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RNA molecules live short lives
To date, there have been two main scientific methods used to measure the half-life of RNA. As the research team led by Attila Becskei PhD, at the Biozentrum, University of Basel, Switzerland, has now discovered, these conventional methods can be quite imprecise and deliver inconsistent results. Becskei's team has found a new method that demonstrates RNA molecules do not last for an average of 20 minutes but rather only two minutes. "This was a challenging endeavor, because no one knew in advance which method yields the correct results," explained Becskei.
Knowing the half-life of RNA is significant for scientific studies on the cell cycle. The whole process of cell division depends on the right amount of proteins being available at the right time. If RNAs are not available in the right concentrations at a given phase of the cell cycle, errors occur.
Becskei measured the half-life of RNA using the "gene control method" well known, but to date not used to measure the half-life of RNA molecules. This is because the method requires complex genetic engineering and is time-consuming. Only one RNA molecule at a time can be studied. Therefore, a single gene of DNA is regulated in such a way that production of RNA can be switched on and off. "Hence, this method only provides a result for one RNA molecule, although results are quite accurate," emphasizes Becskei.
The experiments were repeated for some 50 different genes, showing that 80 percent of all RNAs undergo rapid turnover, living less than 2 minutes. Only about 20 percent live longer to between 5 and 10 minutes.
"These results are astounding, if you consider that until now it was assumed that on average RNAs survived 20 minutes in the cell."
Essentially two main methods are used by scientists to measure the half-life of RNA molecules. In one called "transcriptional inhibition," a substance is introduced into the cell that inhibits RNA production from all genes. "If, however, the production of all RNAs is inhibited — other processes in the cell are also altered and the cell stops functioning. This distorts results," says Becskei.
The second method "in vivo labeling" also has its downside as RNAs are first labeled and then observed to see how long they can be traced within the cell. However, the process of labelling with modified molecules can also interfere with cell function and produce false results. So both methods have drawbacks.
Comparing their method with the two existing ones, the group found the highest correlation between Becskei's method and a unique variant of the "in-vivo labeling" method. In most attempts, both methods classified the same RNAs as stable and unstable, even when mean half-life estimates differed. The team will continue testing both methods to ascertain which has the most accurate and reliable results. However, the quick mRNA degradation was still considerably faster in comparison to previous estimates, with a median half-life of about 2 min.
The work is published in the journal Science Advances.
The rates of mRNA synthesis and decay determine the mRNA expression level. The two processes are under coordinated control, which makes the measurements of these rates challenging, as evidenced by the low correlation among the methods of measurement of RNA half-lives. We developed a minimally invasive method, multiplexed gene control, to shut off expression of genes with controllable synthetic promoters. The method was validated by measuring the ratios of the nascent to mature mRNA molecules and by measuring the half-life with endogenous promoters that can be controlled naturally or through inserting short sequences that impart repressibility. The measured mRNA half-lives correlated highly with those obtained with the metabolic pulse-labeling method in yeast. However, mRNA degradation was considerably faster in comparison to previous estimates, with a median half-life of around 2 min. The half-life permits the estimation of promoter-dependent and promoter-independent transcription rates. The dynamical range of the promoter-independent transcription rates was larger than that of the mRNA half-lives. The rapid mRNA turnover and the broad adjustability of promoter-independent transcription rates are expected to have a major impact on stochastic gene expression and gene network behavior.
Authors: Antoine Baudrimont, Sylvia Voegeli, Eduardo Calero Viloria, Fabian Stritt, Marine Lenon, Takeo Wada, Vincent Jaquet and Attila Becskei†
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RNA molecules [far RIGHT] live an average of two minutes before they are eliminated.
Image Credit: University of Basel, Biozentrum