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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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. 

Abstract

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.

Introduction

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....

Protocol

1. Buffers and media

  1. 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.......

Representative Results

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 .......

Discussion

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.

Acknowledgements

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....

Materials

NameCompanyCatalog NumberComments
1.5 mL microcentrifuge tubeSarstedt72.690.001
15 mL centrifuge tubesFalcon352070
2 mL microcentrifuge tubeStarstedt72.691
2100 Bioanalyzer InstrumentAgilentG2939BARNA quantity and quality
250 mL culture flaskDominique Dutscher2515074Bacterial cultures
50 mL centrifuge tubesFalcon352051Culture centrifugation
Absolute ethanolVWR Chemicals20821.321RNA extraction and purification
Allegra X-12R CentrifugeBeckman CoulterBacterial pelleting
Ampicilin (amp)Sigma-AldrichA9518-5GGrowth medium
Amylose resinNew England BioLabsE8021SMS2-affinity purification
Anti-dioxigenin AP Fab fragmentSigma Aldrich11093274910Northern blot assays
Autoradiography cassetteThermoFisher Scientific50-212-726Northern blot assays
BamHIThermoFisher ScientificER0051Plasmid construction
BHI (Brain Heart Infusion) BrothSigma-Aldrich53286Growth medium
Blocking reagentSigma Aldrich11096176001Northern blot assays
CDP-StarSigma Aldrich11759051001Northern blot assays (substrate)
Centrifuge 5415 REppendorfRNA extraction and purification
ChloroformDominique Dutscher508320-CERRNA extraction and purification
DIG-RNA labelling mixSigma-Aldrich11277073910Northern blot assays
DNase IRoche4716728001DNase treatment
Erythromycin (ery)Sigma-AldrichFluka 45673Growth medium
FastPrep deviceMP Biomedicals116004500Mechanical lysis
Guanidium ThiocyanateSigma-AldrichG9277-250GNorthern blot assays
Hybridization Hoven HybrigeneTechneFHB4DDNorthern blot assays
Hybridization tubesTechneFHB16Northern blot assays
Isoamyl alcoholFisher ScientificA/6960/08RNA extraction and purification
LB (Lysogeny Broth)Sigma-AldrichL3022Growth medium
Lysing Matrix B BulkMP Biomedicals6540-428Mechanical lysis
MicroPulser ElectroporatorBioRad1652100Plasmid construction
Milli-Q water deviceMilliporeZ00QSV0WWUltrapure water
NanoDrop spectrophotometerThermoFisher ScientificRNA/DNA quantity and quality
Nitrocellulose membraneDominique Dutsher10600002Northern blot assays
Phembact NeutrePHEM TechnologiesBAC03-5-11205Cleaning and decontamination
PhenolCarl Roth38.2RNA extraction and purification
Phusion High-Fidelity DNA PolymeraseNew England BiolabsM0530Plasmid construction
pMBP-MS2Addgene65104MS2-MBP production
Poly-Prep chromatography columnBioRad7311550MS2-affinity purification
PstIThermoFisher ScientificER0615Plasmid construction
Qubit 3 FluorometerInvitrogen15387293RNA quantity
RNAPro SolutionMP Biomedicals6055050Mechanical lysis
ScriptSeq Complete KitIlluminaBB1224Preparation of cDNA librairies
Spectrophotometer Genesys 20ThermoFisher Scientific11972278Bacterial cultures
SpeedVac Savant vacuum deviceThermoFisher ScientificDNA120RNA extraction and purification
Stratalinker UV Crosslinker 1800Stratagene400672Northern blot assays
T4 DNA ligaseThermoFisher ScientificEL0014Plasmid construction
TBE (Tris-Borate-EDTA)EuromedexET020-CNorthern blot assays
ThermalCycler T100BioRad1861096Plasmid construction
Tween 20Sigma AldrichP9416-100MLNorthern blot assays
X-ray film processorhu.qHQ-350XTNorthern blot assays
X-ray films Super RX-NFujiFilm4741019318Northern blot assays

References

  1. Carrier, M. C., Lalaouna, D., Masse, E. Broadening the Definition of Bacterial Small RNAs: Characteristics and Mechanisms of Action. Annual Review of Microbiology. 72, 141-161 (2018).
  2. Hör, J., Matera, G., Vogel, J., Gottesman, S., Storz, G.

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