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 standard procedures 10. If no selection is required, prepare rich media such as YPD. Alternatively, prepare selective media if the yeast cells are transformed with plasmids and selection is required. Autoclave the media.</li>
	<li>Grow the yeast cells at 28 &#176;C, which is the permissive temperature for this yeast strain. Do this in two steps:
	<ol>
		<li>For the first O/N, grow the yeast cells in 5 ml of growth media to saturation.</li>
		<li>Set up the second O/N at the end of the second day. For the second O/N, inoculate different amounts of yeast cells from the first O/N into 100 to 150 ml of growth media (ranging from 100 &#181;l to 1 ml, 2 ml or more, depending on when the yeast cells need to be harvested the following day). Do this step to ensure that one of the cultures is at the correct OD600 the following day.</li>
	</ol>
	</li>
</ol>
2. Harvest the Yeast Cells
<ol>
	<li>Prepare to harvest the yeast cells . The timing for this step is critical; label all the required tubes and gather all required equipment and materials in one place. Preheat a water bath to 39 &#176;C and preheat two 15 ml test tubes of the growth media at 28 &#176;C and at 60 &#176;C. 
	NOTE: The 15 ml growth media volume can be varied depending on the number of time points the cells are to be harvested.</li>
	<li>Harvest the yeast cells when they reach an OD600 of 0.4 to 0.7. Transfer the cells to 4 - 6 sterile 50 ml screw cap bottles. Centrifuge at 7,500 x g in a high speed centrifuge for 5 min at RT.</li>
	<li>Discard the supernatant and resuspend the pellets in the 15 ml growth media that was equilibrating at 28 &#176;C. Pool the yeast cells in one sterile 250 ml flask and equilibrate them for ~5 min at the 28 &#176;C growth temperature.</li>
	<li>After the yeast cells have equilibrated at the growth temperature, immediately add the 15 ml of the growth media equilibrated at 60 &#176;C to raise the temperature to 39 &#176;C (the nonpermissive temperature) and immediately place the flask in the 39 &#176;C water bath. Make sure that the culture medium remains in the 39 &#176;C water bath throughout the remaining cell harvesting steps to ensure that transcription is turned off.</li>
	<li>Harvest the cells at different times after placing the flask in the 39 &#176;C water bath. For the first time point, harvest the cells immediately after placing the flask at 39 &#176;C. Harvest a 3 ml aliquot and distribute the 3 ml into two, 1.5 ml microcentrifuge tubes. Pellet the cells for 10 sec in a mini centrifuge or picofuge with rapid deceleration and pour out the supernatant.</li>
	<li>Immediately freeze the pellet in a dry ice/ethanol bath or in liquid nitrogen. Designate the first time point the cells are harvested at as the zero time point.</li>
	<li>Experimentally determine the time points the cells are harvested at thereafter by a pilot experiment, depending on the mRNAs of interest. Normally, harvest yeast cells at the following time points: 0, 3, 6, 9, 12, 18, 25 and 35 min (Figure 2B). If the mRNA half-life cannot be determined using the above mentioned time points, adjust these time points. For example, for mRNAs with anticipated short half-lives, use shorter times points, (i.e., 0, 1, 2, 3, 4, 6, 9, 12 and 18 min) and for mRNAs with anticipated long half-lives, extend the time points.</li>
	<li>After the cells are harvested, store them in a -80 &#176;C freezer until RNA extraction is carried out.</li>
</ol>
3. Extract RNA from the Yeast Cells
<ol>
	<li>For the RNA extraction portion of the protocol, use RNase free techniques to prevent degradation of the RNA by RNases. Prepare RNase free solutions, glassware and plastic ware to be used in the extraction of the RNA.</li>
	<li>Extract the RNA from the yeast cells according to standard protocols. Typically, use the hot phenol method 12. Alternatively, use kits that can be used to extract RNA from yeast cells.</li>
	<li>Determine the quantity and purity of the RNA, normally by measuring the absorbance at A260 and A280 of 2 &#181;l of RNA using a Nanodrop. Determine the concentration of the RNA from the A260. Based on the concentration, dilute the RNA to 1 &#181;g/&#181;l using DEPC treated water. 
	NOTE: The ratio of the A260/A280 provides information on the purity of the RNA. An RNA sample is pure if the A260/A280 ratio is 2.0 &#177; 0.1.</li>
</ol>
4. Northern Blot Analysis
NOTE: Use northern blots to quantify mRNA levels and obtain information on the size of the transcripts. In addition, use northern blots to detect mRNAs that produce multiple isoforms of the same mRNA.
<ol>
	<li>Prepare a 1.0% agarose-formaldehyde gel. Run equal amounts of the sample RNA on the agarose formaldehyde gel by electrophoresis according to standard protocols 12. Run a RNA ladder alongside the RNA samples and use it to determine the sizes of the RNA that are detected on the northern blot. Before transferring the RNA separated on the agarose formaldehyde gel to a membrane, cut the lane with the RNA ladder from the gel and visualize using ethidium bromide. 
	NOTE: Alternatively, the whole gel can be stained with ethidium bromide before transfer of the RNA.</li>
	<li>After the RNA samples have migrated to an appropriate distance on the gel, transfer the RNA to a membrane using RNase free techniques. Use one of several protocols available to transfer RNA to membranes 12-13. To transfer the RNA to a membrane, cut the membrane to approximately the same size as the gel. Transfer the RNA according to standard protocols 12.</li>
	<li>UV cross-link the RNA to the membrane using a UV crosslinker following the manufacturer&#8217;s instructions. Alternatively, bake the membrane in an oven set to 80 &#176;C for 1 hr. Store the membrane at -20 &#176;C in a plastic bag indefinitely until it is hybridized to specific probes. 
	NOTE: The dry membrane can also be stored at RT between filter papers.</li>
</ol>
5. Hybridize Probes Complementary to the RNA of Interest to the Membrane
NOTE: One way to detect mRNA on the membrane is to hybridize 32P labeled DNA probes. CAUTION: Researchers working with 32P need to use protective procedures to prevent contamination. Follow institutional guidelines on use of radioactive material.
<ol>
	<li>Prepare the DNA probe by labeling 25 - 50 ng of the DNA to be hybridized to the membrane according to standard protocols 12. Generate the DNA fragment by PCR or plasmid digestion. Determine the specific activity of the DNA probe by counting 1 &#181;l of the radiolabelled DNA probe after the unincorporated nucleotides are removed 12.</li>
	<li>Preheat prehybridization/hybridization buffer at 42 &#176;C. Use ~10 ml of the prehybridization/hybridization buffer per 100 cm2 of the membrane 12.</li>
	<li>Hybridize 1 - 5 x 106 cpm of radiolabeled DNA probe per ml of hybridization buffer to the membrane O/N in a hybridization oven to detect the mRNA of interest 12. Make sure that during the hybridization the hybridization oven rotation is in a direction that causes the entire membrane to be exposed to the hybridization solution, to ensure proper hybridization.</li>
	<li>Wash the membrane two times at RT for 15 min with 50 ml of 2x SSPE and one time at 65 &#176;C with 50 ml of 2x SSPE/2% SDS for 15 min 12. Wrap the membrane in plastic wrap to ensure that it does not dry out or contaminate the Phosphor screen.</li>
	<li>Using a Geiger counter, estimate the intensity of the radioactivity on the membrane. 
	NOTE: This estimate ensures that the membrane is exposed to the Phosphor screen for the appropriate amount of time. Use longer exposure times for membranes with low amounts of radioactivity.</li>
	<li>For northern blots, use a loading control to confirm that equal amounts of RNA are loaded on each spot. To do this, strip the membrane and reprobe it with an RNA that is not affected by the process being examined. Strip the membrane by placing it in a glass tray containing 200 ml of boiling stripping solution (0.1% SDS/0.01 X SSC) for 2 min. Pour out the stripping solution and repeat the procedure five times 12. 
	NOTE: An example of an RNA used as a loading control is SCR1. SCR1 is an RNA polymerase III transcript that is stable and abundant. SCR1 RNA abundance should not change significantly during short periods of RNA polymerase II inhibition.</li>
</ol>
6. Quantify the RNA that is Bound to the Membrane
NOTE: To quantify the amount of radioactivity on membrane, expose the membrane to a Phosphor screen. After the appropriate exposure time, scan the Phosphor screen using a phosphorimager.
<ol>
	<li>Scan the Phosphor screen using the instructions provided by the manufacturer of the phosphorimager.</li>
	<li>Quantify the mRNA at each time point. Quantify mRNA levels using ImageQuant software or related software. Normalize the mRNA at each time point to the loading control. If SCR1 was used as the loading control, normalize the half-life northerns to SCR1.</li>
	<li>The half-life of the mRNA is calculated by dividing the amount of mRNA remaining at each time point by the amount of mRNA present at time point 0 (the initial time point). Graph the percent mRNA remaining versus time on a semilogarithmic plot (Figure 2C). Calculate mRNA half-lives by the following equation: 
	t1/2 = -0.693/k (negative 0.693)</li>
	<li>k is the slope of the best fit line and t1/2 is the mRNA half-life.</li>
</ol>

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	<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. 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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. 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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><tbody><tr><td>High Speed Centrifuge</td><td>Eppendorf</td><td>22628169</td><td></td></tr><tr><td>Mini Centrifuge</td><td>Fisher Scientific</td><td>05-090-100</td><td></td></tr><tr><td>Genescreen Plus membrane</td><td>PerkinElmer</td><td>50-905-0169</td><td></td></tr><tr><td>Hybridization Oven</td><td>Fisher Scientific</td><td>95-0030-01</td><td></td></tr><tr><td>Nanodrop spectrophotometer</td><td>Thermo Scientific</td><td>ND-8000</td><td></td></tr><tr><td>Phosphor screen</td><td>GE Healthcare Life Sciences</td><td>28-9564-78</td><td></td></tr><tr><td>Typhoon phosphorimager</td><td>GE Healthcare Life Sciences</td><td>29004080</td><td></td></tr><tr><td>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&amp;deg; for 1 hour</td></tr><tr><td>NorthernMax prehybridization/hybridization buffer&amp;nbsp;</td><td>Life Technologies</td><td>AM8677</td><td></td></tr><tr><td>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><td></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 ...

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Translation: RNA to Protein

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12.5K Views.  Baylor University.  Het steady state niveau van specifieke mRNA's wordt bepaald door de snelheid van synthese en afbraak van het mRNA. Genoom-brede mRNA degradatie tarieven of de vervalsnelheid van specifieke mRNA's kan worden gemeten door het bepalen van mRNA halfwaardetijden. Dit protocol richt zich op het meten van mRNA vervalsnelheden in Saccharomyces cerevisiae. 

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

Measuring mRNA Levels Over Time During the Yeast S. cerevisiae Hypoxic Response

Complex changes in gene expression typically mediate a large portion of a cellular response. Each gene may change expression with unique kinetics as the gene is regulated by the particular timing of one of many stimuli, signaling pathways or secondary effects. In order to capture the entire gene expression response to hypoxia in the yeast S. cerevisiae, RNA-seq analysis was used to monitor the mRNA levels of all genes at specific times after exposure to hypoxia. Hypoxia was established by growing cells in ~100% N2 gas. Importantly, unlike other hypoxic studies, ergosterol and unsaturated fatty acids were not added to the media because these metabolites affect gene expression. Time points were chosen in the range of 0 - 4 h after hypoxia because that period captures the major changes in gene expression. At each time point, mid-log hypoxic cells were quickly filtered and frozen, limiting exposure to O2 and concomitant changes in gene expression. Total RNA was extracted from cells and used to enrich for mRNA, which was then converted to cDNA. From this cDNA, multiplex libraries were created and eight or more samples were sequenced in one lane of a next-generation sequencer. A post-sequencing pipeline is described, which includes quality base trimming, read mapping and determining the number of reads per gene. DESeq2 within the R statistical environment was used to identify genes that change significantly at any one of the hypoxic time points. Analysis of three biological replicates revealed high reproducibility, genes of differing kinetics and a large number of expected O2-regulated genes. These methods can be used to study how the cells of various organisms respond to hypoxia over time and adapted to study gene expression during other cellular responses.

Measuring mRNA Levels Over Time During the Yeast S. cerevisiae Hypoxic Response

Here, we present a protocol using RNA-seq to monitor mRNA levels over time during the hypoxic response of S. cerevisiae cells. This method can be adapted to analyze gene expression during any cellular response.

Complex changes in gene expression typically mediate a large portion of a cellular response. Each gene may change expression with unique kinetics as the ...

Genetics

Determining Genome-wide Transcript Decay Rates in Proliferating and Quiescent Human Fibroblasts

Quiescence is a temporary, reversible state in which cells have ceased cell division, but retain the capacity to proliferate. Multiple studies, including ours, have demonstrated that quiescence is associated with widespread changes in gene expression. Some of these changes occur through changes in the level or activity of proliferation-associated transcription factors, such as E2F and MYC. We have demonstrated that mRNA decay can also contribute to changes in gene expression between proliferating and quiescent cells. In this protocol, we describe the procedure for establishing proliferating and quiescent cultures of human dermal foreskin fibroblasts. We then describe the procedures for inhibiting new transcription in proliferating and quiescent cells with Actinomycin D (ActD). ActD treatment represents a straightforward and reproducible approach to dissociating new transcription from transcript decay. A disadvantage of ActD treatment is that the time course must be limited to a short time frame because ActD affects cell viability. Transcript levels are monitored over time to determine transcript decay rates. This procedure allows for the identification of genes and isoforms that exhibit differential decay in proliferating versus quiescent fibroblasts.

We describe a protocol for generating proliferating and quiescent primary human dermal fibroblasts, monitoring transcript decay rates, and identifying differentially decaying genes.

Quiescence is a temporary, reversible state in which cells have ceased cell division, but retain the capacity to proliferate. Multiple studies, including ...

Genome-wide Quantification of Translation in Budding Yeast by Ribosome Profiling

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription reflect changes in protein synthesis, but many exceptions have been observed. Recently, a technique called ribosome profiling (or Ribo-Seq) has emerged as a powerful method that allows identification, with high accuracy, which regions of mRNA are translated into proteins and quantification of translation at the genome-wide level. Here, we present a generalized protocol for genome-wide quantification of translation using Ribo-Seq in budding yeast. In addition, combining Ribo-Seq data with mRNA abundance measurements allows us to simultaneously quantify translation efficiency of thousands of mRNA transcripts in the same sample and compare changes in these parameters in response to experimental manipulations or in different physiological states. We describe a detailed protocol for generation of ribosome footprints using nuclease digestion, isolation of intact ribosome-footprint complexes via sucrose gradient fractionation, and preparation of DNA libraries for deep sequencing along with appropriate quality controls necessary to ensure accurate analysis of in vivo translation.

Translational regulation plays an important role in the control of protein abundance. Here, we describe a high-throughput method for quantitative analysis of translation in the budding yeast Saccharomyces cerevisiae.

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription ...

Biochemistry

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in mRNA synthesis has been shown to be compensated by a simultaneous decrease in mRNA degradation to restore normal steady-state levels. Hence, the genome-wide quantification of mRNA synthesis, independently from mRNA decay, is the best direct reflection of RNA polymerase II transcriptional activity. Here, we discuss a method using non-perturbing metabolic labeling of nascent RNAs in Saccharomyces cerevisiae (S. cerevisiae). Specifically, the cells are cultured for 6 min with a uracil analog, 4-thiouracil, and the labeled newly transcribed RNAs are purified and quantified to determine the synthesis rates of all individual mRNA. Moreover, using labeled Schizosaccharomyces pombe cells as internal standard allows comparing mRNA synthesis in different S. cerevisiae strains. Using this protocol and fitting the data with a dynamic kinetic model, the corresponding mRNA decay rates can be determined.

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

The protocol described here is based on the genome-wide quantification of newly synthesized mRNA purified from yeast cells labeled with 4-thiouracil. This method allows to measure mRNA synthesis uncoupled from mRNA decay and, thus, provides an accurate measurement of RNA polymerase II transcription.

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in ...

A Suppressor Screen for the Characterization of Genetic Links Regulating Chronological Lifespan in Saccharomyces cerevisiae

Aging is the time dependent deterioration of an organism&#8217;s normal biological processes that increases the probability of death. Many genetic factors contribute to alterations in the normal aging process. These factors intersect in complex ways, as evidenced by the wealth of documented links identified and conserved in many organisms. Most of these studies focus on loss-of-function, null mutants that allow for rapid screening of many genes simultaneously. There is much less work that focuses on characterizing the role that overexpression of a gene in this process. In the present work, we present a straightforward methodology to identify and clone genes in the budding yeast, Saccharomyces cerevisiae, for study in suppression of the short-lived chronological lifespan phenotype seen in many genetic backgrounds. This protocol is designed to be accessible to researchers from a wide variety of backgrounds and at various academic stages. The SIR2 gene, which codes for a histone deacetylase, was selected for cloning in the pRS315 vector, as there have been conflicting reports on its effect on the chronological lifespan. SIR2 also plays a role in autophagy, which results when disrupted via the deletion of several genes, including the transcription factor ATG1. As a proof of principle, we clone the SIR2 gene to perform a suppressor screen on the shortened lifespan phenotype characteristic of the autophagy deficient atg1&#916; mutant and compare it to an otherwise isogenic, wild type genetic background.

A Suppressor Screen for the Characterization of Genetic Links Regulating Chronological Lifespan in Saccharomyces cerevisiae

Here is a protocol to identify genetic interactions through an increased copy number suppressor screen in Saccharomyces cerevisiae. This method allows researchers to identify, clone, and test suppressors in short-lived yeast mutants. We test the effect of the copy number increase of SIR2 on lifespan in an autophagy null mutant.

Aging is the time dependent deterioration of an organism&#8217;s normal biological processes that increases the probability of death. Many genetic factors ...

Monitoring Protein-RNA Interaction Dynamics In Vivo at High Temporal Resolution Using &#967;CRAC

The interaction between RNA-binding proteins (RBPs) and their RNA substrates exhibits fluidity and complexity. Within its lifespan, a single RNA can be bound by many different RBPs that will regulate its production, stability, activity, and degradation. As such, much has been done to understand the dynamics that exist between these two types of molecules. A particularly important breakthrough came with the emergence of &#8216;cross-linking and immunoprecipitation&#8217; (CLIP). This technique allowed stringent investigation into which RNAs are bound by a particular RBP. In short, the protein of interest is UV cross-linked to its RNA substrates in vivo, purified under highly stringent conditions, and then the RNAs covalently cross-linked to the protein are converted into cDNA libraries and sequenced. Since its conception, many derivative techniques have been developed in order to make CLIP amenable to particular fields of study. However, cross-linking using ultraviolet light is notoriously inefficient. This results in extended exposure times that make the temporal study of RBP-RNA interactions impossible. To overcome this issue, we recently designed and built much-improved UV irradiation and cell harvesting devices. Using these new tools, we developed a protocol for time-resolved analyses of RBP-RNA interactions in living cells at high temporal resolution: Kinetic CRoss-linking and Analysis of cDNAs (&#967;CRAC). We recently used this technique to study the role of yeast RBPs in nutrient stress adaptation. This manuscript provides a detailed overview of the &#967;CRAC method and presents recent results obtained with the Nrd1 RBP.

Kinetic cross-linking and analysis of cDNA is a method that allows investigation of the dynamics of protein-RNA interactions in living cells at high temporal resolution. Here the protocol is described in detail, including the growth of yeast cells, UV cross-linking, harvesting, protein purification, and next generation sequencing library preparation steps.

The interaction between RNA-binding proteins (RBPs) and their RNA substrates exhibits fluidity and complexity. Within its lifespan, a single RNA can be ...

In Vivo Monitoring of Transcriptional Activity During Metabolic Transition Using a Bioluminescent Reporter in Yeast

Sequential sugar consumption, from a preferred sugar source to a less preferred one, represents a critical metabolic adaptation in yeast, which is particularly relevant for survival in fluctuating environments such as those found in beer fermentation. However, sugar transitions are an environmental variable that is challenging to predict and detect, impacting the outcome of beer fermentations. This protocol describes an in vivo system to monitor transcriptional activation associated with the glucose-to-maltose metabolic shift in Saccharomyces eubayanus that applies to different wild Saccharomyces yeast strains. 
The system employs an episomal bioluminescent transcriptional reporter for maltose metabolism, focusing on MAL32, since it provides a good readout for metabolic shifts, as studied in S. cerevisiae. For this, yeast strains were transformed with plasmids containing the MAL32 regulatory region from S. eubayanus, controlling the expression of a gene encoding for a destabilized version of firefly luciferase1, and a hygromycin resistance gene used exclusively during transformation to ensure plasmid acquisition. Following selection, transformed yeast cells can be cultured under non-selective conditions, as the episomal plasmid remains stable in culture conditions for up to 7 days. 
This system was validated under a complex sugar environment in microfermentation assays, confirming the effectiveness of the luciferase reporter in informing metabolic transitions. Samples were collected regularly and analyzed with a luminometer, providing continuous insights into yeast responses. While broadly applicable, this protocol is particularly valuable for assessing yeast performance under fermentation conditions, where metabolic changes pose a significant challenge. Additionally, this methodology can be adapted by selecting alternative promoters to explore a broader range of responses to environmental changes, allowing characterization as well as optimization of wild yeast strains for diverse industrial applications.

This protocol employs a bioluminescent reporter, allowing measurements of transcriptional activity in Saccharomyces eubayanus to monitor the glucose-to-maltose transition, enabling real-time analysis of metabolic adaptations and supporting strain optimization for industrial fermentation under diverse conditions.

Sequential sugar consumption, from a preferred sugar source to a less preferred one, represents a critical metabolic adaptation in yeast, which is ...

Combining Magnetic Sorting of Mother Cells and Fluctuation Tests to Analyze Genome Instability During Mitotic Cell Aging in Saccharomyces cerevisiae

Saccharomyces cerevisiae has been an excellent model system for examining mechanisms and consequences of genome instability. Information gained from this yeast model is relevant to many organisms, including humans, since DNA repair and DNA damage response factors are well conserved across diverse species. However, S. cerevisiae has not yet been used to fully address whether the rate of accumulating mutations changes with increasing replicative (mitotic) age due to technical constraints. For instance, measurements of yeast replicative lifespan through micromanipulation involve very small populations of cells, which prohibit detection of rare mutations. Genetic methods to enrich for mother cells in populations by inducing death of daughter cells have been developed, but population sizes are still limited by the frequency with which random mutations that compromise the selection systems occur. The current protocol takes advantage of magnetic sorting of surface-labeled yeast mother cells to obtain large enough populations of aging mother cells to quantify rare mutations through phenotypic selections. Mutation rates, measured through fluctuation tests, and mutation frequencies are first established for young cells and used to predict the frequency of mutations in mother cells of various replicative ages. Mutation frequencies are then determined for sorted mother cells, and the age of the mother cells is determined using flow cytometry by staining with a fluorescent reagent that detects bud scars formed on their cell surfaces during cell division. Comparison of predicted mutation frequencies based on the number of cell divisions to the frequencies experimentally observed for mother cells of a given replicative age can then identify whether there are age-related changes in the rate of accumulating mutations. Variations of this basic protocol provide the means to investigate the influence of alterations in specific gene functions or specific environmental conditions on mutation accumulation to address mechanisms underlying genome instability during replicative aging.

Combining Magnetic Sorting of Mother Cells and Fluctuation Tests to Analyze Genome Instability During Mitotic Cell Aging in Saccharomyces cerevisiae

Mutation rates in young Saccharomyces cerevisiae cells measured through fluctuation tests are used to predict mutation frequencies for mother cells of different replicative ages. Magnetic sorting and flow cytometry are then used to measure actual mutation frequencies and age of mother cells to identify any deviations from predicted mutation frequencies.

Saccharomyces cerevisiae has been an excellent model system for examining mechanisms and consequences of genome instability. Information gained from this ...

Reporter-based Growth Assay for Systematic Analysis of Protein Degradation

Protein degradation by the ubiquitin-proteasome system (UPS) is a major regulatory mechanism for protein homeostasis in all eukaryotes. The standard approach to determining intracellular protein degradation relies on biochemical assays for following the kinetics of protein decline. Such methods are often laborious and time consuming and therefore not amenable to experiments aimed at assessing multiple substrates and degradation conditions. As an alternative, cell growth-based assays have been developed, that are, in their conventional format, end-point assays that cannot quantitatively determine relative changes in protein levels.
Here we describe a method that faithfully determines changes in protein degradation rates by coupling them to yeast cell-growth kinetics. The method is based on an established selection system where uracil auxotrophy of URA3-deleted yeast cells is rescued by an exogenously expressed reporter protein, comprised of a fusion between the essential URA3 gene and a degradation determinant (degron). The reporter protein is designed so that its synthesis rate is constant whilst its degradation rate is determined by the degron. As cell growth in uracil-deficient medium is proportional to the relative levels of Ura3, growth kinetics are entirely dependent on the reporter protein degradation.
This method accurately measures changes in intracellular protein degradation kinetics. It was applied to: (a) Assessing the relative contribution of known ubiquitin-conjugating factors to proteolysis (b) E2 conjugating enzyme structure-function analyses (c) Identification and characterization of novel degrons. Application of the degron-URA3-based system transcends the protein degradation field, as it can also be adapted to monitoring changes of protein levels associated with functions of other cellular pathways.

Here we describe a robust biological assay for quantifying the relative rate of proteolysis by the ubiquitin-proteasome system. The assay readout is yeast growth rate in liquid culture, which is dependent on the cellular levels of a reporter protein comprising a degradation signal fused to an essential metabolic marker.

Protein degradation by the ubiquitin-proteasome system (UPS) is a major regulatory mechanism for protein homeostasis in all eukaryotes. The standard ...

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic requirements for eukaryotic protein degradation was initially established in Saccharomyces cerevisiae. Classical analyses of protein degradation have relied on biochemical pulse-chase and cycloheximide-chase methodologies. While these techniques provide sensitive means for observing protein degradation, they are laborious, time-consuming, and low-throughput. These approaches are not amenable to rapid or large-scale screening for mutations that prevent protein degradation. Here, a yeast growth-based assay for the facile identification of genetic requirements for protein degradation is described. In this assay, a reporter enzyme required for growth under specific selective conditions is fused to an unstable protein. Cells lacking the endogenous reporter enzyme but expressing the fusion protein can grow under selective conditions only when the fusion protein is stabilized (i.e. when protein degradation is compromised). In the growth assay described here, serial dilutions of wild-type and mutant yeast cells harboring a plasmid encoding a fusion protein are spotted onto selective and non-selective medium. Growth under selective conditions is consistent with degradation impairment by a given mutation. Increased protein abundance should be biochemically confirmed. A method for the rapid extraction of yeast proteins in a form suitable for electrophoresis and western blotting is also demonstrated. A growth-based readout for protein stability, combined with a simple protocol for protein extraction for biochemical analysis, facilitates rapid identification of genetic requirements for protein degradation. These techniques can be adapted to monitor degradation of a variety of short-lived proteins. In the example presented, the His3 enzyme, which is required for histidine biosynthesis, was fused to Deg1-Sec62. Deg1-Sec62 is targeted for degradation after it aberrantly engages the endoplasmic reticulum translocon. Cells harboring Deg1-Sec62-His3 were able to grow under selective conditions when the protein was stabilized.

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

This article describes a yeast growth-based assay for the determination of genetic requirements for protein degradation. It also demonstrates a method for rapid extraction of yeast proteins, suitable for western blotting to biochemically confirm degradation requirements. These techniques can be adapted to monitor degradation of a variety of proteins.

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

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