Name: Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity (Video) | JoVE Journal
Uploaded: 2018-10-22T13:00:00.000+00:00
Duration: 9 min 21 s
Description: Learn metabolic labeling with 4-thiouracil to quantify newly synthesized mRNA in Saccharomyces cerevisiae. This method accurately measures RNA polymerase II transcription activity by separating synthesis from decay.

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5 minutes

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 mL of YPD medium and let it grow up until the OD600 is around 0.8.</li>
	<li>In parallel, inoculate a single colony of S. pombe cells from a fresh plate onto 50 mL of YES medium (0.5% yeast extract; 250 mg/L adenine, histidine, uracil, leucine, and lysine; 3% glucose) and grow the cells overnight at 32 &#176;C with constant agitation (150 rpm).</li>
	<li>Measure the OD600 of the S. pombe overnight culture and dilute the culture to an OD600 of approximately 0.1 in 500 mL of YES medium and let it grow up until the OD600 is approximately 0.8.</li>
	<li>For each S. cerevisiae anchor-away 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 the cells overnight at 30 &#176;C with constant agitation (150 rpm).</li>
	<li>The next morning, measure the OD600, dilute the culture to an OD600 of approximately 0.1 in 100 mL of YPD medium and let it grow until the OD600&#8776; 0.8.</li>
	<li>Add 100 &#181;L of rapamycin to the culture from a stock solution of 1 mg/mL (final rapamycin concentration of 1 &#181;g/mL) and let the cells incubate at 30 &#176;C with constant agitation for the time necessary for the protein of interest to be conditionally depleted from the nucleus (usually, 30 min is adequate). For the control, use a similar yeast culture, but instead of adding rapamycin, add the equivalent volume of dimethyl sulfoxide (DMSO).</li>
</ol>
2. 4tU Labeling with S. pombe as a Spike-in (Counting)
<ol>
	<li>Prepare a fresh solution of 2 M 4-thiouracil. Once prepared, keep it at room temperature and away from light.
	<ol>
		<li>Accurately weigh 64.1 mg of 4-thiouracil for each S. cerevisiae culture and dissolve it in 250 &#181;L of dimethylformamide (DMF) or in 250 &#181;L of DMSO.</li>
		<li>For the S. pombe culture to be used as spike-in, weigh 320.5 mg of 4-thiouracil and dissolve it in 1,250 &#181;L of DMSO.</li>
	</ol>
	</li>
	<li>Add the 4-thiouracil solution to S. cerevisiae and S. pombe cultures for a final concentration of 5 mM and incubate them for 6 min with constant agitation at 30 &#176;C and 32 &#176;C, respectively.</li>
	<li>After 6 min, remove a small aliquot of each culture for cell counting. Count the cells using an automatic cell counter or a Neubauer chamber.</li>
	<li>Collect the cells through centrifugation (2,500 x g) for 5 min at 4 &#176;C.</li>
	<li>Discard the supernatant, wash the cells with ice-cold 1x PBS, and centrifuge again (2,500 x g, 4 &#176;C, 5 min).</li>
	<li>Calculate the total number of cells in each of the S. cerevisiae and S. pombe samples.</li>
	<li>Resuspend the cells in 5 mL of ice-cold 1x PBS and mix S. cerevisiae with S. pombe cells with a 3:1 ratio.</li>
	<li>Centrifuge the cells (2,500 x g, 4 &#176;C, 5 min), remove the PBS, flash-freeze the cells in liquid N2, and store the sample at -80 &#176;C until further use.</li>
</ol>
3. RNA Extraction and DNase Treatment
<ol>
	<li>Thaw the cells on ice for approximately 20&#8211;30 min.</li>
	<li>Proceed with the RNA extraction using a yeast-RNA extraction kit (Table of Materials) with a few adaptations.</li>
	<li>Per each sample, pour 750 &#181;L of ice-cold Zirconia beads into a 1.5-mL screw cap tube supplied with the kit. Keep in mind that per each tube, RNA from up to 109 cells can be efficiently extracted. Hence, prepare the number of tubes necessary for each sample. For example, an S. cerevisiae culture of 100 mL (OD600&#8776; 0.8) can render around 2 x 109 to 3 x 109 cells, leading up to a total of 2.7 x 109 to 4 x 109 cells in total (after the spike-in with a third of the S. pombe cells). In this case, up to 3 - 4 reaction tubes per each sample/condition/mutant/replicate would be required.</li>
	<li>Per each 1 x 109 cells, add 480 &#181;L of the lysis buffer provided with the kit, 48 &#181;L of 10% SDS, and 480 &#181;L of phenol:chloroform:isoamyl alcohol (25:24:1, v/v/v).</li>
	<li>Mix the cells using a vortex mixer and transfer them to the tubes containing the ice-cold Zirconia beads.</li>
	<li>Accommodate the tubes on a vortex mixer adaptor, turn the vortex at maximum speed, and beat for 10 min to lyse the yeast cells (in a room at 4 &#176;C). Alternatively, perform lysis of the cells in an automatic bead-beater.</li>
	<li>Centrifuge tubes at 16,000 x g for 5 min at room temperature and carefully collect the upper phase (RNA-containing phase) to a fresh 15 mL falcon tube. Typically, the volume recovered per each tube is around 500&#8211;600 &#181;L.</li>
	<li>To the 15 mL tubes containing the partially purified RNA, add the binding buffer provided with the kit and mix thoroughly. Per each 100 &#181;L of RNA solution, add 350 &#181;L of binding buffer (i.e., when the aqueous-phase solution volume is 600 &#181;L, 2.1 mL of the binding buffer should be added).</li>
	<li>To the previous mixture, add the 100% ethanol and mix thoroughly. Per each 100 &#181;L of RNA solution, add 235 &#181;L of 100% ethanol (i.e., when the aqueous-phase solution volume is 600 &#181;L, add 1.41 mL of ethanol).</li>
	<li>Apply up to 700 &#181;L of the mixture from step 3.9 to a filter cartridge assembled in a collection tube, both provided with the kit.</li>
	<li>Centrifuge for 1 min at 16,000 x g. If the centrifugation duration was not enough for the total volume to pass through the filter, repeat the centrifugation for 30 s.</li>
	<li>Discard the flow-through and reuse the same collection tube. Add another 700 &#181;L of RNA-binding buffer-ethanol solution to the filter and centrifuge again at 16,000 x g for 1 min.</li>
	<li>Discard the flow-through and repeat steps 3.11 and 3.12 until the RNA solution is finished.</li>
	<li>Wash the filter 2x with 700 &#181;L of washing solution 1. Collect the washing solution via centrifugation at 16,000 x g for 1 min and always keep the collection tube.</li>
	<li>Wash the filter 2x with 500 &#181;L of washing solution 2. Collect the washing solution via centrifugation at 16,000 x g for 1 min and always keep the collection tube.</li>
	<li>Centrifuge the tubes once again at 16,000 x g for 1 min to completely dry the filter.</li>
	<li>Transfer the filter cartridge to the final collection tube (RNA-appropriate tube) and elute RNA with 50 &#181;L of DEPC-treated, RNase-free H2O (preheated to 100 &#176;C).</li>
	<li>Centrifuge for 1 min at 16,000 x g.</li>
	<li>Elute RNA again (to the same tube) with 50 &#181;L of preheated DEPC-treated, RNase-free H2O. Make sure that all the volume has passed through the filter; otherwise, centrifuge for longer periods.</li>
	<li>If multiple tubes for one single sample are used, pool them all in one tube.</li>
	<li>Quantify and check the purity of the sample using the appropriate equipment. 
	NOTE: While 4tU is only incorporated within newly synthesized RNA, there is a chance of minor contamination with DNA. For that reason, it is always advisable to treat the samples with DNase I. For that, use the reagents provided with the RNA-extraction kit (Table of Materials) following the manufacturer&#39;s recommendations.</li>
</ol>
4. Thiol-specific Biotinylation of Newly Synthesized RNA
<ol>
	<li>Adjust the concentration of the RNA obtained with section 3 of the protocol to 2 mg/mL. Aliquot 200 &#181;g of total RNA, heat it for 10 min at 60 &#176;C, and immediately chill it on ice for 2 min.</li>
	<li>To the RNA aliquot, add the reagents mentioned below in the following order: 600 &#181;L of DEPC-treated, RNase-free H2O, 100 &#181;L of biotinylation buffer (100 mM Tris-HCl [pH 7.5] and 10 mM EDTA, in DEPC-treated, RNase-free H2O), and 200 &#181;L of biotin-HPDP from a stock of 1 mg/mL biotin-HPDP in DMSO or DMF.
	<ol>
		<li>In some situations, the biotin-HPDP solution tends to precipitate, likely due to its low solubility in water. In this situation, increase the volume of DMSO/DMF up to 40% of the reaction volume (to the RNA sample, add 400 &#181;L of DEPC-treated H2O, 100 &#181;L of biotinylation buffer, and 400 &#181;L of biotin-HPDP from a 0.5 mg/mL stock).</li>
	</ol>
	</li>
	<li>Incubate the sample at room temperature and protected from light for 3 h, with gentle agitation.</li>
	<li>After incubation, add an approximately equal volume of chloroform to the tubes and mix vigorously.</li>
	<li>Spin the sample at 13,000 x g for 5 min, at 4 &#176;C. This step allows the removal of excess biotin that did not biotinylate the RNA. Alternatively, perform this step using phase-lock tubes (heavy). For that, spin down the phase-lock tubes for 1 min at 13,000 x g, add the RNA mixture and an equal amount of chloroform, mix them vigorously, and centrifuge them at 13,000 x g for 5 min, at 4 &#176;C.</li>
	<li>Carefully transfer the upper phase into new 2 mL tubes.</li>
	<li>Add one-tenth of the volume of 5 M NaCl and mix the sample.</li>
	<li>Add an equal volume of isopropanol, mix the sample thoroughly, and spin it at 13,000 x g for at least 30 min, at 4 &#176;C.</li>
	<li>Cautiously remove the supernatant and add 1 mL of ice-cold 75% ethanol.</li>
	<li>Spin at 13,000 x g for 10 min, at 4 &#176;C.</li>
	<li>Carefully remove supernatant, quick-spin the tube, and remove the remaining ethanol solution. Make sure that the RNA pellet does not dry.</li>
	<li>Suspend the RNA in 100 &#181;L of DEPC-treated, RNase-free H2O.</li>
</ol>
5. Purification of Newly Synthesized Fraction from Total and Unlabeled RNA Using Streptavidin-coated Magnetic Beads
<ol>
	<li>Heat the biotinylated RNA for 10 min at 65 &#176;C and then chill the samples on ice for 5 min.</li>
	<li>Add 100 &#181;L of streptavidin-coated magnetic beads to the biotinylated RNA (at a final volume of 200 &#181;L). Specifically, it is recommended to use the beads indicated in the Table of Materials, since, after conversations with other laboratories, these seemed to be the more consistent and reliable.</li>
	<li>Incubate the sample with slight shaking for 90 min, at room temperature.</li>
	<li>Place the columns provided with the kit (Table of Materials) in the magnetic stand.</li>
	<li>Add 900 &#181;L of room-temperature washing buffer (100 mM Tris-HCl [pH 7.5], 10 mM EDTA, 1 M NaCl, and 0.1% Tween 20, in DEPC-treated, RNase-free H2O) to the columns (pre-run and equilibrate).</li>
	<li>Apply beads/RNA mixture (200 &#181;L) to the columns.</li>
	<li>Collect the flow-through in 1.5-mL tubes and apply it again to the same magnetic column. If necessary, keep this flow-through as it represents the unlabeled RNA fraction.</li>
	<li>Wash the columns 5x with increasing volumes of washing buffer (600, 700, 800, 900, and 1,000 &#181;L).</li>
	<li>Elute the newly synthesized RNA with 200 &#181;L of 0.1 M DTT.</li>
	<li>Perform a second elution, 3 min later, with an equal volume of 0.1 M DTT.</li>
	<li>After eluting the RNA, add 0.1 volumes of 3 M NaOAc (pH 5.2), 3 volumes of ice-cold 100% ethanol, and 2 &#181;L of 20 mg/mL glycogen (RNA-grade) and let the RNA precipitate overnight, at -20 &#176;C.</li>
	<li>Recover the RNA by centrifugation (13,000 x g for 10 min, at 4 &#176;C) and resuspend it in 15 &#181;L of DEPC-treated, RNase-free H20. The proportion of labeled-to-total RNA is expected to be around 2% - 4% (usually more toward the lower end), rendering approximately 2.0 &#181;g of newly synthesized RNA. This quantity is enough to do several qPCR experiments, as well for microarray/sequencing analyses.</li>
</ol>
6. RT-qPCR Validation of the Different Fractions
<ol>
	<li>Synthesize cDNA from 2 &#181;g of total RNA or 10 &#181;L of labeled RNA using random hexamers and the reverse transcriptase of choice, according to the manufacturer&#39;s instructions (Table of Materials).</li>
	<li>Amplify the cDNA by real-time qPCR using a standard protocol (Table of Materials). 
	NOTE: All samples should be run in triplicate from a minimum of two biological replicates. Correct all raw values for the expression of S. pombe tubulin.</li>
</ol>
7. Microarray Hybridization
<ol>
	<li>Hybridize RNA samples onto preferred microarray chips according to the manufacturer&#39;s instructions (for this specific protocol, see Table of Materials). Briefly, prepare biotinylated cRNA targets from 150 ng of RNA using the Premier RNA Amplification Kit (Table of Materials), according to the manufacturer&#39;s instructions. Hybridize 4 mg of fragmented cRNAs for 16 h at 45 &#176;C and 60 rpm on microarray chips.</li>
	<li>Wash, stain, and scan the chips using the indicated station and scanner (Table of Materials). Extract the raw data (CEL Intensity files) from the scanned images using the command console (AGCC, version 4.1.2).</li>
	<li>Further process the CEL files with Expression Console software version 1.4.1 to calculate probe set signal intensities, using the statistics-based algorithms MAS 5.0 with default settings and global scaling as normalization method. 
	NOTE: The trimmed mean target intensity of each chip was arbitrarily set to 100. Perform all experiments using at least two independent biological replicates. Normalize raw data to the S. pombe signal and calculate fold changes in total and newly synthesized RNA levels.</li>
</ol>
8. Data Analysis Using an Existing R Pipeline
<ol>
	<li>Calculate synthesis and decay rates using a pipeline and R/Bioconductor package publicly available, as previously described8,10.</li>
</ol>

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<li>Schulz, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transcriptome+surveillance+by+selective+termination+of+noncoding+RNA+synthesis%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Transcriptome surveillance by selective termination of noncoding RNA synthesis.</a> Cell. 155 (5), 1075-1087 (2013).</li>
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The authors have nothing to disclose.

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.

<table><tbody><tr><td>4-Thiouracil</td><td>Sigma-Aldrich</td><td>Cat# 440736</td><td></td></tr><tr><td>Rapamycin</td><td>Euromedex</td><td>Cat# SYN-1185</td><td></td></tr><tr><td>Countess II FL Automated Cell Counter</td><td>ThermoFisher</td><td>N/A</td><td></td></tr><tr><td>RiboPure RNA Purification kit, yeast</td><td>ThermoFisher</td><td>Cat# AM1926</td><td></td></tr><tr><td>NanoDrop 2000 Spectrophotometer</td><td>ThermoFisher</td><td>ND-2000</td><td></td></tr><tr><td>TURBO DNA-free Kit</td><td>ThermoFisher</td><td>AM1907</td><td></td></tr><tr><td>EZ-Link HPDP Biotin</td><td>ThermoFisher</td><td>Cat# 21341</td><td></td></tr><tr><td>Thiolutin</td><td>Abcam</td><td>ab143556</td><td></td></tr><tr><td>&amp;micro;MACS Streptavidin kit</td><td>Miltenyi Biotec</td><td>Cat# 130-074-101</td><td></td></tr><tr><td>Transcriptor Reverse Transcriptase</td><td>Roche</td><td>03 531 295 001</td><td></td></tr><tr><td>SYBR Green I Master</td><td>Roche</td><td>4707516001</td><td></td></tr><tr><td>GeneChip Yeast Genome 2.0</td><td>ThermoFisher</td><td>900555</td><td></td></tr><tr><td>GeneChip Fluidics Station 450</td><td>ThermoFisher</td><td>00-0079</td><td></td></tr><tr><td>GeneChip Scanner 3000 7G</td><td>ThermoFisher</td><td>00-0210</td><td></td></tr></tbody></table>

saccharomyces cerevisiae metabolic labeling with 4-thiouracil and the quantification of newly synthesized mrna as a proxy for rna polymerase ii activity

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...

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 ...

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JoVE Journal

Genetics

Total cellular RNA provides a poor template for studying short-term changes in RNA synthesis and decay as well as the kinetics of RNA processing. Here, we describe metabolic labeling of newly transcribed RNA with 4-thiouridine followed by thiol-specific biotinylation and purification of newly transcribed RNA allowing to overcome these limitations.

Metabolic Labeling of Newly Transcribed RNA for High Resolution Gene Expression Profiling of RNA Synthesis, Processing and Decay in Cell Culture

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae.  Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

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

This protocol describes the combinatorial use of ChIP-seq, 4sU-seq, total RNA-seq, and ribosome profiling for cell lines and primary cells. It enables tracking changes in transcription-factor binding, de novo transcription, RNA processing, turnover and translation over time, and displaying the overall course of events in activated and/or rapidly changing cells.

Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation

The use of thiolated uracil to sensitively and specifically purify newly transcribed RNA from the yeast Saccharomyces cerevisiae.

Extremely Rapid and Specific Metabolic Labelling of RNA In Vivo with 4-Thiouracil (Ers4tU)

Baker’s yeast mitochondrial genome encodes eight polypeptides. The goal of the current protocol is to label all of them and subsequently visualize them as separate bands.

Labelling and Visualization of Mitochondrial Genome Expression Products in Baker's Yeast Saccharomyces cerevisiae

Here, a protocol is presented for the metabolic labeling of yeast with 14C-acetic acid, which is coupled with thin layer chromatography for the separation of neutral lipids.

Analysis of Neutral Lipid Synthesis in Saccharomyces cerevisiae by Metabolic Labeling and Thin Layer Chromatography

We describe two complementary protocols to accurately determine S-phase duration in S. cerevisiae using EdU, a thymidine analog, which is incorporated in vivo and detected using Click chemistry by microscopy and flow cytometry. It allows for the easy characterization of the duration of DNA replication and overlooked replication defects in mutants.

Determination of S-Phase Duration Using 5-Ethynyl-2'-deoxyuridine Incorporation in Saccharomyces cerevisiae

RNA secondary structure has primarily been observed in mature RNA with structure probing methods. Co-transcriptional Structure Tracking sequencing (CoSTseq) unifies nuclear run-on, which has been used to study Polymerase position on nascent RNA, with structure probing. CoSTseq thereby enables observation of RNA secondary structure in RNA under active transcription.

Comparative RNA Structure Analysis of Nascent and Mature Transcripts in Saccharomyces cerevisiae

Learn metabolic labeling with 4-thiouracil to quantify newly synthesized mRNA in Saccharomyces cerevisiae. This method accurately measures RNA polymerase II transcription activity by separating synthesis from decay.

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity (Video) | JoVE Journal

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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