Research in the author&#8217;s laboratory is supported by the Texas Higher Education Coordinating Board&#8217;s Norman Hackerman Advanced Research Program and start-up funds from Baylor University.

1. Growth of Yeast Cells
<ol>
	<li>Select the appropriate yeast strains to be utilized for the mRNA decay rate measurements. To inhibit transcription using the temperature sensitive allele of RNA polymerase II, use yeast strains harboring the rpb1-1 mutation 1. Obtain this yeast strain from a laboratory that already has one, or generate it in the laboratory using standard techniques if a specific genetic background is required9.</li>
	<li>Using sterile technique, prepare yeast media using...

<ol>
	<li>Nonet, M., Scafe, C., Sexton, J., Young, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eukaryotic+RNA+polymerase+conditional+mutant+that+rapidly+ceases+mRNA+synthesis.">Eukaryotic RNA polymerase conditional mutant that rapidly ceases mRNA synthesis.</a> Mol. Cell. Biol. 7, 1602-1616 (1987).</li><li>Santiago, T. C., Purvis, I. J., Bethany, A. J., Brown, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relationship+between+mRNA+stability+and+length+in+Saccharomyces+cerevisiae.">The relationship between mRNA stability and length in Saccharomyces cerevisiae.</a> Nucleic Acids Res. 14 (21), 8347-8360 (1986).</li><li>Jimenez, A., Tipper, D. J., Davies, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mode+of+action+of+thiolutin,+an+inhibitor+of+macromolecular+synthesis+in+Saccharomyces+cerevisiae.">Mode of action of thiolutin, an inhibitor of macromolecular synthesis in Saccharomyces cerevisiae.</a> Antimicrob Agents Chemother. 3 (6), 729-738 .</li><li>Herrick, D., Parker, R., Jacobson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+Comparison+of+stable+and+unstable+mRNAs+in+Saccharomyces+cerevisiae.">Identification and Comparison of stable and unstable mRNAs in Saccharomyces cerevisiae.</a> Mol Cell Biol. 10 (5), 2269-2284 .</li><li>Grigull, J., Mnaimneh, S., Pootoolal, J., Robinson, M. D., Hughes, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+Analysis+of+mRNA+Stability+Using+Transcription+Inhibitors+and+Microarrays+Reveals+Postranscriptional+Control+of+Ribosome+Biogenesis+Factors.">Genome-wide Analysis of mRNA Stability Using Transcription Inhibitors and Microarrays Reveals Postranscriptional Control of Ribosome Biogenesis Factors.</a> Mol Cell Biol. 24 (12), 5534-5547 (2004).</li><li>Tani, H., Akimitsu, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+technology+for+determining+RNA+stability+in+mammalian+cells.">Genome-wide technology for determining RNA stability in mammalian cells.</a> RNA Biology. 9 (10), 1233-1238 (2012).</li><li>Pelechano, V., Perez-Ortin, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transcriptional+inhibitor+thiolutin+blocks+mRNA+degradation+in+yeast.">The transcriptional inhibitor thiolutin blocks mRNA degradation in yeast.</a> Yeast. 25, 85-92 (2008).</li><li>Kervestin, S., Jacobson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NMD:+a+multifaceted+response+to+premature+translational+termination.">NMD: a multifaceted response to premature translational termination.</a> Nat Rev Mol Cell Biol. 13, 700-712 (2012).</li><li>Kolodziej, P. A., Woychik, N., Liao, S. M., Young, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+Polymerase+II+subunit+composition,+stoichiometry+and+phosphorylation.">RNA Polymerase II subunit composition, stoichiometry and phosphorylation.</a> Mol Cell Biol. 10 (5), 1270-1275 (1990).</li><li>Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., Struhl, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Current Protocols in Molecular Biology. , (1998).</li><li>Kebaara, B. W., Nazarenus, T., Taylor, R., Atkin, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+background+affects+relative+nonsense+mRNA+accumulation+in+wild-type+and+upf+mutant+yeast+strains+Current+Genetics).">Genetic background affects relative nonsense mRNA accumulation in wild-type and upf mutant yeast strains Current Genetics).</a> 43, 171-177 (2003).</li><li>Kebaara, B. W., Baker, K. E., Patefield, K. D., Atkin, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+Nonsense-Mediated+mRNA+Decay+in+Saccharomyces+cerevisiae.">Analysis of Nonsense-Mediated mRNA Decay in Saccharomyces cerevisiae.</a> Current Protocols in Cell Biology. 27 (27.3), 27.3.1-27.3.39 (2012).</li><li>Hu, W., Coller, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+measuring+mRNA+Decay+Rate+in+Saccharomycescerevisiae.">Methods for measuring mRNA Decay Rate in Saccharomycescerevisiae.</a> Methods in Enzymology. 530, 137-155 (2013).</li></ol>

The authors declare that they have no competing financial interest.

Inhibition of mRNA synthesis and monitoring mRNA turnover in the absence of new synthesis is a method that is frequently used to measure mRNA decay rates. In S. cerevisiae, measurement of mRNA decay rates by inhibiting transcription using the temperature sensitive allele of RNA polymerase II is one of the most frequently used methods. This method specifically inhibits RNA polymerase II. The most critical steps for determination of mRNA decay rates using this technique are: 1) Prior to harvesting the yeast cells,...

The transcription and decay of specific mRNA are crucial determinants of gene expression. The rate of synthesis and decay of specific mRNAs determines the steady-state level of that particular mRNA. The steady state levels of mRNAs govern the abundance of mRNAs and determine how much of each mRNA is available for protein synthesis. Measurements of mRNA half-lives are used extensively to determine the decay rate of mRNAs. Specific mRNAs decay at different rates that are related to features of the mRNA, the function of the protein encoded by the mRNA and the environmental conditions. Depending on the technique utilized to determine mRNA decay rates, decay rate measurements can be determined either globally or for individual transcripts. In the yeast S. cerevisiae, the techniques that are most commonly used to measure global mRNA decay rates include utilizing a yeast strain harboring the temperature sensitive allele of RNA polymerase II and chemical transcriptional inhibitors such as thiolutin and 1,10-phenanthroline 1-5. These methods can also be utilized to measure individual mRNA decay rates 4. Other methods can also be utilized to measure mRNA decay rates. These methods include approach to steady state labeling or utilization of mRNA molecules that are expressed from a regulated promoter that is expressed only in select conditions. Each of these techniques has certain advantages and limitations. The technique described here utilizes the temperature sensitive allele of RNA polymerase II. This method uses S. cerevisiae as the model, but can been modified and utilized in other systems using specific transcriptional inhibition techniques 6.
mRNA half-life measurements using the temperature sensitive allele of RNA polymerase II are extensively used for both genome-wide and individual measurements of mRNA decay 4-5. This technique requires the use of a specific yeast strain that harbors a temperature sensitive allele of RNA polymerase II, rpb1-11. The rationale for this technique is that exposure of the temperature sensitive yeast strain to the nonpermissive temperature inhibits mRNA synthesis. Subsequently, decay of the preexisting mRNA is monitored at different time points after transcription has been inhibited. The disappearance of the preexisting mRNA is monitored by extracting RNA from the yeast cells at different time points after transcription has been turned off. The time points at which the yeast cells are harvested are predetermined by a pilot experiment, and depend on the transcripts and system being investigated. Quicker time points are used for short-lived transcripts, while longer time points are used for longer lived transcripts. Afterwards, the decay of the mRNA is monitored by either northern blot analysis, quantitative PCR or RNAseq.
Measurement of mRNA half-lives using a temperature sensitive allele of RNA polymerase II has its advantages. First, this technique is easy and straight forward. Second, once the yeast strain is acquired or generated in the laboratory the mRNA half-life measurements can be determined in different growth conditions; enabling determination of environmental influence on mRNA decay. Third, mRNA decay rates can be monitored genome-wide. Use of other transcription inhibition techniques also has advantages and limitations. For example, use of an inducible promoter requires subcloning to generate an mRNA that is under the control of the regulated promoter. Thiolutin is not readily available and is expensive when available. In addition, thiolutin&#8217;s mode of action is not completely understood and it has been reported to affect other cellular processes including inhibiting mRNA decay 7. Alternatively, 1,10-phenanthroline, is more readily available. Furthermore, all of the techniques used to inhibit transcription can perturb cellular function and can affect different mRNAs in distinct ways. An investigator needs to determine the most appropriate method to use in their experimental conditions to attain the most reliable results. To determine which method is most suitable for their application, a researcher needs to identify the transcripts and function of the proteins encoded by the transcripts being investigated. The most reliable mRNA decay rate measurements are those that are determined using multiple techniques and show the same decay rate. No single technique is always the best, and the most appropriate technique depends on the specific situation.
Numerous studies in S. cerevisiae have measured mRNA decay rates in various conditions and genetic backgrounds. The conditions that mRNA decay rates are measured in depend on the specific experiment being investigated. Measuring mRNA decay rates in different cellular environments determines whether the conditions being examined preferentially affect the decay rates of specific mRNAs. The decay rates of mRNAs can also vary depending on the yeast strain being used. For example, mRNA decay rates can be determined in wild-type yeast cells and yeast cells with a nonfunctional nonsense-mediated mRNA degradation (NMD) pathway. This mRNA degradation pathway is found in all eukaryotic organisms that have been examined so far and it triggers the degradation of mRNAs that prematurely terminate translation 8. NMD was initially identified as a pathway that degrades mRNAs with premature termination codons or nonsense codons, but is now recognized as a pathway that also regulates the expression of non-nonsense containing natural mRNAs. mRNAs that are targets of the pathway are rapidly degraded in yeast cells with a functional NMD pathway and stabilized in yeast cells with a nonfunctional NMD pathway. Thus, the half-lives of mRNAs that are direct targets of this pathway are shorter in wild-type yeast cells compared to yeast cells with a nonfunctional NMD pathway.

<table border="0" cellpadding="0" cellspacing="0" width="1077">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:272px;">Name of Material/Equipment</td>
			<td style="width:181px;">Company</td>
			<td style="width:147px;">Catalog Number</td>
			<td style="width:479px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">High Speed Centrifuge</td>
			<td>Eppendorf</td>
			<td>22628169</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mini Centrifuge</td>
			<td>Fisher Scientific</td>
			<td>05-090-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Genescreen Plus membrane</td>
			<td>PerkinElmer</td>
			<td>50-905-0169</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hybridization Oven</td>
			<td>Fisher Scientific</td>
			<td>95-0030-01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nanodrop spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>ND-8000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphor screen</td>
			<td>GE Healthcare Life Sciences</td>
			<td>28-9564-78</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Typhoon phosphorimager</td>
			<td>GE Healthcare Life Sciences</td>
			<td>29004080</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">UV cross-linker</td>
			<td>GE Healthcare Life Sciences</td>
			<td>80-6222-31</td>
			<td>Alternatively the membrane can be baked in an oven set to 80&#176; for 1 hour</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:272px;">NorthernMax prehybridization/hybridization buffer&#160;</td>
			<td>Life Technologies</td>
			<td>AM8677</td>
			<td></td>
		</tr>
		<tr height="20">
			<td colspan="2" height="20" style="height:20px;">Yeast strains harboring the rpb1-1 mutation</td>
			<td></td>
			<td>Yeast strains can be obtained from a laboratory or created with a specific background</td>
		</tr>
	</tbody>
</table>

measurement of mrna decay rates in saccharomyces cerevisiae using rpb1-1 strains

mRNA steady state levels vary depending on environmental conditions. Regulation of the steady state accumulation levels of an mRNA ensures that the correct amount of protein is synthesized for the cell&#8217;s specific growth conditions. One approach for measuring mRNA decay rates is inhibiting transcription and subsequently monitoring the disappearance of the already present mRNA. The rate of mRNA decay can then be quantified, and an accurate half-life can be determined utilizing several techniques. In S. cerevisiae, protocols that measure mRNA half-lives have been developed and include inhibiting transcription of mRNA using strains that harbor a temperature sensitive allele of RNA polymerase II, rpb1-1. Other techniques for measuring mRNA half-lives include inhibiting transcription with transcriptional inhibitors such as thiolutin or 1,10-phenanthroline, or alternatively, by utilizing mRNAs that are under the control of a regulatable promoter such as the galactose inducible promoter and the TET-off system. Here, we describe measurement of S. cerevisiae mRNA decay rates using the temperature sensitive allele of RNA polymerase II. This technique can be used to measure mRNA decay rates of individual mRNAs or genome-wide.

The ability of this protocol to accurately measure mRNA decay rates depends on inhibition of transcription, the harvesting of yeast cells at the appropriate time points, and utilization of RNase free techniques while extracting RNA and northern blotting. Probing for two control mRNAs known to be unstable and stable, respectively, provides confidence that the experiment worked. For example, this can be accomplished by probing with a probe that detects both the CYH2 pre-mRNA and mRNA. Figure 2B sh...

Watch this Scientific Journal Video about Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains at JoVE.com

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

The steady state level of specific mRNAs is determined by the rate of synthesis and decay of the mRNA. Genome-wide mRNA degradation rates or the decay rates of specific mRNAs can be measured by determining mRNA half-lives. This protocol focuses on measurement of mRNA decay rates in Saccharomyces cerevisiae.

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

mRNA steady state levels vary depending on environmental conditions. Regulation of the steady state accumulation levels of an mRNA ensures that the ...

measurement-mrna-decay-rates-saccharomyces-cerevisiae-using-rpb1-1

Research

Education

JoVE Journal

JoVE Core

Molecular Biology

Biology

Unit 1

Translation: RNA to Protein

SCIENTISTS IN ACTION

12.4K vues.  Baylor University.  Le niveau de l'état d'équilibre des ARNm spécifiques est déterminée par le taux de synthèse et de la décomposition de l'ARNm. Taux de dégradation de l'ARNm pangénomiques ou les taux d'ARNm spécifiques de désintégration peuvent être mesurés en déterminant ARNm demi-vies. Ce protocole met l'accent sur ​​la mesure du taux d'ARNm de désintégration dans Saccharomyces cerevisiae. 