Name: ms2-affinity purification coupled with rna sequencing in gram-positive bacteria
Uploaded: 2021-02-23T15:00:00
Description: Watch this Scientific Journal Video about MS2-Affinity Purification Coupled with RNA Sequencing in Gram-Positive Bacteria at JoVE.com

This work was supported by the &#8220;Agence Nationale de la Recherche&#8221; (ANR, Grant ANR-16-CE11-0007-01, RIBOSTAPH, and ANR-18-CE12- 0025-04, CoNoCo, to PR). It has also been published under the framework of the labEx NetRNA ANR-10-LABX-0036 and of ANR-17-EURE- 0023 (to PR), as funding from the state managed by ANR as part of the investments for the future program. DL was supported by the European Union&#39;s Horizon 2020 research and innovation program under the Marie Sk&#322;odowska-Curie Grant Agreement No. 753137-SaRNAReg. Work in E. Mass&#233; Lab has been supported by operating grants from the Canadian Institutes of Health Research (CIHR), the Natural Sciences and Engineering Research Council of Canada (NSERC), and the National institutes of Health NIH Team Grant R01 GM092830-06A1.

1. Buffers and media
<ol>
	<li>For MAPS experiments, prepare the following buffers and media: 
	- Buffer A (150 mM KCl, 20 mM Tris-HCl pH 8, 1 mM MgCl2 and 1 mM DTT) 
	- Buffer E (250 mM KCl, 20 mM Tris-HCl pH 8, 12 mM maltose, 0.1% Triton, 1 mM MgCl2 and 1 mM DTT) 
	- RNA loading buffer (0.025% xylene cyanol and 0.025% bromophenol blue in 8 M urea) 
	- Brain Heart Infusion (BHI) medium (12.5 g of calf brain, 10 g of peptone, 5 g of beef heart, 5 g of NaCl, 2.5...

<ol>
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C., Masse, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DsrA+regulatory+RNA+represses+both+hns+and+rbsD+mRNAs+through+distinct+mechanisms+in+Escherichia+coli.">DsrA regulatory RNA represses both hns and rbsD mRNAs through distinct mechanisms in Escherichia coli.</a> Molecular Microbiology. 98 (2), 357-369 (2015).</li><li>Lalaouna, D., Prevost, K., Laliberte, G., Houe, V., Masse, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contrasting+silencing+mechanisms+of+the+same+target+mRNA+by+two+regulatory+RNAs+in+Escherichia+coli.">Contrasting silencing mechanisms of the same target mRNA by two regulatory RNAs in Escherichia coli.</a> Nucleic Acids Research. 46 (5), 2600-2612 (2018).</li><li>Lalaouna, D., Eyraud, A., Devinck, A., Prevost, K., Masse, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GcvB+small+RNA+uses+two+distinct+seed+regions+to+regulate+an+extensive+targetome.">GcvB small RNA uses two distinct seed regions to regulate an extensive targetome.</a> Molecular Microbiology. 111 (2), 473-486 (2019).</li><li>Silva, I. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SraL+sRNA+interaction+regulates+the+terminator+by+preventing+premature+transcription+termination+of+rho+mRNA.">SraL sRNA interaction regulates the terminator by preventing premature transcription termination of rho mRNA.</a> Proceedings of the National Academy of Sciences. 116 (8), 3042-3051 (2019).</li><li>Lalaouna, D., Masse, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+sRNA+interacting+with+a+transcript+of+interest+using+MS2-affinity+purification+coupled+with+RNA+sequencing+(MAPS)+technology.">Identification of sRNA interacting with a transcript of interest using MS2-affinity purification coupled with RNA sequencing (MAPS) technology.</a> Genomics Data. 5, 136-138 (2015).</li><li>Tomasini, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RNA+targetome+of+Staphylococcus+aureus+non-coding+RNA+RsaA:+impact+on+cell+surface+properties+and+defense+mechanisms.">The RNA targetome of Staphylococcus aureus non-coding RNA RsaA: impact on cell surface properties and defense mechanisms.</a> Nucleic Acids Research. 45 (11), 6746-6760 (2017).</li><li>Bronesky, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multifaceted+small+RNA+modulates+gene+expression+upon+glucose+limitation+in+Staphylococcus+aureus.">A multifaceted small RNA modulates gene expression upon glucose limitation in Staphylococcus aureus.</a> The EMBO Journal. 38 (6), (2019).</li><li>Lalaouna, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RsaC+sRNA+modulates+the+oxidative+stress+response+of+Staphylococcus+aureus+during+manganese+starvation.">RsaC sRNA modulates the oxidative stress response of Staphylococcus aureus during manganese starvation.</a> Nucleic Acids Research. 47 (1), 9871-9887 (2019).</li><li>Carrier, M. 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A modified protocol for Gram-positive bacteria 
The initial protocol of MAPS was developed to study sRNA interactome in the model organism E. coli20,30. Here, we describe a modified protocol which is suitable for the characterization of sRNA-dependent regulatory networks in the opportunistic human pathogen S. aureus and is certainly transposable to other Gram-positive bacteria, pathogenic or not.
<p class="jove_content"...

Hundreds, perhaps even thousands of small regulatory RNAs (sRNAs) have been identified in most bacterial genomes, but the functions of the vast majority of them remain uncharacterized. Overall, sRNAs are short non-coding molecules, playing major roles in bacterial physiology and adaptation to fluctuating environments1,2,3. Indeed, these macromolecules are at the center of numerous intricate regulatory networks, impacting metabolic pathways, stress responses but also virulence and antibiotic resistance. Logically, their synthesis is triggered by specific environment stimuli (e.g., nutrient starvation, oxidative or membrane stresses). Most sRNAs regulate multiple target mRNAs at the post-transcriptional level through short and non-contiguous base pairing. They usually prevent translation initiation by competing with ribosomes for translation initiation regions4. The formation of sRNA:mRNA duplexes also often results in the active degradation of the target mRNA by recruitment of specific RNases.
The characterization of an sRNA targetome (i.e., the whole set of its target RNAs) allows the identification of the metabolic pathways in which it intervenes and the potential signal it answers to. Consequently, the functions of a specific sRNA can generally be inferred from its targetome. For this purpose, several in silico prediction tools have been developed such as IntaRNA and CopraRNA5,6,7. They notably rely on sequence complementarity, pairing energy and accessibility of the potential interaction site to determine putative sRNA partners. However, prediction algorithms do not integrate all factors influencing base-pairing in vivo such as the involvement of RNA chaperones8 favoring sub-optimal interactions or the co-expression of both partners. Due to their inherent limitations, the false positive rate of prediction tools remains high. Most experimental large-scale approaches are based on the co-purification of sRNA:mRNA couples interacting with a tagged RNA binding protein (RBP)6,9. For example, the RNA Interaction by Ligation and sequencing (RIL-seq) method identified RNA duplexes co-purified with RNA chaperones such as Hfq and ProQ in Escherichia coli10,11. A similar technology called UV-Crosslinking, Ligation And Sequencing of Hybrids (CLASH) was applied to RNase E- and Hfq-associated sRNAs in E. coli12,13. Despite the well-described roles of Hfq and ProQ in sRNA-mediated regulation in multiple bacteria8,14,15, sRNA-based regulation seems to be RNA chaperone-independent in several organisms like S. aureus16,17,18. Even if the purification of RNA duplexes in association with RNases is feasible as demonstrated by Waters and coworkers13, this remains tricky as RNases trigger their rapid degradation. Hence, the MS2-Affinity Purification coupled with RNA Sequencing (MAPS) approach19,20 constitutes a solid alternative in such organisms.
Unlike above-mentioned methods, MAPS uses a specific sRNA as bait to capture all interacting RNAs and hence does not rely on the involvement of an RBP. The entire process is depicted in Figure 1. In brief, the sRNA is tagged at the 5&#8217; with the MS2 RNA aptamer that is specifically recognized by the MS2 coat protein. This protein is fused with the maltose binding protein (MBP) to be immobilized on an amylose resin. Therefore, MS2-sRNA and its RNA partners are retained on the affinity chromatography column. After elution with maltose, co-purified RNAs are identified using high-throughput RNA sequencing followed by bioinformatic analysis (Figure 2). The MAPS technology ultimately draws an interacting map of all potential interactions occurring in vivo.
MAPS technology was originally implemented in the non-pathogenic Gram-negative bacterium E. coli21. Remarkably, MAPS helped identify a tRNA-derived fragment specifically interacting with both RyhB and RybB sRNAs and preventing any sRNA transcriptional noise to regulate mRNA targets in non-inducing conditions. Thereafter, MAPS has been successfully applied to other E. coli sRNAs like DsrA22, RprA23, CyaR23 and GcvB24 (Table 1). In addition to confirming previously known targets, MAPS extended the targetome of these well-known sRNAs. Recently, MAPS has been performed in Salmonella Typhimurium and revealed that SraL sRNA binds to rho mRNA, coding for a transcription termination factor25. Through this pairing, SraL protects rho mRNA from the premature transcription termination triggered by Rho itself. Interestingly, this technology is not restricted to sRNAs and can be applied to any type of cellular RNAs as exemplified by the use of a tRNA-derived fragment26 and a 5&#8217;-untranslated region of mRNA22 (Table 1).
MAPS method has been also adapted to the pathogenic Gram-positive bacterium S. aureus19. Specifically, the lysis protocol has been widely modified to efficiently break cells due to a thicker cell wall than Gram-negative bacteria and to maintain RNA integrity. This adapted protocol already unravelled the interactome of RsaA27, RsaI28 and RsaC29. This approach gave insights into the crucial role of these sRNAs in regulatory mechanisms of cell surface properties, glucose uptake, and oxidative stress responses.
The protocol developed and implemented in E. coli in 2015 has been recently described in great detail30. Here, we provide the modified MAPS protocol, which is particularly suitable for studying sRNA regulatory networks in Gram-positive (thicker cell wall) bacteria whether non-pathogenic or pathogenic (safety precautions).

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">1.5 mL microcentrifuge tube</td>
			<td style="width:187px;">Sarstedt</td>
			<td style="width:119px;">72.690.001</td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">15 mL centrifuge tubes</td>
			<td style="width:187px;">Falcon</td>
			<td style="width:119px;">352070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">2 mL microcentrifuge tube</td>
			<td style="width:187px;">Starstedt</td>
			<td style="width:119px;">72.691</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">2100 Bioanalyzer Instrument</td>
			<td style="width:187px;">Agilent</td>
			<td style="width:119px;">G2939BA</td>
			<td>RNA quantity and quality</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">250 mL culture flask</td>
			<td style="width:187px;">Dominique Dutscher</td>
			<td style="width:119px;">2515074</td>
			<td>Bacterial cultures</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">50 mL centrifuge tubes</td>
			<td style="width:187px;">Falcon</td>
			<td style="width:119px;">352051</td>
			<td>Culture centrifugation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Absolute ethanol</td>
			<td style="width:187px;">VWR Chemicals</td>
			<td style="width:119px;">20821.321</td>
			<td>RNA extraction and purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Allegra X-12R Centrifuge</td>
			<td style="width:187px;">Beckman Coulter</td>
			<td style="width:119px;"></td>
			<td>Bacterial pelleting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Ampicilin (amp)</td>
			<td style="width:187px;">Sigma-Aldrich</td>
			<td style="width:119px;">A9518-5G</td>
			<td>Growth medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Amylose resin</td>
			<td style="width:187px;">New England BioLabs</td>
			<td style="width:119px;">E8021S</td>
			<td>MS2-affinity purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Anti-dioxigenin AP Fab fragment</td>
			<td style="width:187px;">Sigma Aldrich</td>
			<td style="width:119px;">11093274910</td>
			<td>Northern blot assays</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Autoradiography cassette</td>
			<td style="width:187px;">ThermoFisher Scientific</td>
			<td style="width:119px;">50-212-726</td>
			<td>Northern blot assays</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">BamHI</td>
			<td style="width:187px;">ThermoFisher Scientific</td>
			<td style="width:119px;">ER0051</td>
			<td>Plasmid construction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">BHI (Brain Heart Infusion) Broth</td>
			<td style="width:187px;">Sigma-Aldrich</td>
			<td style="width:119px;">53286</td>
			<td>Growth medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Blocking reagent</td>
			<td style="width:187px;">Sigma Aldrich</td>
			<td style="width:119px;">11096176001</td>
			<td>Northern blot assays</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">CDP-Star</td>
			<td style="width:187px;">Sigma Aldrich</td>
			<td style="width:119px;">11759051001</td>
			<td>Northern blot assays (substrate)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Centrifuge 5415 R</td>
			<td style="width:187px;">Eppendorf</td>
			<td style="width:119px;"></td>
			<td>RNA extraction and purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Chloroform</td>
			<td style="width:187px;">Dominique Dutscher</td>
			<td style="width:119px;">508320-CER</td>
			<td>RNA extraction and purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">DIG-RNA labelling mix</td>
			<td style="width:187px;">Sigma-Aldrich</td>
			<td style="width:119px;">11277073910</td>
			<td>Northern blot assays</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">DNase I</td>
			<td style="width:187px;">Roche</td>
			<td style="width:119px;">4716728001</td>
			<td>DNase treatment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Erythromycin (ery)</td>
			<td style="width:187px;">Sigma-Aldrich</td>
			<td>Fluka 45673</td>
			<td>Growth medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FastPrep device</td>
			<td style="width:187px;">MP Biomedicals</td>
			<td style="width:119px;">116004500</td>
			<td>Mechanical lysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Guanidium Thiocyanate</td>
			<td style="width:187px;">Sigma-Aldrich</td>
			<td style="width:119px;">G9277-250G</td>
			<td>Northern blot assays</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Hybridization Hoven Hybrigene</td>
			<td style="width:187px;">Techne</td>
			<td style="width:119px;">FHB4DD</td>
			<td>Northern blot assays</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Hybridization tubes</td>
			<td style="width:187px;">Techne</td>
			<td style="width:119px;">FHB16</td>
			<td>Northern blot assays</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoamyl alcohol</td>
			<td style="width:187px;">Fisher Scientific</td>
			<td style="width:119px;">A/6960/08</td>
			<td>RNA extraction and purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">LB (Lysogeny Broth)</td>
			<td style="width:187px;">Sigma-Aldrich</td>
			<td style="width:119px;">L3022</td>
			<td>Growth medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lysing Matrix B Bulk</td>
			<td style="width:187px;">MP Biomedicals</td>
			<td style="width:119px;">6540-428</td>
			<td>Mechanical lysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">MicroPulser Electroporator</td>
			<td style="width:187px;">BioRad</td>
			<td style="width:119px;">1652100</td>
			<td>Plasmid construction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Milli-Q water device</td>
			<td style="width:187px;">Millipore</td>
			<td style="width:119px;">Z00QSV0WW</td>
			<td>Ultrapure water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">NanoDrop spectrophotometer</td>
			<td style="width:187px;">ThermoFisher Scientific</td>
			<td style="width:119px;"></td>
			<td>RNA/DNA quantity and quality</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Nitrocellulose membrane</td>
			<td style="width:187px;">Dominique Dutsher</td>
			<td style="width:119px;">10600002</td>
			<td>Northern blot assays</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Phembact Neutre</td>
			<td style="width:187px;">PHEM Technologies</td>
			<td style="width:119px;">BAC03-5-11205</td>
			<td>Cleaning and decontamination</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Phenol</td>
			<td style="width:187px;">Carl Roth</td>
			<td style="width:119px;">38.2</td>
			<td>RNA extraction and purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Phusion High-Fidelity DNA Polymerase</td>
			<td style="width:187px;">New England Biolabs</td>
			<td style="width:119px;">M0530</td>
			<td>Plasmid construction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">pMBP-MS2</td>
			<td style="width:187px;">Addgene</td>
			<td style="width:119px;">65104</td>
			<td>MS2-MBP production</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Poly-Prep chromatography column</td>
			<td>BioRad</td>
			<td style="width:119px;">7311550</td>
			<td>MS2-affinity purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PstI</td>
			<td style="width:187px;">ThermoFisher Scientific</td>
			<td style="width:119px;">ER0615</td>
			<td>Plasmid construction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Qubit 3 Fluorometer</td>
			<td style="width:187px;">Invitrogen</td>
			<td style="width:119px;">15387293</td>
			<td>RNA quantity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">RNAPro Solution</td>
			<td style="width:187px;">MP Biomedicals</td>
			<td style="width:119px;">6055050</td>
			<td>Mechanical lysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">ScriptSeq Complete Kit</td>
			<td style="width:187px;">Illumina</td>
			<td style="width:119px;">BB1224</td>
			<td>Preparation of cDNA librairies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Spectrophotometer Genesys 20</td>
			<td style="width:187px;">ThermoFisher Scientific</td>
			<td style="width:119px;">11972278</td>
			<td>Bacterial cultures</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">SpeedVac Savant vacuum device</td>
			<td style="width:187px;">ThermoFisher Scientific</td>
			<td style="width:119px;">DNA120</td>
			<td>RNA extraction and purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Stratalinker UV Crosslinker 1800</td>
			<td style="width:187px;">Stratagene</td>
			<td style="width:119px;">400672</td>
			<td>Northern blot assays</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">T4 DNA ligase</td>
			<td style="width:187px;">ThermoFisher Scientific</td>
			<td style="width:119px;">EL0014</td>
			<td>Plasmid construction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">TBE (Tris-Borate-EDTA)</td>
			<td style="width:187px;">Euromedex</td>
			<td style="width:119px;">ET020-C</td>
			<td>Northern blot assays</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">ThermalCycler T100</td>
			<td style="width:187px;">BioRad</td>
			<td style="width:119px;">1861096</td>
			<td>Plasmid construction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween 20</td>
			<td style="width:187px;">Sigma Aldrich</td>
			<td style="width:119px;">P9416-100ML</td>
			<td>Northern blot assays</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">X-ray film processor</td>
			<td style="width:187px;">hu.q</td>
			<td style="width:119px;">HQ-350XT</td>
			<td>Northern blot assays</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">X-ray films Super RX-N</td>
			<td style="width:187px;">FujiFilm</td>
			<td style="width:119px;">4741019318</td>
			<td>Northern blot assays</td>
		</tr>
	</tbody>
</table>

ms2-affinity purification coupled with rna sequencing in gram-positive bacteria

Although small regulatory RNAs (sRNAs) are widespread among the bacterial domain of life, the functions of many of them remain poorly characterized notably due to the difficulty of identifying their mRNA targets. Here, we described a modified protocol of the MS2-Affinity Purification coupled with RNA Sequencing (MAPS) technology, aiming to reveal all RNA partners of a specific sRNA in vivo. Broadly, the MS2 aptamer is fused to the 5&#8217; extremity of the sRNA of interest. This construct is then expressed in vivo, allowing the MS2-sRNA to interact with its cellular partners. After bacterial harvesting, cells are mechanically lysed. The crude extract is loaded into an amylose-based chromatography column previously coated with the MS2 protein fused to the maltose binding protein. This enables the specific capture of MS2-sRNA and interacting RNAs. After elution, co-purified RNAs are identified by high-throughput RNA sequencing and subsequent bioinformatic analysis. The following protocol has been implemented in the Gram-positive human pathogen Staphylococcus aureus and is, in principle, transposable to any Gram-positive bacteria. To sum up, MAPS technology constitutes an efficient method to deeply explore the regulatory network of a particular sRNA, offering a snapshot of its whole targetome. However, it is important to keep in mind that putative targets identified by MAPS still need to be validated by complementary experimental approaches.

The representative results originate from the study of RsaC targetome in S. aureus29. RsaC is an unconventional 1,116 nt-long sRNA. Its 5&#8217; end contains several repeated regions while its 3&#8217; end (544 nt) is structurally independent and contains all predicted interaction sites with its mRNA targets. The expression of this sRNA is induced when manganese (Mn) is scarce, which is often encountered in the context of host immune response. Using MAPS technology, we identified several ...

Watch this Scientific Journal Video about MS2-Affinity Purification Coupled with RNA Sequencing in Gram-Positive Bacteria at JoVE.com

MS2-Affinity Purification Coupled with RNA Sequencing in Gram-Positive Bacteria

MAPS technology has been developed to scrutinize the targetome of a specific regulatory RNA in vivo. The sRNA of interest is tagged with a MS2 aptamer enabling the co-purification of its RNA partners and their identification by RNA sequencing. This modified protocol is particularly suited for Gram-positive bacteria.&#160;

Although small regulatory RNAs (sRNAs) are widespread among the bacterial domain of life, the functions of many of them remain poorly characterized ...

MS2-affinity purification coupled with RNA sequencing or MAPS has been developed to identify the whole targetome of any RNA of interest in bacteria. This technology makes it possible to identify any kind of sRNA partners independently of the mechanism of regulation. The characterization of sRNA-dependent regulatory networks will help understand better virulence factors production, biofilm formation, and survival of the bacteria within the host cells, and ultimately fight against staphylococcal infections.This protocol can be applied to any pathogenic or non-pathogenic gram-positive bacteria. Demonstrating the procedure will be Noemie Mercier, a PhD student from Dr.Pascal Romby's laboratory. To begin, grow one colony of strains carrying either pCN51 P3 MS2 sRNA or pCN51 P3 MS2 plasmids in three milliliters of BHI medium supplemented with 10 micrograms per microliter erythromycin in duplicates.Dilute each overnight culture in 50 milliliters of fresh BHI medium supplemented with erythromycin to reach an OD 600 of 0.05. Grow the cultures at 37 degrees Celsius with shaking at 180 RPM for six hours. Transfer each culture into a 50 milliliter centrifuge tube and centrifuge the tubes at 2, 900 times G for 15 minutes at four degrees Celsius, then discard the supernatant, freeze and store them at minus 80 degrees Celsius or keep the pellets on ice and directly perform mechanical cell lysis.To perform mechanical cell lysis, resuspend pellets in five milliliters of buffer A and transfer the resuspended cells to 15 milliliter centrifuge tubes with 3.5 grams of silica beads. Insert the tubes in a mechanical cell lysis instrument and run a cycle of 40 seconds at four meters per second. Centrifuge the tubes at 2, 900 times G for 15 minutes, then recover the supernatant and keep it on ice.Make sure that the supernatant is clear, repeating the centrifugation if it is not. Remove the column tip, then put a chromatography column in a column rack and wash the column with ultrapure water. Add 300 microliters of amylose resin and wash the column with 10 milliliters of buffer A.Dilute 1, 200 picamoles of MBP-MS2 protein in six milliliters of buffer A and load it into the column.Wash the column with 10 milliliters of buffer A.Next, perform MS2-affinity purification. Load the cell lysate into the column and collect the flow-through fraction in a clean collection tube. Wash the column three times with 10 milliliters of buffer A and collect the wash fraction, then elute the column with one milliliter of buffer E and collect the elution fraction in a two milliliter microtube.Keep all collected fractions on ice until RNA extraction. Use one milliliter of each fraction for RNA extraction. Add one volume of phenol to the RNA and mix vigorously, then centrifuge the mixture at 16, 000 times G for 10 minutes at 20 degrees Celsius.Transfer the upper phase to a clean two milliliter microtube. Add one volume of chloroform isoamyl alcohol and repeat the centrifugation, then transfer the upper phase to a new tube. Add 2.5 volumes of 100%cold ethanol and 0.1 volume of three molar sodium acetate at pH 5.2 and precipitate overnight at minus 20 degrees Celsius.On the next day, centrifuge the tubes for 15 minutes at 16, 000 times G and four degrees Celsius. Slowly remove the ethanol with a pipette, taking care not to disturb the pellet. Add 500 microliters of 80%cold ethanol and centrifuge for five minutes.Discard the ethanol by pipetting it slowly, then dry the pellet using a vacuum concentrator for five minutes on run mode. Resuspend the pellet in an appropriate volume of ultrapure water. This protocol was used to study the RsaC targetome in S.aureus.Several mRNAs interacting directly with RsaC were identified using MAPS technology, revealing its crucial role in oxidative stress and metal-related responses. To confirm the MS2 sRNA constructs and visualize their pattern of expression, cells were harvested after two, four, and six hours of growth in BHI medium. RNA was extracted and Northern blot analysis was performed using the RsaC-specific DIG probe.The level of endogenous RsaC significantly increased after six hours of growth. Importantly, the level of MS2-RsaC 544 and MS2-RsaC 1, 116 were comparable to endogenous RsaC at six hours. Affinity purification was performed and RNAs were extracted from the crude extract, flow-through, and elution fractions in the wild-type strain expressing the MS2 tag alone and the mutant RsaC strain expressing the MS2-RsaC 544 construct.The 1, 116 nucleotide long endogenous RsaC was enriched in the elution fraction, but interacted non-specifically with the affinity column due to its length and complex secondary structure. The MS2-RsaC 544 was highly enriched in the elution fraction demonstrating that it was successfully retained by the MS2-MBP fusion protein. A bioinformatic analysis revealed putative mRNA targets according to the fold change between MS2 sRNA and MS2 control, indicating that sodA was the first putative target.A Northern blot analysis performed sodA-specific DIG probe after MS2-affinity purification shows that sodA was efficiently co-enriched with MS2-RsaC 544 compared to the MS2 control. When attempting this protocol, keep in mind that the addition of the MS2 tag can affect sRNA confirmation, stability, or function. This should be carefully monitored.MAPS provides a snapshot of sRNA-RNA targets pairing. Further experimental validation will unravel their function. This technique represents a powerful tool to build sRNA regulatory networks in any bacteria.

Research

JoVE Journal

Biochemistry

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Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

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Single-step Purification of Macromolecular Complexes Using RNA Attached to Biotin and a Photo-cleavable Linker

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Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

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Translating Ribosome Affinity Purification (TRAP) for RNA Isolation from Endothelial Cells In Vivo

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Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis ...

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

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