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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.
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’ 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.
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....
1. Buffers and media
The representative results originate from the study of RsaC targetome in S. aureus29. RsaC is an unconventional 1,116 nt-long sRNA. Its 5’ end contains several repeated regions while its 3’ 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 .......
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.
This work was supported by the “Agence Nationale de la Recherche” (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's Horizon 2020 research and innovation program under the Marie Skłodowska-Curie Grant Agreement No. 753137-SaRNAReg. Work in E. Massé Lab has been supported by operating grants from the Canadian Institutes of Health Research (CIHR), the Natural Scie....
Name | Company | Catalog Number | Comments |
1.5 mL microcentrifuge tube | Sarstedt | 72.690.001 | |
15 mL centrifuge tubes | Falcon | 352070 | |
2 mL microcentrifuge tube | Starstedt | 72.691 | |
2100 Bioanalyzer Instrument | Agilent | G2939BA | RNA quantity and quality |
250 mL culture flask | Dominique Dutscher | 2515074 | Bacterial cultures |
50 mL centrifuge tubes | Falcon | 352051 | Culture centrifugation |
Absolute ethanol | VWR Chemicals | 20821.321 | RNA extraction and purification |
Allegra X-12R Centrifuge | Beckman Coulter | Bacterial pelleting | |
Ampicilin (amp) | Sigma-Aldrich | A9518-5G | Growth medium |
Amylose resin | New England BioLabs | E8021S | MS2-affinity purification |
Anti-dioxigenin AP Fab fragment | Sigma Aldrich | 11093274910 | Northern blot assays |
Autoradiography cassette | ThermoFisher Scientific | 50-212-726 | Northern blot assays |
BamHI | ThermoFisher Scientific | ER0051 | Plasmid construction |
BHI (Brain Heart Infusion) Broth | Sigma-Aldrich | 53286 | Growth medium |
Blocking reagent | Sigma Aldrich | 11096176001 | Northern blot assays |
CDP-Star | Sigma Aldrich | 11759051001 | Northern blot assays (substrate) |
Centrifuge 5415 R | Eppendorf | RNA extraction and purification | |
Chloroform | Dominique Dutscher | 508320-CER | RNA extraction and purification |
DIG-RNA labelling mix | Sigma-Aldrich | 11277073910 | Northern blot assays |
DNase I | Roche | 4716728001 | DNase treatment |
Erythromycin (ery) | Sigma-Aldrich | Fluka 45673 | Growth medium |
FastPrep device | MP Biomedicals | 116004500 | Mechanical lysis |
Guanidium Thiocyanate | Sigma-Aldrich | G9277-250G | Northern blot assays |
Hybridization Hoven Hybrigene | Techne | FHB4DD | Northern blot assays |
Hybridization tubes | Techne | FHB16 | Northern blot assays |
Isoamyl alcohol | Fisher Scientific | A/6960/08 | RNA extraction and purification |
LB (Lysogeny Broth) | Sigma-Aldrich | L3022 | Growth medium |
Lysing Matrix B Bulk | MP Biomedicals | 6540-428 | Mechanical lysis |
MicroPulser Electroporator | BioRad | 1652100 | Plasmid construction |
Milli-Q water device | Millipore | Z00QSV0WW | Ultrapure water |
NanoDrop spectrophotometer | ThermoFisher Scientific | RNA/DNA quantity and quality | |
Nitrocellulose membrane | Dominique Dutsher | 10600002 | Northern blot assays |
Phembact Neutre | PHEM Technologies | BAC03-5-11205 | Cleaning and decontamination |
Phenol | Carl Roth | 38.2 | RNA extraction and purification |
Phusion High-Fidelity DNA Polymerase | New England Biolabs | M0530 | Plasmid construction |
pMBP-MS2 | Addgene | 65104 | MS2-MBP production |
Poly-Prep chromatography column | BioRad | 7311550 | MS2-affinity purification |
PstI | ThermoFisher Scientific | ER0615 | Plasmid construction |
Qubit 3 Fluorometer | Invitrogen | 15387293 | RNA quantity |
RNAPro Solution | MP Biomedicals | 6055050 | Mechanical lysis |
ScriptSeq Complete Kit | Illumina | BB1224 | Preparation of cDNA librairies |
Spectrophotometer Genesys 20 | ThermoFisher Scientific | 11972278 | Bacterial cultures |
SpeedVac Savant vacuum device | ThermoFisher Scientific | DNA120 | RNA extraction and purification |
Stratalinker UV Crosslinker 1800 | Stratagene | 400672 | Northern blot assays |
T4 DNA ligase | ThermoFisher Scientific | EL0014 | Plasmid construction |
TBE (Tris-Borate-EDTA) | Euromedex | ET020-C | Northern blot assays |
ThermalCycler T100 | BioRad | 1861096 | Plasmid construction |
Tween 20 | Sigma Aldrich | P9416-100ML | Northern blot assays |
X-ray film processor | hu.q | HQ-350XT | Northern blot assays |
X-ray films Super RX-N | FujiFilm | 4741019318 | Northern blot assays |
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