Impact of Methods on the Measurement of mRNA Turnover

Abstract

The turnover of the RNA molecules is determined by the rates of transcription and RNA degradation. Several methods have been developed to study RNA turnover since the beginnings of molecular biology. Here we summarize the main methods to measure RNA half-life: transcription inhibition, gene control, and metabolic labelling. These methods were used to detect the cellular activity of the mRNAs degradation machinery, including the exo-ribonuclease Xrn1 and the exosome. On the other hand, the study of the differential stability of mature RNAs has been hampered by the fact that different methods have often yielded inconsistent results. Recent advances in the systematic comparison of different method variants in yeast have permitted the identification of the least invasive methodologies that reflect half-lives the most faithfully, which is expected to open the way for a consistent quantitative analysis of the determinants of mRNA stability.

Keywords: 4-thiouracil; 4sU; NMD; Saccharomyces cerevisiae; exponential decay; nonsense mediated decay; posttranscriptional regulation; rpb1-1; splicing.

Figure 1

Main classes of methods to study RNA stability. (a) Scheme of the molecular mechanism affected by the specific method procedures. In transcriptional inhibition, the RNA polymerase is inactivated; the expression of all genes is reduced. In the gene control method, a transcriptional activator dissociates from a specific promoter, shutting off the expression of the specific gene under the control of this promoter. For labelling of the RNAs, modified nucleotides are introduced into the cell (red dots), which are then incorporated into the RNA; (b) Time course of the experiments to determine RNA half-lives. Inhibition of transcription of the gene(s) is triggered at t = 0 in transcriptional inhibition and gene control methods. There are two subclasses of the in vivo labelling. In the approach to equilibrium method, a pulse of modified nucleotides is applied and the increase of the labelled mRNA is monitored. In the pulse–chase method, the RNA is first labeled (pulse period). During the chase period starting at t = 0, the labeled nucleotides are washed out and replaced with unlabeled nucleotides and the decline of the labelled RNA is monitored.

Figure 2

Impact of the speed of transcriptional inhibition on the measured mRNA half-lives. (a) Two different scenarios are shown for the inhibition of transcription. In the ideal case, the inhibition occurs instantaneously down to a baseline level (full line). In suboptimal cases, inhibition of transcription ensues with slow kinetics (dashed line); (b) The decay of RNA upon instantaneous (full lines) and slow (dashed lines) inhibition of transcription. The precursor mRNA (orange), containing the intron, can be used to monitor the inhibition kinetics since the precursor is converted rapidly to mature mRNA (purple) by splicing. The half-life of the mature mRNA (purple) will appear much longer with the slow inhibition kinetics and there is also a longer lag period before the mRNA level starts to decline.