Electrophoretic Mobility Shift Assay (EMSA) for the Study of RNA-Protein Interactions: The IRE/IRP Example

Summary

Here we present a protocol to analyze RNA/protein interactions. The electrophoretic mobility shift assay (EMSA) is based on the differential migration of RNA/protein complexes and free RNA during native gel electrophoresis. By using a radiolabeled RNA probe, RNA/protein complexes can be visualized by autoradiography.

Abstract

RNA/protein interactions are critical for post-transcriptional regulatory pathways. Among the best-characterized cytosolic RNA-binding proteins are iron regulatory proteins, IRP1 and IRP2. They bind to iron responsive elements (IREs) within the untranslated regions (UTRs) of several target mRNAs, thereby controlling the mRNAs translation or stability. IRE/IRP interactions have been widely studied by EMSA. Here, we describe the EMSA protocol for analyzing the IRE-binding activity of IRP1 and IRP2, which can be generalized to assess the activity of other RNA-binding proteins as well. A crude protein lysate containing an RNA-binding protein, or a purified preparation of this protein, is incubated with an excess of³² P-labeled RNA probe, allowing for complex formation. Heparin is added to preclude non-specific protein to probe binding. Subsequently, the mixture is analyzed by non-denaturing electrophoresis on a polyacrylamide gel. The free probe migrates fast, while the RNA/protein complex exhibits retarded mobility; hence, the procedure is also called “gel retardation” or “bandshift” assay. After completion of the electrophoresis, the gel is dried and RNA/protein complexes, as well as free probe, are detected by autoradiography. The overall goal of the protocol is to detect and quantify IRE/IRP and other RNA/protein interactions. Moreover, EMSA can also be used to determine specificity, binding affinity, and stoichiometry of the RNA/protein interaction under investigation.

Introduction

The EMSA was originally developed to study the association of DNA-binding proteins with target DNA sequences^1,2. The principle is similar for RNA/protein interactions³, which is the focus of this article. Briefly, RNA is negatively charged and will migrate towards the anode during non-denaturing electrophoresis in polyacrylamide (or agarose) gels. Migration within the gel depends on the size of the RNA, which is proportional to its charge. Specific binding of a protein to RNA alters its mobility, and the complex migrates slower compared to the free RNA. This is mainly due to an increase in the molecular mass, but also to alterations in the charge....

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Protocol

Experimental procedures with mice were approved by the Animal Care Committee of McGill University (protocol 4966).

1. Preparation of Protein Extracts from Cultured Cells

1. Wash cultured cells twice with 10 ml of ice-cold phosphate buffered saline (PBS).
2. Scrape adherent cells with either a rubber policeman or a plastic cell scraper in 1 ml of ice-cold PBS, transfer suspension into a 1.5 ml microcentrifuge tube.
3. Spin in a microcentrifuge for 5 min at 700 x g, at 4 °C. Aspirate PBS.
4. Add 100 μl of ice-cold cytoplasmic lysis buffer (Table 1....

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Results

A radiolabeled IRE probe was prepared, as described in sections 3 and 4 of the protocol. The sequence of the probe was 5'-GGGCGAAUUC GAGCUCGGUA CCCGGGGAUC CUGCUUCAAC AGUGCUUGGA CGGAUCCU-3'; the bolded nucleotides represent an unpaired C residue and the loop, which are critical IRE features. The specific radioactivity of the probe was 4.5 x 10⁹ cpm/μg of RNA.

To assess the effects of iron perturbations on IRE-binding activity, murine RAW264.7 macrop.......

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Discussion

Herein, we describe a protocol that has been developed to study the IRE-binding activities of IRP1 and IRP2, and we show representative data. By using different probes, this protocol can also be adjusted for the study of other RNA-binding proteins. A critical step is the size of the probe. Usage of long probes, which is common when the exact binding site is unknown, can result in RNA/protein complexes that do not migrate differently than the free RNA. In this case, it is advisable to remove unbound RNA by treatment with .......

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Materials

Name	Company	Catalog Number	Comments
leupeptin	SIGMA	L2884
PMSF	SIGMA	78830
BioRad Protein Assay	BIORAD	500-0006
T7 RNA polymerase	Thermoscientific	EPO111
RNase Inhibitor	Invitrogen	15518-012
UTP [alpha-32P]	Perkin-Elmer	NEG507H
Scintillation liquid	Beckman Coulter	141349
heparin	SIGMA	H0777
Rnase T1	Thermoscientific	EN0541
Name of the Equipment
Tissue Ruptor	Qiagen	9001271
Scintillation counter	Beckman Coulter	LS6500
Protean II xi Cell	BIORAD	165-1834
20 wells combs	BIORAD	165-1868	1.5 mm thick
1.5 mm spacers	BIORAD	165-1849
PowerPac	BIORAD	164-5070