title: RNA degradation in cell extracts: real-time monitoring by fluorescence resonance energy transfer.
abstract: Short noncoding RNAs are increasingly recognized as key regulators of essential cellular processes such as RNA interference. A better understanding of the processes by which such RNAs are degraded is necessary to expand our knowledge of these processes and our ability to harness them. To this end we have developed a novel fluorescence resonance energy transfer (FRET) assay to monitor in real-time the degradation kinetics of short RNAs by a purified RNase and S100 cytosolic HeLa cell extract. An unstructured RNA is found to be degraded more rapidly than a stem-loop RNA under all conditions tested except for low concentrations of cell extract, showing that secondary structure confers protection against RNase activity. The assay also allows for the quantitative comparison of inhibitors such as Contrad70 and aurin tricarboxylic acid (ATA). Finally, gel electrophoretic FRET analysis confirms that HeLa cell extract is dominated by 5' to 3' exonucleolytic activity.
authors: Department of Chemistry, University of Michigan, Ann Arbor, MI 48109-1055, USA.
authors: Sarah A
authors: SA
authors: Uhler
authors: Dawen
authors: D
authors: Cai
authors: Yunfang
authors: Y
authors: Man
authors: Carina
authors: C
authors: Figge
authors: Nils G
authors: NG
authors: Walter
mesh_terms: D002457:Cell Extracts / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D006163:Ribonuclease T1 / Q000378:metabolism; D013050:Spectrometry, Fluorescence
chemical_list: D002457:Cell Extracts; D009841:Oligonucleotides; D012313:RNA; D006163:Ribonuclease T1