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>
			<td style="width:119px;">Euromedex</td>
			<td style="width:139px;">Cat# SYN-1185</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">Countess II FL Automated Cell Counter</td>
			<td style="width:119px;">ThermoFisher</td>
			<td style="width:139px;">N/A</td>
			<td></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:216px;">RiboPure RNA Purification kit, yeast</td>
			<td style="width:119px;">ThermoFisher</td>
			<td style="width:139px;">Cat# AM1926</td>
			<td></td>
		</tr>
		<tr height="22">
			<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>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">TURBO DNA-free Kit</td>
			<td style="width:119px;">ThermoFisher</td>
			<td style="width:139px;">AM1907</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">EZ-Link HPDP Biotin</td>
			<td style="width:119px;">ThermoFisher</td>
			<td style="width:139px;">Cat# 21341</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Thiolutin</td>
			<td style="width:119px;">Abcam</td>
			<td style="width:139px;">ab143556</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">&#181;MACS Streptavidin kit</td>
			<td style="width:119px;">Miltenyi Biotec</td>
			<td style="width:139px;">Cat# 130-074-101</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Transcriptor Reverse Transcriptase</td>
			<td style="width:119px;">Roche</td>
			<td style="width:139px;">03 531 295 001</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">SYBR Green I Master</td>
			<td style="width:119px;">Roche</td>
			<td style="width:139px;">4707516001</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">GeneChip Yeast Genome 2.0</td>
			<td style="width:119px;">ThermoFisher</td>
			<td style="width:139px;">900555</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">GeneChip Fluidics Station 450</td>
			<td style="width:119px;">ThermoFisher</td>
			<td style="width:139px;">00-0079</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">GeneChip Scanner 3000 7G</td>
			<td style="width:119px;">ThermoFisher</td>
			<td style="width:139px;">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

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.

saccharomyces-cerevisiae-metabolic-labeling-with-4-thiouracil

Research

JoVE Journal

Genetics

Inducible T7 RNA Polymerase-mediated Multigene Expression System, pMGX

Co-expression of multiple proteins is increasingly essential for synthetic biology, studying protein-protein complexes, and characterizing and harnessing biosynthetic pathways. In this manuscript, the use of a highly effective system for the construction of multigene synthetic operons under the control of an inducible T7 RNA polymerase is described. This system allows many genes to be expressed simultaneously from one plasmid. Here, a set of four related vectors, pMGX-A, pMGX-hisA, pMGX-K, and pMGX-hisK, with either the ampicillin or kanamycin resistance selectable marker (A and K) and either possessing or lacking an N-terminal hexahistidine tag (his) are disclosed. Detailed protocols for the construction of synthetic operons using this vector system are provided along with the corresponding data, showing that a pMGX-based system containing five genes can be readily constructed and used to produce all five encoded proteins in Escherichia coli. This system and protocol enables researchers to routinely express complex multi-component modules and pathways in E. coli.

This study describes methods for the T7-mediated co-expression of multiple genes from a single plasmid in Escherichia coli using the pMGX plasmid system.

Co-expression of multiple proteins is increasingly essential for synthetic biology, studying protein-protein complexes, and characterizing and harnessing ...

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident processes. Formaldehyde crosslinking followed by chromatin immunoprecipitation and high-throughput sequencing (X-ChIP-seq) has been used to gain many valuable insights into genome biology. However, X-ChIP-seq has notable limitations linked to crosslinking and sonication. Native ChIP avoids these drawbacks by omitting crosslinking, but often results in poor recovery of chromatin-bound proteins. In addition, all ChIP-based methods are subject to antibody quality considerations. Enzymatic methods for mapping protein-DNA interactions, which involve fusion of a protein of interest to a DNA-modifying enzyme, have also been used to map protein-DNA interactions. We recently combined one such method, chromatin endogenous cleavage (ChEC), with high-throughput sequencing as ChEC-seq. ChEC-seq relies on fusion of a chromatin-associated protein of interest to micrococcal nuclease (MNase) to generate targeted DNA cleavage in the presence of calcium in living cells. ChEC-seq is not based on immunoprecipitation and so circumvents potential concerns with crosslinking, sonication, chromatin solubilization, and antibody quality while providing high resolution mapping with minimal background signal. We envision that ChEC-seq will be a powerful counterpart to ChIP, providing an independent means by which to both validate ChIP-seq findings and discover new insights into genomic regulation.

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

We describe chromatin endogenous cleavage coupled with high-throughput sequencing (ChEC-seq), a chromatin immunoprecipitation (ChIP)-orthogonal method for mapping protein binding sites genome-wide with micrococcal nuclease (MNase) fusion proteins.

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident ...

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 ...

Metabolic Labeling and Profiling of Transfer RNAs Using Macroarrays

Transfer RNAs (tRNA) are abundant short non-coding RNA species that are typically 76 to 90 nucleotides in length. tRNAs are directly responsible for protein synthesis by translating codons in mRNA into amino acid sequences. tRNAs were long considered as house-keeping molecules that lacked regulatory functions. However, a growing body of evidence indicates that cellular tRNA levels fluctuate in correspondence to varying conditions such as cell type, environment, and stress. The fluctuation of tRNA expression directly influences gene translation, favoring or repressing the expression of particular proteins. Ultimately comprehending the dynamic of protein synthesis requires the development of methods able to deliver high-quality tRNA profiles. The method that we present here is named SPOt, which stands for Streamlined Platform for Observing tRNA. SPOt consists of three steps starting with metabolic labeling of cell cultures with radioactive orthophosphate, followed by guanidinium thiocyanate-phenol-chloroform extraction of radioactive total RNAs and finally hybridization on in-house printed macroarrays. tRNA levels are estimated by quantifying the radioactivity intensities at each probe spot. In the protocol presented here we profile tRNAs in Mycobacterium smegmatis mc2155, a nonpathogenic bacterium often used as a model organism to study tuberculosis.

We describe an original transfer RNA analysis platform named SPOt (Streamlined Platform for Observing tRNA). SPOt simultaneously measures cellular levels of all tRNAs in biological samples, in only three steps, and in less than 24 hours.

Transfer RNAs (tRNA) are abundant short non-coding RNA species that are typically 76 to 90 nucleotides in length. tRNAs are directly responsible for ...

Semi-quantitative Detection of RNA-dependent RNA Polymerase Activity of Human Telomerase Reverse Transcriptase Protein

Human telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase, and it elongates telomere through RNA-dependent DNA polymerase activity. Although TERT is named as a reverse transcriptase, structural and phylogenetic analyses of TERT demonstrate that TERT is a member of right-handed polymerases, and relates to viral RNA-dependent RNA polymerases (RdRPs) as well as viral reverse transcriptase. We firstly identified RdRP activity of human TERT that generates complementary RNA stand to a template non-coding RNA and contributes to RNA silencing in cancer cells. To analyze this non-canonical enzymatic activity, we developed RdRP assay with recombinant TERT in 2009, thereafter established in vitro RdRP assay for endogenous TERT. In this manuscript, we describe the latter method. Briefly, TERT immune complexes are isolated from cells, and incubated with template RNA and rNTPs including radioactive rNTP for RdRP reaction. To eliminate single-stranded RNA, reaction products are treated with RNase I, and the final products are analyzed with polyacrylamide gel electrophoresis. Radiolabeled RdRP products can be detected by autoradiography after overnight exposure.

Human telomerase reverse transcriptase (TERT) synthesizes not only telomeric DNA but also double-stranded RNA through RNA-dependent RNA polymerase activity. Here, we describe a newly established assay to detect RNA-dependent RNA polymerase activity of endogenous TERT.

Human telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase, and it elongates telomere through RNA-dependent DNA polymerase ...

Chromatin Immunoprecipitation (ChIP) of Histone Modifications from Saccharomyces cerevisiae

Histone post-translational modifications (PTMs), such as acetylation, methylation and phosphorylation, are dynamically regulated by a series of enzymes that add or remove these marks in response to signals received by the cell. These PTMS are key contributors to the regulation of processes such as gene expression control and DNA repair. Chromatin immunoprecipitation (chIP) has been an instrumental approach for dissecting the abundance and localization of many histone PTMs throughout the genome in response to diverse perturbations to the cell. Here, a versatile method for performing chIP of post-translationally modified histones from the budding yeast Saccharomyces cerevisiae (S. cerevisiae) is described. This method relies on crosslinking of proteins and DNA using formaldehyde treatment of yeast cultures, generation of yeast lysates by bead beating, solubilization of chromatin fragments by micrococcal nuclease, and immunoprecipitation of histone-DNA complexes. DNA associated with the histone mark of interest is purified and subjected to quantitative PCR analysis to evaluate its enrichment at multiple loci throughout the genome. Representative experiments probing the localization of the histone marks H3K4me2 and H4K16ac in wildtype and mutant yeast are discussed to demonstrate data analysis and interpretation. This method is suitable for a variety of histone PTMs and can be performed with different mutant strains or in the presence of diverse environmental stresses, making it an excellent tool for investigating changes in chromatin dynamics under different conditions.

Chromatin Immunoprecipitation ChIP of Histone Modifications from Saccharomyces cerevisiae

Here, we describe a protocol for chromatin immunoprecipitation of modified histones from the budding yeast Saccharomyces cerevisiae. Immunoprecipitated DNA is subsequently used for quantitative PCR to interrogate the abundance and localization of histone post-translational modifications throughout the genome.

Histone post-translational modifications (PTMs), such as acetylation, methylation and phosphorylation, are dynamically regulated by a series of enzymes ...

Methodology for Accurate Detection of Mitochondrial DNA Methylation

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert unmethylated cytosine into uracil, in a single-stranded DNA context. Bisulfite sequencing can be targeted (using PCR) or performed on the whole genome and provides absolute quantification of cytosine methylation at the single base-resolution. Given the distinct nature of nuclear- and mitochondrial DNA, notably in the secondary structure, adaptions of bisulfite sequencing methods for investigating cytosine methylation in mtDNA should be made. Secondary and tertiary structure of mtDNA can indeed lead to bisulfite sequencing artifacts leading to false-positives due to incomplete denaturation poor access of bisulfite to single-stranded DNA. Here, we describe a protocol using an enzymatic digestion of DNA with BamHI coupled with bioinformatic analysis pipeline to allow accurate quantification of cytosine methylation levels in mtDNA. In addition, we provide guidelines for designing the bisulfite sequencing primers specific to mtDNA, in order to avoid targeting undesirable NUclear MiTochondrial segments (NUMTs) inserted into the nuclear genome.

Here, we present a protocol to allow accurate quantification of mitochondrial DNA (mtDNA) methylation. In this protocol, we describe an enzymatic digestion of DNA with BamHI coupled with a bioinformatic analysis pipeline which can be used to avoid overestimation of mtDNA methylation levels caused by the secondary structure of mtDNA.

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert ...

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell type-specific fashion. AS expression patterns are typically analyzed by RT-PCR and RNA-seq using RNA samples isolated from a population of cells. In situ examination of AS expression patterns for a particular biological structure can be carried out by RNA in situ hybridization (ISH) using exon-specific probes. However, this particular use of ISH has been limited because alternative exons are generally too short to design exon-specific probes. In this report, the use of BaseScope, a recently developed technology that employs short antisense oligonucleotides in RNA ISH, is described to analyze AS expression patterns in mouse brain sections. Exon 23a of neurofibromatosis type 1 (Nf1) is used as an example to illustrate that short exon-exon junction probes exhibit robust hybridization signals with high specificity in RNA ISH analysis on mouse brain sections. More importantly, signals detected with exon inclusion- and skipping-specific probes can be used to reliably calculate the percent spliced in values of Nf1 exon 23a expression in different anatomical areas of a mouse brain. The experimental protocol and calculation method for AS analysis are presented. The results indicate that BaseScope provides a powerful new tool to assess AS expression patterns in situ.

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

An in situ hybridization (ISH) protocol that uses short antisense oligonucleotides to detect alternative pre-mRNA splicing patterns in mouse brain sections is described.

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell ...

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 polymerase chain reaction (nested RT-PCR) was developed to detect the conserved region of the nucleoprotein (N) gene of lyssaviruses. The method applies reverse transcription (RT) using viral RNA as template and oligo (dT)15 and random hexamers as primers to synthesize the viral complementary DNA (cDNA). Then, the viral cDNA is used as a template to amplify an 845 bp N gene fragment in first-round PCR using outer primers, followed by second-round nested PCR to amplify the final 371 bp fragment using inner primers. This method can detect different genetic clades of rabies viruses (RABV). The validation, using 9,624 brain specimens from eight domestic animal species in 10 years of clinical rabies diagnoses and surveillance in China, showed that the method has 100% sensitivity and 99.97% specificity in comparison with the direct fluorescent antibody test (FAT), the gold standard method recommended by the World Health Organization (WHO) and the World Organization for Animal Health (OIE). In addition, the method could also specifically amplify the targeted N gene fragment of 15 other approved and two novel lyssavirus species in the 10th Report of the International Committee on Taxonomy of Viruses (ICTV) as evaluated by a mimic detection of synthesized N gene plasmids of all lyssaviruses. The method provides a convenient alternative to FAT for rabies diagnosis and has been approved as a National Standard (GB/T36789-2018) of China.

A pan-lyssavirus nested reverse transcription polymerase chain reaction has been developed to detect specifically all known lyssaviruses. Validation using rabies brain samples of different animal species showed that this method has a sensitivity and specificity equivalent to the gold standard fluorescent antibody test and could be used for routine rabies diagnosis.

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

Eukaryotic mRNA synthesis is a complex biochemical process requiring transcription of a DNA template into a precursor RNA by the multi-subunit enzyme RNA polymerase II and co-transcriptional capping and splicing of the precursor RNA to form the mature mRNA. During mRNA synthesis, the RNA polymerase II elongation complex is a target for regulation by a large collection of transcription factors that control its catalytic activity, as well as the capping, splicing, and 3&#8217;-processing enzymes that create the mature mRNA. Because of the inherent complexity of mRNA synthesis, simpler experimental systems enabling isolation and investigation of its various co-transcriptional stages have great utility.
In this article, we describe one such simple experimental system suitable for investigating co-transcriptional RNA capping. This system relies on defined RNA polymerase II elongation complexes assembled from purified polymerase and artificial transcription bubbles. When immobilized via biotinylated DNA, these RNA polymerase II elongation complexes provide an easily manipulable tool for dissecting co-transcriptional RNA capping and mechanisms by which the elongation complex recruits and regulates capping enzyme during co-transcriptional RNA capping. We anticipate this system could be adapted for studying recruitment and/or assembly of proteins or protein complexes with roles in other stages of mRNA maturation coupled to the RNA polymerase II elongation complex.

Here, we describe the assembly of RNA polymerase II (Pol II) elongation complexes requiring only short synthetic DNA and RNA oligonucleotides and purified Pol II. These complexes are useful for studying mechanisms underlying co-transcriptional processing of transcripts associated with the Pol II elongation complex.

Eukaryotic mRNA synthesis is a complex biochemical process requiring transcription of a DNA template into a precursor RNA by the multi-subunit enzyme RNA ...