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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 and possibly conformation. Utilizing a labeled RNA as probe allows easy monitoring of the “gel retardation” or “bandshift”. Usage of ³²P-labeled RNA probes is very common and offers high sensitivity. The migration of RNA/protein complexes and free RNA are detected by autoradiography. Drawbacks are the short half-life of ³²P (14.29 days), the gradual deterioration of the quality of the probe due to radiolysis, the requirement of a radioactivity license and infrastructure for radioactivity work, and potential biosafety concerns. Therefore, alternative non-isotopic methods for labeling the RNA probe have been developed, for instance with fluorophores or biotin, which enable detection by fluorescent or chemiluminescent imaging^4,5. Limitations of these methods are the higher cost and often reduced sensitivity compared to isotopic labeling, and the potential of non-isotopic labels to interfere with the RNA/protein interaction. Non-denaturing polyacrylamide gels are suitable for most EMSA applications and are commonly used. On occasion, agarose gels may pose an alternative for the analysis of large complexes.

The major advantage of EMSA is that it combines simplicity, sensitivity, and robustness⁴. The assay can be completed within a few hours and does not require sophisticated instrumentation. RNA/protein interactions can be detected by EMSA at concentrations as low as 0.1 nM or less, and within a broad range of binding conditions (pH 4.0 - 9.5, monovalent salt concentration 1 - 300 mM, and temperature 0 - 60 °C).

RNA/protein complex formation can also be studied by the filter-binding assay. This is a simple, fast, and inexpensive procedure based on the retention of RNA/protein complexes in a nitrocellulose filter, while a free RNA probe passes through⁶. Compared to EMSA, it is limited in cases where the RNA probe contains multiple binding sites, or the crude extract contains more than one RNA-binding proteins that bind to the probe at the same site. While multiple RNA/protein interactions will escape detection by the filter-binding assay, they can be readily visualized by EMSA. In some cases, visualization is even possible when two RNA/protein complexes co-migrate (for instance, human IRP1/IRE and IRP2/IRE complexes), by adding an antibody against one of the RNA-binding proteins to the EMSA reaction, yielding further retardation on the gel (“supershift”)⁷.

The EMSA has been widely used to study IRP1 and IRP2, which are post-transcriptional regulators of iron metabolism^8-10. They operate by binding to IREs, phylogenetically conserved hairpin structures within the UTRs of several mRNAs¹¹. IREs were first discovered in the mRNAs encoding ferritin¹² and transferrin receptor 1 (TfR1)¹³, proteins of iron storage and uptake, respectively. Later on, IREs were found in erythroid-specific aminolevulinate synthase (ALAS2)¹⁴, mitochondrial aconitase¹⁵, ferroportin¹⁶, divalent metal transporter 1 (DMT1)¹⁷, hypoxia inducible factor 2α (HIF2α)¹⁸, and other mRNAs^19-21. The prototype H- and L-ferritin mRNAs contain one IRE in their 5’ UTR, while TfR1 mRNA contains multiple IREs in its 3’ UTR. IRE/IRP interactions specifically inhibit ferritin mRNA translation by sterically blocking its association of the 43S ribosomal subunit; moreover, they stabilize TfR1 mRNA against endonucleolytic cleavage. IRP1 and IRP2 share extensive sequence similarity and exhibit high IRE-binding activity in iron-starved cells. In iron-replete cells, IRP1 assembles a cubane Fe-S cluster that converts it to cytosolic aconitase at the expense of its IRE-binding activity, while IRP2 undergoes proteasomal degradation. Thus, the IRE/IRP interactions depend upon the cellular iron status, but are also regulated by other signals, such as H₂O₂, nitric oxide (NO) or hypoxia. Here, we describe the protocol for assessing IRE-binding activity from crude cell and tissue extracts by EMSA. We used a ³²P-labeled H-ferritin IRE probe that was generated by in vitro transcription from a plasmid DNA template (I-12.CAT), where the IRE sequence was originally introduced in sense orientation downstream of the T7 RNA polymerase site by cloning of annealed synthetic oligonucleotides ²².

Protokół

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) per 10⁷ cells, and pipette up and down.
5. Incubate on ice for 20 min.
6. Spin for 10 min at full speed in a microcentrifuge at 4 °C.
7. Discard pellet. Transfer supernatant into new 1.5 ml microcentrifuge tube and keep on ice.
8. Determine protein concentration (usually 1 - 10 μg/μl) using the Bradford assay²³.
9. Aliquot and store cell extracts at -80 °C until use.

2. Preparation of Protein Extracts from Mouse Liver and Spleen

1. Euthanize a mouse with CO₂ inhalation.
2. Lay the euthanized animal on a clean pad over a dissecting board. Open the abdomen with scissors.
3. Dissect the liver and the spleen by using scissors and forceps, and rinse each tissue in approximately 50 ml ice-cold PBS.
4. Immediately cut tissues into small pieces with a scalpel (for example: approximately 1 - 2 mm³).
5. Without delay, put pieces of tissues in a fresh cryotube and then snap freeze them in liquid nitrogen. Store snap-frozen tissue aliquots at -80 °C until use.
6. Homogenize one piece of frozen tissue (approximately 1 - 2 mm³) in 0.25 - 0.5 ml of ice-cold cytoplasmic lysis buffer (Table 1) with a tissue homogenizer for 10 sec.
7. Transfer homogenate to 1.5 ml microcentrifuge tube and chill on ice for 20 min.
8. Spin for 10 min at full speed in a microcentrifuge at 4 °C.
9. Discard pellet and transfer supernatant into new 1.5 ml microcentrifuge tube. Keep on ice.
10. Determine protein concentration (usually 1 - 10 μg/μl) using the Bradford assay²³.
11. Aliquot and store cell extracts at -80 °C until use.

3. Preparation of Radiolabeled IRE-probe

1. Linearize the IRE-containing plasmid I-12.CAT²² by incubating at 37 °C for 1 hr with the restriction endonuclease XbaI (1 U per μg of plasmid), which cleaves downstream of the IRE sequence. The linearized plasmid will be used as template for in vitro transcription.
2. Set up an in vitro transcription reaction in a total volume of 20 μl. Use the stock solutions shown in Table 2 and add: 1 μl linearized plasmid template, 4 μl transcription buffer; 1 μl mix of ATP/CTP/GTP mix, 10 μl [α-³²P]-UTP, 2 μl dithiothreitol, 1 μl RNase inhibitor and 1 μl T7 RNA polymerase. Mix by pipetting up and down.
3. Incubate at 40 °C for 1 hr²⁴.

4. Purification of Radiolabeled IRE-probe

1. Terminate in vitro transcription reaction by adding 1 μl of 0.5 M EDTA, pH 8. Mix by pipetting up and down.
2. Add 10 μl of 10 mg/ml tRNA, as carrier for better precipitation. Mix by pipetting up and down.
3. Add 82.5 μl of 3 M ammonium acetate. Mix by vortexing.
4. Add 273 μl of ethanol. Mix by vortexing.
5. Let stand at RT for 5 min.
6. Spin for 10 min at full speed in a microcentrifuge at RT. Discard supernatant.
7. Wash pellet with 100 μl of 70% ethanol.
8. Spin for 10 min at full speed in a microcentrifuge at RT. Discard supernatant.
9. Air dry pellet for 10 min.
10. Resuspend pellet in 100 μl of double distilled, previously autoclaved H₂O.
11. Quantify radioactivity in a liquid scintillation counter, aliquot radiolabeled IRE probe and store at -80 °C until use. Frozen aliquots can be used for up to 3 weeks.

5. Preparation of a native polyacrylamide gel for EMSA

1. Assemble the gel (16 x 16 cm) by using 1.5 mm spacers and comb.
2. To prepare a 6% native polyacrylamide gel, use the stock solutions shown in Table 3. Mix 7.5 ml of 40% acrylamide:bisacrylamide, 5 ml of 5x TBE and 37.5 ml double distilled H₂O.
3. Add 0.5 ml of 10% freshly prepared ammonium persulfate (APS) and 25 μl tetramethylethylenediamine (TEMED).
4. Immediately pour the acrylamide solution to the gel and let it polymerize. Wait for approximately 30 min.
5. Assemble the electrophoresis apparatus with the gel, fill the tanks with 0.5x TBE and connect with the power supply.

6. Electrophoretic Mobility Shift Assay

1. Dilute 25 μg of protein extract from cells or tissues with cytoplasmic lysis buffer (Table 1) in a total volume of in 10 μl (lower protein concentrations may also be used). Keep on ice. If necessary, add 1 μl of 1:4 diluted 2-mercaptoethanol (2-ME) to activate dormant IRP1 (final concentration: 2%)²⁵.
2. Dilute the radiolabeled IRE probe in double distilled H₂O to 200,000 cpm/μl, heat denature at 95 °C for 1 min, and cool down at RT for at least 5 min.
3. Set up an EMSA reaction by adding 1 μl of radiolabeled IRE probe to the protein extract.
4. Incubate for 20 min at RT.
5. Add 1 μl of 50 mg/ml heparin to the reaction (to inhibit non-specific protein interactions with the probe⁹) and continue the incubation for another 10 min. Aliquot the stock solution of heparin (50 mg/ml) and store at -80 °C.
6. When using long radiolabeled probes (>60 nucleotides), add 1 μl RNase T1 (1 U/μl) and incubate for 10 min at RT to reduce non-specific protein binding to the probe, and to allow better separation of the RNA/protein complex during electrophoresis.
7. Add 3 μl of loading buffer (80% glycerol + bromophenol blue), mix and load on the 6% non-denaturing polyacrylamide gel.
8. Run the gel for 60 min at 130 V (5 V/cm).
9. Transfer the gel onto a large filter paper and dry.
10. Expose to a film and develop autoradiography. Exposure time may range from 1 hr (or less) up to O/N.

Wyniki

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 RAW...

Dyskusje

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 ...

Materiały

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