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

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

Research output: Contribution to journalReview article

1 Citation (Scopus)

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

Fingerprint

RNA Stability
Dactinomycin
Transcription
Cultured Cells
Messenger RNA
Untranslated RNA
Cells
RNA-Binding Proteins
Artifacts
Insects
History
Research Personnel
Genome
Guidelines
Costs and Cost Analysis
Genes
Growth
Costs

Keywords

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

PubMed: MeSH publication types

  • Journal Article
  • Review
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, N.I.H., Extramural

Cite this

Inhibiting transcription in cultured metazoan cells with actinomycin D to monitor mRNA turnover. / Lai, Wi S.; Arvola, Rene M.; Goldstrohm, Aaron; Blackshear, Perry J.

In: Methods, Vol. 155, 15.02.2019, p. 77-87.

Research output: Contribution to journalReview article

Lai, Wi S. ; Arvola, Rene M. ; Goldstrohm, Aaron ; Blackshear, Perry J. / Inhibiting transcription in cultured metazoan cells with actinomycin D to monitor mRNA turnover. In: Methods. 2019 ; Vol. 155. pp. 77-87.
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