Name: saccharomyces cerevisiae metabolic labeling with 4-thiouracil and the quantification of newly synthesized mrna as a proxy for rna polymerase ii activity
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We thank Laszlo Tora for his support and V. Fisher, K. Schumacher, and F. El Saafin for their discussions. T.B. was supported by a Marie Curie-ITN fellowship (PITN-GA-2013-606806, NR-NET) and the Fondation ARC. This work was supported by funds from the Agence Nationale de la Recherche (ANR-15-CE11-0022 SAGA2). This study was also supported by ANR-10-LABX-0030-INRT, a French State fund managed by the Agence Nationale de la Recherche under the frame program Investissements d'Avenir ANR-10-IDEX-0002-02.

1. Cell Culturing and Rapamycin Depletion of a SAGA Subunit
<ol>
	<li>For each S. cerevisiae strain and replicate, including wild-type or control strains, inoculate a single colony from a fresh plate onto 5 mL of YPD medium (2% Peptone, 1% yeast extract, and 2% glucose).</li>
	<li>Grow S. cerevisiae cells overnight at 30 &#176;C with constant agitation (150 rpm).</li>
	<li>Measure the optical density at 600 nm (OD600) and dilute the culture to an OD600 of approximately 0.1 in 100...

<ol>
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While genome-wide tools to analyze changes in transcription are still improving, the sole analysis of the transcriptome through the quantification of steady-state levels of RNA might not accurately reflect changes in RNA polymerase II activity. Indeed, mRNA levels are regulated not only by RNA synthesis but also by their maturation and degradation. To measure mRNA synthesis uncoupled from mRNA degradation, distinct protocols have been developed in recent years for the analysis of nascent transcription in both yeast and m...

Cells respond to endogenous and exogenous cues, through the dynamic alteration of their gene expression program. In recent years, a tremendous development of genome-wide methodologies allows the precise and comprehensive description of transcriptome changes in different conditions. In most transcriptomic studies, microarray hybridization or high-throughput sequencing are used to quantify RNA levels from a total steady-state RNA fraction. Transcriptional changes under a specific perturbation can display a wide range of possible outcomes, with either specific gene expression changes or a large spectrum of genes being either up- or downregulated. Gene expression is the result of a fine-tuned equilibrium &#8212; or steady-state &#8212; between RNA synthesis by RNA polymerases and other processes affecting RNA levels. RNA polymerase II transcription, including its three distinct phases (initiation, elongation, and termination), is highly and intricately associated with mRNA processing, cytoplasmic export, translation, and degradation.
Several recent studies demonstrated that mRNAs synthesis and decay are coupled mechanisms and showed that transcriptional effects upon mutation or under stimuli can be overlooked when quantifying total steady-state RNA. First, the detection of transcriptional changes through the analyses of steady-state levels of mRNA always depends on mRNAs half-life. Once the perturbation is introduced, the steady-state levels of mRNAs with long half-lives will be much less affected than those of mRNAs with short half-lives. Therefore, the detectability of the changes in RNA synthesis is strongly biased in favor of short-lived transcripts, while the analysis of longer-lived mRNA species might fail to reveal dynamic changes in transcription rate. Second, several reports have shown that, both in yeast and mammals, global changes in transcription might be overlooked when analyzing the steady-state levels of mRNA. This is likely due to the mechanisms that link mRNA synthesis and degradation resulting in mRNA buffering. This prompted the development of new protocols to quantify mRNA synthesis uncoupled from degradation, through the analysis of newly transcribed mRNA. In recent years, several alternatives have been presented, including global run-on sequencing (GRO-seq)1, and native elongation transcript sequencing (NET-seq)2,3. Here, we are presenting a protocol initially developed in mammalian cells4,5,6 and then adapted to yeast7,8,9,10,11, which is based on RNA labeling with a thiolated nucleoside or base analog, 4-thiouridine (4sU) or 4-thiouracil (4tU), respectively.
This method specifically purifies newly transcribed RNA from the cells in which RNA are pulse-labeled with 4sU with virtually no interference in the cell homeostasis. Hence, once the cells are exposed to 4sU, the molecule is rapidly uptaken, phosphorylated to 4sU-triphosphate, and incorporated in RNAs being transcribed. Once pulse-labeled, it is possible to extract total cellular RNA (corresponding to steady-state levels of RNA), and, subsequently, the 4sU-labeled RNA fraction is thiol-specifically modified, leading to the formation of a disulfide bond between biotin and the newly transcribed RNA4,5. However, 4sU can only be uptaken by the cells expressing a nucleoside transporter, like the human equilibrative nucleoside transporter (hENT1), preventing its immediate use in budding or fission yeast. While one could express hENT1 in either S. pombe or S. cerevisiae, an easier approach can be achieved using the modified base 4tU, since yeast cells can take up 4tU, without the need of expression of a nucleoside transporter10,11,12,13. In fact, the metabolism of 4tU requires the activity of the enzyme uracil phosphoribosyltransferase (UPRT). In several organisms, including yeast but not mammals, UPRT is essential for a pyrimidine salvage pathway, recycling uracil to uridine monophosphate.
An important bias in transcriptomic studies can be introduced by the normalization between different samples analyzed in parallel. Indeed, many deviating factors can affect the comparative analysis of the transcriptome of mutant and wild-type strains: the efficiency of cell lysis, differences in the extraction and recovery of RNA, and variances in scanner calibration for microarray analyses, among others. As discussed above, such variations can be particularly misleading when global effects on RNA polymerase II transcription are expected. An elegant mean to accurately compare mRNA synthesis rates between different samples was designed by using the distantly related fission yeast Schizosaccharomyces pombe as an internal standard. For that, a fixed number of labeled S. pombe cells is added to the S. cerevisiae samples, either wild-type or mutant cells, prior to cell lysis and RNA extraction10. Subsequently, both steady-state and newly synthesized RNAs from S. pombe and S. cerevisiae are quantified either by RT-qPCR or via the use of microarray chips or high-throughput sequencing10. Combining these data with kinetic modeling, absolute rates of mRNA synthesis and decay in budding yeast can be measured.
In the framework of this manuscript, we will show how the analysis of newly transcribed RNA allowed to reveal a global role for the coactivator complexes SAGA and TFIID in RNA polymerase II transcription in budding yeast14,15,16. Importantly, past studies quantified steady-state mRNA levels in S. cerevisiae and suggested that SAGA plays a predominant function on a limited set of yeast genes which are strongly affected by mutations in SAGA but relatively resistant to TFIID mutations17,18,19. Surprisingly, the SAGA enzymatic activities were shown to act on the whole transcribed genome, suggesting a broader role for this co-activator in RNA polymerase II transcription. Decreased RNA polymerase II recruitment at most expressed genes was observed upon the inactivation of SAGA or TFIID, suggesting that these coactivators work together at most genes. Hence, the quantification of newly transcribed mRNA revealed that SAGA and TFIID are required for the transcription of nearly all genes by RNA polymerase II14,15,16. The implementation of compensatory mechanisms emerges as a way for the cells to cope with a global decrease in mRNA synthesis which is buffered by a simultaneous global decrease in mRNA degradation. SAGA adds to the list of factors having a global effect on RNA polymerase II transcription, such as RNA Pol II subunits10, the Mediator coactivator complex20, the general transcription factor TFIIH21,22, and indirectly, elements of the mRNA degradation machinery9,10,23. Such compensatory events were universally observed in SAGA mutants, accounting for the modest and limited changes in steady-state mRNA levels despite a global and severe decrease in mRNA synthesis14. Similar analyses were also performed in a BRE1 deletion strain, resulting in a complete loss of histone H2B ubiquitination. Interestingly, a much milder but consistent global effect on RNA polymerase II transcription could be detected in the absence of Bre1, indicating that metabolic labeling of newly transcribed RNA in yeast can detect and quantify a wide range of changes in mRNA synthesis rates.

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			<td height="23" style="height:23px;width:216px;">4-Thiouracil</td>
			<td style="width:119px;">Sigma-Aldrich</td>
			<td style="width:139px;">Cat# 440736</td>
			<td style="width:167px;"></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Rapamycin</td>
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			<td height="35" style="height:35px;width:216px;">Countess II FL Automated Cell Counter</td>
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			<td height="36" style="height:36px;width:216px;">RiboPure RNA Purification kit, yeast</td>
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			<td style="width:139px;">Cat# AM1926</td>
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			<td height="22" style="height:22px;width:216px;">NanoDrop 2000 Spectrophotometer</td>
			<td style="width:119px;">ThermoFisher</td>
			<td style="width:139px;">ND-2000</td>
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			<td height="22" style="height:22px;width:216px;">TURBO DNA-free Kit</td>
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			<td style="width:139px;">AM1907</td>
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			<td height="22" style="height:22px;width:216px;">EZ-Link HPDP Biotin</td>
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			<td height="22" style="height:22px;width:216px;">Thiolutin</td>
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			<td height="22" style="height:22px;width:216px;">&#181;MACS Streptavidin kit</td>
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			<td style="width:119px;">Roche</td>
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			<td height="22" style="height:22px;width:216px;">SYBR Green I Master</td>
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			<td height="22" style="height:22px;width:216px;">GeneChip Yeast Genome 2.0</td>
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			<td style="width:139px;">00-0210</td>
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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.

When performing metabolic labeling of newly transcribed RNA, several aspects need to be controlled: the time and efficiency of the labeling, the spike-in proportion, the extraction protocol, and the biotinylation efficacy (including signal-to-noise ratio), among others. These conditions have been extensively and methodically shown by others7,10,11. Here we mainly focus on the interpretation and i...

Watch this Scientific Journal Video about Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity at Jo

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.

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 ...

The main advantage of this technique is that it reflects RNA polymerase II activity by measuring newly-synthesized mRNA which are not influenced by RNA degradation. This protocol was originally described using microarray hybridization, but can easily be adopted to high-throughout sequencing of newly-transcribed mRNA. Demonstrating this procedure will be Tiago Baptista, a former grad student from our laboratory.After inoculating the S.cerevisiae cells, grow them overnight at 30 degrees Celsius with constant agitation at 150 RPM. The next day, measure the optical density at 600 nanometers. Dilute the culture to an OD600 of approximately 0.1 in 100 milliliters of YPD medium.Grow up the cells until the OD600 is around 0.8. After this, measure the OD600 of the S.pombe overnight culture. Dilute this culture to an OD600 of approximately 0.1 in 500 milliliters of YAS medium and let it grow up until the OD600 is approximately 0.8.First, prepare a fresh solution of two molar four-thiouracil. Keep the prepared solution at room temperature and protected from light. Add the four-thiouracil solution to the S.cerevisiae and S.pombe cultures such that the final concentration is five millimolar.Incubate the S.cerevisiae and S.pombe cultures at 30 degrees Celsius and 32 degrees Celsius respectively with constant agitation for six minutes. After this, remove a small aliquot of each culture and count the cells using either an automatic cell counter or a Neubauer chamber. Centrifuge at 2, 500 times g and four degrees Celsius for five minutes to collect the cells.Discard the supernatant and wash the cells with ice cold one times PBS. Centrifuge again at 2, 500 times g and four degrees Celsius for five minutes. Next, resuspend the cells in five milliliters of ice cold one times PBS.Mix the S.cerevisiae and S.pombe cells at a ratio of three to one. Centrifuge the mixed samples at 2, 500 times g and four degrees Celsius for five minutes. Then remove the PBS and flash freeze the cells in liquid nitrogen.Store the sample at negative 80 degrees Celsius until ready to use. When ready to proceed, thaw the cells on ice for approximately 20 to 30 minutes. Use an adapted yeast RNA extraction kit to extract the RNA as outlined in the text protocol.After this, transfer the filter cartridge to the final collection tube. Elute the RNA with 50 microliters of DEPC-treated RNase-free water that was preheated to 100 degrees Celsius. Centrifuge at 16, 000 times g for one minute.Then use 50 microliters of preheated DEPC-treated RNase-free water to elute the RNA again to the same tube. Centrifuge at 16, 000 times g for one minute making sure that the entire sample volume has passed through the filter. If it has not, centrifuge again for a longer period of time.When finished, quantify and check the purity of the sample using the appropriate equipment. Use DEPC-treated nuclease-free water to adjust the concentration of the previously acquired RNA to two milligrams per milliliter. Aliquot 200 micrograms of total RNA and heat it at 60 degrees Celsius for 10 minutes.Immediately chill it on ice for two minutes. Add 600 microliters of DEPC-treated RNase-free water. Add 100 microliters of biotinylation buffer and then add 200 microliters of biotin HPDP.If the biotin HPDP solution precipitates out of the solution, increase the volume of DMSO or DMF up to 40%of the reaction volume as outlined in the text protocol. Protect the sample from light and incubate it at room temperature with gentle agitation for three hours. After this, add an approximately equal volume of chloroform to the tubes and mix vigorously.Centrifuge at 13, 000 times g and four degrees Celsius for five minutes. Next, carefully transfer the upper phase into new two milliliter tubes. Add an amount of five molar sodium chloride equal to approximately 1/10 of the sample volume.Then mix the sample. First, heat the biotinylated RNA at 65 degrees Celsius for 10 minutes. Then chill the samples on ice for five minutes.Add 100 microliters of Streptavidin-coated magnetic beads to the biotinylated RNA. Incubate at room temperature with slight shaking for 90 minutes. Place the columns provided with the kit in the magnetic stand.Next, add 900 microliters of room temperature washing buffer to the columns. Apply the 200 microliter bead and RNA mixture to the columns. Collect the flow-through in 1.5 milliliter tubes and then apply it again to the same magnetic column.Keep this flow-through if necessary as it represents the unlabeled RNA fraction. To begin RT-qPCR validation of the different fractions, first synthesize cDNA as outlined in the text. Then amplify the cDNA by real-time qPCR using a standard protocol.The measured levels of transcripts in fractions purified from wild-type cells cultured with and without four-thiouracil confirmed this procedure specifically purifies labeled RNA. Analysis of the spt20 delta strain reveals that the quantity of total steady-state RNA is largely unchanged or is only mildly reduced for the tested genes. Similar results are seen for strains deleted for bre1.However, analysis of newly-transcribed RNA in the spt20 delta strain reveals a three to five-fold decrease in mRNA synthesis for all tested genes. Meanwhile, the loss of bre1 leads to a more discreet but still visible decrease. Upon inducible depletion of spt7, a structural subunit of the saga complex, newly-transcribed mRNA levels are reduced to an extent similar to the deletion strain.When total RNA levels are measured by genome-wide analysis, only a few genes are seen to have their expression levels altered upon deletion of spt20 either by up regulating or down regulating. However, analysis of the newly-transcribed RNA reveals that the levels of over 4, 000 genes are significantly reduced at least two-fold upon deletion of spt20 which suggests a global positive effect of saga on RNA polymerase II transcription in budding yeasts. Though this method can provide insight in transcription in Saccharomyces cerevisiae, it was only applied with slight variations to By using this protocol, we could detect a global decrease in mRNA synthesis which was compensated by a global decrease in mRNA degradation.

Research

JoVE Journal

Genetics

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Evaluation of a Universal Nested Reverse Transcription Polymerase Chain Reaction for the Detection of Lyssaviruses

To detect rabies virus and other member species of the genus Lyssavirus within the family Rhabdoviridae, the pan-lyssavirus nested reverse transcription ...

Artificial RNA Polymerase II Elongation Complexes for Dissecting Co-transcriptional RNA Processing Events

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