TY - JOUR
T1 - Inhibiting transcription in cultured metazoan cells with actinomycin D to monitor mRNA turnover
AU - Lai, Wi S.
AU - Arvola, Rene M.
AU - Goldstrohm, Aaron C.
AU - Blackshear, Perry J.
N1 - Publisher Copyright:
© 2019 Elsevier Inc.
PY - 2019/2/15
Y1 - 2019/2/15
N2 - 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.
AB - 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.
KW - Drosophila cells
KW - Post-transcriptional gene expression
KW - RNA-binding proteins
KW - Transcription shut-off
KW - mRNA turnover
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U2 - 10.1016/j.ymeth.2019.01.003
DO - 10.1016/j.ymeth.2019.01.003
M3 - Review article
C2 - 30625384
AN - SCOPUS:85061293217
SN - 1046-2023
VL - 155
SP - 77
EP - 87
JO - Methods
JF - Methods
ER -