Abstract
Gene expression is regulated through multiple steps at both transcriptional and post-transcriptional levels. The net abundance of mature mRNA species in cells is determined by the balance between transcription and degradation. Thus, the regulation of mRNA stability is a key post-transcriptional event that can greatly affect the net level of mRNAs in cells. The mRNA stability within cells can be measured indirectly by analyzing the mRNA half-life following transcription inhibition, where changes in mRNA levels are assumed to reflect mRNA degradation. Determination of mRNA half-life as a measure of mRNA stability is useful in understanding gene expression changes and underlying mechanisms regulating the level of transcripts at different physiological conditions or developmental stages. The protocol described here presents the analysis of mRNA decay as a measure for determining mRNA stability after transcriptional inhibition with Actinomycin D treatment in control and SRSF3 depleted mouse induced pluripotent stem cells (iPSC).
Keywords: mRNA stability, Actinomycin D, mRNA decay, Transcription inhibitors, mRNA half-life
Background
Determining the stability of mRNA within cells provides an important measure for understanding post-transcriptional gene regulation and the potential role of RNA-protein interactions in the process. Under any specific condition such as following extracellular stimuli or gene knockdown the stability of mRNAs may change due to enhanced degradation or extended half-lives (Shyu et al., 1989). Therefore, to assess mRNA stability, direct measurements of decay rates of endogenous mRNAs have been performed in a number of ways, including kinetic labeling techniques and the use of transcriptional inhibitors (Chen et al., 2008). One of the simplest techniques of measuring mRNA stability is by inhibiting transcription in vivo with transcription inhibitors and measuring the mRNA kinetics. Actinomycin D is a transcription inhibitor which intercalates into DNA. Actinomycin D forms a very stable complex with DNA, preventing the unwinding of the DNA double-helix, thus inhibiting the DNA-dependent RNA polymerase activity. Actinomycin D is widely used in mRNA stability assays to inhibit the synthesis of new mRNA, allowing the assessment of mRNA decay by measuring mRNA abundance following transcription inhibition (Avendano and Menéndez, 2008). At low concentrations, Actinomycin D inhibits transcription without significantly affecting DNA replication or protein synthesis (Berg et al., 2002). Other transcription inhibitors such as 5,6-dichloro-1β-1-ribofuranosylbenzimidazole (DRB) which interacts directly with the RNA polymerase II have also been successfully used in similar assays (Harrold et al., 1991). Please refer to Bensaude (2011) for more detailed information on different transcription inhibitors that can be used and their specific properties. More advanced techniques such as the use of inducible promoters to control transient transcription have presented advantages over the potential cytotoxic effects of Actinomycin D or other transcription inhibitors in the analysis of mRNA decay (Chen et al., 2008). However, the advantage of Actinomycin D assay is that it does not require the construction and introduction of exogenous genes into cells, and provides a way of measuring stability changes of endogenous mRNAs (Chen et al., 2008). We have established serine-arginine-rich splicing factor 3 (SRSF3)-RNA interactions as a critical means to co-ordinate gene expression in pluripotent cells (Ratnadiwakara et al., 2018). Further, SRSF3 has been reported to regulate mRNA levels including its own mRNA abundance via alternative splicing coupled to nonsense mediated decay (NMD) (Anko et al., 2012). To determine if SRSF3 affects the production or stability of NMD-sensitive transcript variants in pluripotent cells, we determined mRNA half-lives of SRSF3 target mRNAs in Actinomycin D treated control and SRSF3-depleted pluripotent stem cells (Ratnadiwakara et al., 2018). Several techniques such as Northern blot analysis, in situ hybridization and quantitative PCR can be used to determine the mRNA half-life after transcription inhibition. We used quantitative PCR which allows rapid and sensitive measurement of half-lives of mRNAs across a broad range of expression levels, including low abundant mRNAs. The protocol described here can be used to successfully measure mRNA decay in pluripotent stem cells.
Materials and Reagents
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Acknowledgments
MLA was supported by National Health and Medical Research Council (NHMRC) GNT1043092 and GNT1138870, Aatos and Jane Erkko Foundation and Monash Biomedicine Discovery Fellowship.
Competing interests
The authors have no conflicts of interest or competing interests.
References
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Glad to hear that you find the protocol useful! Actinomycin D is not a selective PolII inhibitor, so the transcription of any RNA is affected (including PolI and PolII transcripts) making it hard to choose a transcript for normalisation. Therefore, we only normalised to the first data point to determine the rate of decay. FOr this reason, it was important to run multiple independent experiments (biological replicates). There are more specific PolII/III inhibitors such as alpha-amanitin where normalization to a PolI transcript would be more appropriate. We also analysed transcripts with previously established decay rates as controls (in our case c-Myc).