutes is required. We developed an improved approach to separate total cellular RNA into newly transcribed and preexisting RNA following 10–15 min of metabolic labeling. Employing new computational tools for array normalization and half-life determination we simultaneously study short-term RNA synthesis and decay as well as their impact on cellular transcript levels. As an example we studied the response of fibroblasts to type I and II interferons (IFN). Analysis of RNA transcribed within 15–30 min at different times during the first three hours of interferon-receptor activation resulted in a >10-fold increase in microarray sensitivity and provided a comprehensive profile of the kinetics of IFN-mediated changes in gene expression. We identify a previously undisclosed highly connected network of short-lived transcripts selectively down-regulated by IFNγ in between 30 and 60 min after IFN treatment showing strong associations with cell cycle and apoptosis, indicating novel mechanisms by which IFNγ affects these pathways.Keywords
This publication has 28 references indexed in Scilit:
- Microarray analysis of newly synthesized RNA in cells and animalsProceedings of the National Academy of Sciences of the United States of America, 2007
- Complex Modulation of Cell Type-Specific Signaling in Response to Type I InterferonsImmunity, 2006
- Type I Interferons in Host DefenseImmunity, 2006
- Mechanisms of type-I- and type-II-interferon-mediated signallingNature Reviews Immunology, 2005
- Involvement of MicroRNA in AU-Rich Element-Mediated mRNA InstabilityCell, 2005
- Linear Models and Empirical Bayes Methods for Assessing Differential Expression in Microarray ExperimentsStatistical Applications in Genetics and Molecular Biology, 2004
- p38 Mitogen-Activated Protein Kinase-Dependent and -Independent Signaling of mRNA Stability of AU-Rich Element-Containing TranscriptsMolecular and Cellular Biology, 2003
- Methods Issue on Nuclear Structure and ChromatinMethods, 1999
- The c-fos transcript is targeted for rapid decay by two distinct mRNA degradation pathways.Genes & Development, 1989
- Incorporation of 6‐Thioguanosine and 4‐Thiouridine into RNAJBIC Journal of Biological Inorganic Chemistry, 1978