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Methods. 2019 Jan 6. pii: S1046-2023(18)30248-2. doi: 10.1016/j.ymeth.2019.01.003. [Epub ahead of print]

Inhibiting transcription in cultured metazoan cells with actinomycin D to monitor mRNA turnover.

Author information

1
Signal Transduction Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, United states.
2
Department of Biological Chemistry, University of Michigan, Ann Arbor, MI, United states.
3
Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN, United states.
4
Signal Transduction Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, United states; Departments of Medicine and Biochemistry, Duke University School of Medicine, Durham, NC 27710, United states. Electronic address: Black009@niehs.nih.gov.

Abstract

Decay of transcribed mRNA is a key determinant of steady state mRNA levels in cells. Global analysis of mRNA decay in cultured cells has revealed amazing heterogeneity in rates of decay under normal growth conditions, with calculated half-lives ranging from several minutes to many days. The factors that are responsible for this wide range of decay rates are largely unknown, although our knowledge of trans-acting RNA binding proteins and non-coding RNAs that can control decay rates is increasing. Many methods have been used to try to determine mRNA decay rates under various experimental conditions in cultured cells, and transcription inhibitors like actinomycin D have probably the longest history of any technique for this purpose. Despite this long history of use, the actinomycin D method has been criticized as prone to artifacts, and as ineffective for some promoters. With appropriate guidelines and controls, however, it can be a versatile, effective technique for measuring endogenous mRNA decay in cultured mammalian and insect cells, as well as the decay of exogenously-expressed transcripts. It can be used readily on a genome-wide level, and is remarkably cost-effective. In this short review, we will discuss our utilization of this approach in these cells; we hope that these methods will allow more investigators to apply this useful technique to study mRNA decay under the appropriate conditions.

KEYWORDS:

Drosophila cells; RNA-binding proteins; mRNA turnover; post-transcriptional gene expression; transcription shut-off

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