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

Wi S. Lai, Rene M. Arvola, Aaron C. Goldstrohm, Perry J. Blackshear

Research output: Contribution to journalReview articlepeer-review

4 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)77-87
Number of pages11
JournalMethods
Volume155
DOIs
StatePublished - Feb 15 2019

Bibliographical note

Funding Information:
We thank Drs. Michael Fessler and Donald Cook for constructive comments on the manuscript. This work was supported in part by the Intramural Research Program of the National Institute of Environmental Health Sciences , NIH (WSL and PJB). It was also supported by grant R01GM105707 from the National Institute of General Medical Sciences , National Institutes of Health (ACG). René M. Arvola was supported by graduate research fellowship DGE 1256260 from the National Science Foundation and the University of Michigan Genetics Training Program NRSA 5T32GM007544 .

Funding Information:
We thank Drs. Michael Fessler and Donald Cook for constructive comments on the manuscript. This work was supported in part by the Intramural Research Program of the National Institute of Environmental Health Sciences, NIH (WSL and PJB). It was also supported by grant R01GM105707 from the National Institute of General Medical Sciences, National Institutes of Health (ACG). René M. Arvola was supported by graduate research fellowship DGE 1256260 from the National Science Foundation and the University of Michigan Genetics Training Program NRSA 5T32GM007544.

Publisher Copyright:
© 2019 Elsevier Inc.

Keywords

  • Drosophila cells
  • Post-transcriptional gene expression
  • RNA-binding proteins
  • Transcription shut-off
  • mRNA turnover

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