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

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 environments^1,2,3. 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.......

Discussion

A modified protocol for Gram-positive bacteria
The initial protocol of MAPS was developed to study sRNA interactome in the model organism E. coli^20,30. 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.

Materials

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