What is the full form of EMSA

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

Summary

Here we present a protocol to investigate RNA / protein interactions. Electrophoretic Mobility Shift Assay (EMSA) on the differential migration of RNA / protein complexes and free RNA during native gel electrophoresis. By using a radioactively labeled RNA probe, RNA / protein complexes can be visualized by autoradiography.

Abstract

RNA / protein interactions are critical to post-transcriptional regulatory pathways. Among the best characterized are cytosolic RNA-binding proteins Iron regulatory proteins, IRP1 and irp2. They bind to iron responsive elements (IRES) within the untranslated regions (UTR) of multiple target mRNAs to allow mRNA translation or stability control. IRE / IRP interactions have been extensively studied by EMSA. Here we describe the EMSA protocol for the analysis of the IRE-binding activity of IRP1 and irp2, which can be used to generalize to assess the activity of other RNA-binding proteins and. A crude protein lysate containing an RNA binding protein, or a purified preparation of the protein, is made with an excess of 32 P-labeled RNA probe incubated, resulting in complex formation. Heparin is added to exclude non-specific protein from probe binding. The mixture is then analyzed by non-denaturing electrophoresis on a polyacrylamide gel. The free probe wanders quickly, while the RNA / protein complex exhibits delayed mobility; hence the procedure is also called "gel retardation" or "band shift" assay. After completion of the electrophoresis, the gel is dried and RNA / protein complexes and free probe are detected by autoradiography. The overall goal of the protocol is to detect and quantify IRE / IRP and other RNA / protein interactions. In addition, EMSA can be used to determine the specificity, affinity, and stoichiometry of the RNA / protein interaction being studied.

Introduction

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The EMSA was originally developed to identify the association of DNA-binding proteins with DNA target sequences 1,2 to study. The principle is similar for RNA / protein interactions 3, which is the focus of this article. In short, RNA will be 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 changes its mobility and the complex migrates more slowly than the free RNA. This is mainly due to an increase in the molecular mass, but also changes in the charge and possibly conformation. Using a labeled RNA as a probe enables easy monitoring of the "gel retardation" or "band shift". use of 32 P-labeled RNA probes is very common and offers high sensitivity. The migration of RNA / protein complexes and free RNA can be detected by autoradiography. Disadvantages are the short half-life of 32 P (14.29 days), the gradual deterioration in the quality of the probe by radiolysis, the requirement for 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 example with fluorophores or biotin, detection by fluorescence or chemiluminescence imaging ​​4,5 enable. Limitations of these methods are the higher cost and often decreased sensitivity to isotopic labeling, and the potential of non-isotopic labels to interfere with the RNA / protein interaction. Non-denaturing polyacrylamide gels are EMSA for most applications and are widely used. Occasionally, agarose gels can be an alternative for the analysis of large complexes.

The great advantage of the EMSA is that it combines simplicity, sensitivity and robustness 4 </ Sup>. The test can be completed within hours and does not require sophisticated instrumentation. RNA / protein interactions can be recognized by EMSA at concentrations as low as 0.1 nM or less, and over a wide range of binding conditions (pH 4.0 to 9.5, monovalent salt concentration from 1 to 300 mM, and temperature 0 - 60 ° C).

RNA / protein complex formation can also be examined by the filter binding assay. This is a simple, quick, and inexpensive method based on keeping the RNA / protein complexes in a nitrocellulose filter while a free RNA probe passes through 6 passes through. Compared to EMSA, in cases where the RNA probe contains multiple binding sites or the crude extract contains multiple RNA binding proteins that bind to the probe, it is limited to the same site. While multiple RNA / protein interactions will escape detection by the filter binding test, they can be easily visualized by EMSA. In some cases, visualization is possible on the evening before, when two RNA / protein complexes co-migrate (for example human IRP1 / IRE and irp2 / IRE complexes), by adding an antibody against one of the RNA binding proteins to the EMSA reaction , which leads to further retardation on the gel ("Supershift") 7.

The EMSA has been widely used to identify IRP1 and irp2, which are classified as transcriptional regulators of iron metabolism 8-10 are to be studied. They work by binding to IREs phylogenetically conserved hairpin structures within the UTRs of several mRNAs 11. IREs were first found in the ferritin mRNAs 12 and Transferrin receptor 1 (TfR1) 13, Proteins encoded by iron storage and uptake, respectively, discovered. Later IREs became in erythroid specific aminolevulinate synthase (ALAS2) 14, mitochondrial aconitase 15. Ferroportin 16,divalent metal transporter 1 (DMT1) 17,Hypoxia-inducible factor 2 found 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 their 3' UTR. IRE / IRP interactions specifically inhibit ferritin mRNA translation by sterically blocking the association of the 43S ribosomal subunit; In addition, they stabilize TfR1 mRNA against endonucleolytic cleavage. IRP1 and irp2 share extensive sequence similarity and exhibit high IRE-binding activity in iron-supplied cells. In iron-supplied cells, IRP1 assembles a cubane Fe-S cluster, which converts it to cytosolic aconitase at the expense of IRE-binding activity, while irp2 undergoes proteasomal degradation. Thus, the IRE / IRP interaction depends on the cellular iron status, but also on other signals, such as H 2 O 2, Nitric oxide (NO) or hypoxia. Here we describe the protocol for assessing the IRE-binding activity of crude cell and tissue extracts from EMSA. We used one 32 P-labeled H-ferritin IRE probe through in vitro transcription generated from plasmid DNA template (I-12.CAT), where the IRE sequence was originally generated in sense orientation downstream of the T7 RNA polymerase site by cloning of annealed synthetic oligonucleotides 22.

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Protocol

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

1. Production of protein extracts from cultured cells

  1. Wash cultured cells twice with 10 ml of ice cold phosphate buffered saline (PBS).
  2. Scrape attached cells with either a rubber squeegee or a plastic cell scraper in 1 ml ice-cold PBS, transfer suspension into a 1.5 ml microcentrifuge tube.
  3. In a microcentrifuge for 5 minutes at 700 × g at 4 ° C. Aspirate PBS.
  4. 100 l ice-cold cytoplasmic lysis buffer (Table 1) per 10 7 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 to new 1.5 ml microcentrifuge tubes and keep on ice.
  8. DetHermelin's protein concentration (usually 1-10 µg / µL) using the Bradford assay 23.
  9. Aliquot and store cell extracts at -80 ° C until use.

2. Production of protein extracts from mouse liver and spleen

  1. Euthanize a mouse with CO 2 Inhalation.
  2. Place the euthanized animal on a clean pad over a dissecting board. Open the abdomen with scissors.
  3. Dissect the liver and spleen with scissors and forceps, and rinse each tissue in approximately 50 ml of ice-cold PBS.
  4. Immediately cut tissue into small pieces with a scalpel (e.g. about 1-2 mm 3).
  5. Immediately, put pieces of tissue in a fresh cryovial and then snap freeze them in liquid nitrogen. Store snap frozen tissue aliquots at -80 ° C until use.
  6. Homogenize a piece of frozen tissue (approximately 1 to 2 mm 3) 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 supernatant in new 1.5 ml microcentrifuge tube. Keep on the ice.
  10. Determine protein concentration (usually 1-10 µg / µL) using the Bradford assay 23.
  11. Aliquot and store cell extracts at -80 ° C until use.

3. Manufacture of radiolabelled IRE probe

  1. Linearize the IRE-containing plasmid I-12.CAT 22 by incubation at 37 ° C. for 1 hour with the restriction endonuclease XbaI (1 U per ug plasmid), cleavage behind the IRE sequence. The linearized plasmid is used as a template for the in vitro transcription be used.
  2. Set up a in vitro transcription response in a total volume of 20 μl. Use the in Table 2 given Stock solutions and add: 1 µl of linearized plasmid template, 4 µl of transcription buffer; 1 µ l mixture of ATP / CTP / GTP mix, 10 μl l [α- 32 P] -UTP, 2 µ l dithiothreitol, 1 µl RNase inhibitor and 1 µl T7 RNA polymerase. Mix by pipetting up and down.
  3. Incubate at 40 ° C for 1 h 24.

4. Purification of radiolabelled IRE probe

  1. end up in vitro transcription response 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 to act as a 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 room temperature for 5 min.
  6. Spin for 10 minutes at full speed in a microcentrifuge at RT. Discard the supernatant.
  7. Wash the pellet with 100 μl of 70% ethanol.
  8. Spin for 10 minutes at full speed in a microcentrifuge at RT. Discard the supernatant.
  9. Air dry pellet for 10 min.
  10. Pellet in 100. l double distilled, previously autoclaved H 2 O.
  11. Quantify the radioactivity in a liquid scintillation counter, aliquot radiolabelled IRE probe and store at -80 ° C until use. Frozen aliquots can be used for 3 weeks.

5. Production of a native polyacrylamide gel for EMSA

  1. Assemble the gel (16 x 16 cm) using 1.5 mm spacers and a comb.
  2. To prepare a 6% native polyacrylamide gel, use the in Table 3 Stock solutions shown mix 7.5 ml of 40% acrylamide. Bisacrylamide, 5 ml 5 x TBE and 37.5 ml double-distilled H. 2 O.
  3. 0.5 ml of 10% freshly made ammonium persulfate (APS) and 25 µl of tetramethylethylenediamine (TEMED).
  4. Immediately pour the acrylamide solution to the gel and allow it to polymerize. Wait for about 30 minutes.
  5. Assemble the electrophoresis device with the gel, fill the tanks with 0.5x TBE and connect to the power supply.

6. Electrophoretic Mobility Shift Assay

  1. Dilute 25 µ g protein extract from cells or tissues with the cytoplasmic lysis buffer (Table 1) in a total volume of 10 µl (lower protein concentrations can also be used). Keep on the ice. If necessary, add 1 μl 1: 4 diluted 2-mercaptoethanol (2-ME) to activate dormant IRP1 (final concentration: 2%) 25.
  2. Dilute the radiolabelled IRE probe in double distilled H. 2 O to 200,000 cpm / ul, heat denature at 95 ° C for 1 min and cool at room temperature for at least 5 min.
  3. Set up an EMSA reaction by adding 1 μl of radiolabelled probe to the IRE protein extract.
  4. Incubate for 20 min at RT.
  5. Add 1 μl 50 mg / ml heparin to the reaction (to avoid non-specific protein interactions with the probe 9 inhibit) and the incubation for a further 10 min.Aliquot the stock solution of heparin (50 mg / ml) and at -80 ° C.
  6. If long radiolabeled probes (> 60 nucleotides), addition of 1 µg l RNase T1 (1 U / μl) and incubate for 10 min at RT to reduce non-specific protein binding to the probe and allow better separation of the RNA / protein complex during electrophoresis.
  7. In 3 μl of application buffer (80% glycerin + 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 to a large filter paper and dry.
  10. Expose to a film and develop autoradiography. Exposure time can be from 1 hour (or less) to O / N.

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Representative Results

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A radiolabelled IRE probe was prepared as described in Sections 3 and 4 of the protocol. The sequence of the probe was 5'-GGGCGAAUUC GAGCUCGGUA CCCGGGGAUC CUG C. UUCAA C AGUGC UUGGA CGGAUCCU-3 '; the bold nucleotides represent an unpaired C residue and the loop, which are critical IRE features. The specific radioactivity of the sample was 4.5 x 10 ? cpm / ug RNA.

To assess the effects of disturbances on iron IRE-binding activity, murine macrophages RAW264.7 were untreated, or treated with hemin (iron source) or desferrioxamine (iron chelator). The lysates were prepared and analyzed by EMSA with the IRE probe (8000 cpm / ug protein). Representative data are in Figure 1 shown with shorter and longer exposures of the autoradiograms on the left and right plates, respectively. Murine IRE / IRP1 and IRE / irp2 complexes show different mobility and two different bands can be seen in lane 1, corresponding to the untreated cells. Eisenchela profoundly induces the IRE-binding activities of both IRP1 and irp2 (lane 2), while iron substitution diminishes them (lane 3). Pretreatment with 2% 2-ME completely activates IRP1 (bottom), also in extracts from iron-treated cells. This shows that iron disturbances do not affect the stability of IRP1 and also serves as a charge control. In contrast, the 2-ME pretreatment inhibited the IRE-binding activity of irp2. The fast migration of unspecific bands are influenced by iron.

We next examined IRE-binding activity in the liver of wild-type IRP1 - / - and irp2 - / - mice that had previously been on a standard or iron-fortified diet for one week (Picture 2) fed. The IRP1 - / - and irp2 - / - mice were kindly provided by Dr. MW Hentze (EMBL, Heidelberg) made available. In wild-type liver extracts, IRP1 accounted for most of the IRE-binding activity and IRE / irp2 complexes were barely visible (lanes 1, 2), in agreement with previous observations 26. Irp2 showed high IRE-binding activity in liver extracts from IRP1 - / - mice (lanes 3, 4). No IRE / IRP1 complexes were observed under these conditions. / - - Were no IRE / irp2 complexes formed in liver extracts from irp2 mice (lanes 5, 6). Feeding mice a high-iron diet decreased liver IRE-binding activity in both IRP1 and irp2; this effect is even more dramatic on irp2 (lanes 7-12). Note that after treatment of wild-type or irp2 - / - liver extracts with 2-ME, the IRE-binding activity of IRP1 was expanded to a point where it moved almost all of the IRE probe (this is evident in the shorter exposure of the probe Autoradiograms observed on the left).

Finally, we examined the IRE binding activity in the liver and spleen of wild-type and hjv - / - mice (Figure 3). The hjv - / - mice were kindly provided by Dr. NC Andrews (Duke University, NC) provided.These animals represent a model of hereditary hemochromatosis 27. deficient a disease of systemic iron overload, in which excessive iron accumulates in parenchymal cells, while reticuloendothelial macrophages remain iron 28. As expected, livers of hjv - / - mice (hepatocytes loaded with iron) had a reduced IRE-binding activity compared to the wild type (lanes 1-4). Conversely, IRE-binding activity was higher in spleens hjv - / - mice (containing iron-deficient macrophages, lanes 5-8). Here, too, the 2-ME treatment promoted almost complete displacement of the IRE probe in liver extracts (see shorter exposure of the autoradiograms on the left).

Table 1 Cytoplasmic Lysis Buffer.

1% Triton X100
25 mM Tris-HCl, pH 7.4
40 mM KCl
  1. The solution should beautoclaved and stored at RT.
  2. Protease inhibitors such as 10 & mgr; g / ml leupeptin and 0.1 mM phenyl methanesulfonyl fluoride (PMSF) can be added before use.
  3. Addition of fresh 1 mM dithiotheritol is recommended for the detection of irp2 activity.

Table 2 Stock solutions for the in vitro transcription reaction.

1 µg / µl linearized plasmid template
5x transcription buffer (supplied with T7 RNA polymerase)
20 mM mixture of ATP, CTP and GTP
3000 Ci / mmol [α-32P] -UTP
100 mM dithiothreitol
10 U / µl RNase inhibitor
20 U / µl T7 RNA polymerase


Table 3. On solutions for non-denaturing polyacrylamide gel electrophoresis.

40% acrylamide: bisacrylamide (37.5: 1)
5x TBE (Tris / Borate / EDTA)

For the preparation of 1 liter of 5x TBE, use 54 g of Tris base, 27.5 g of boric acid and 20 ml of 0.5 M EDTA (pH 8.0).


Figure 1. Iron-dependent regulation of IRE-binding activities in RAW264.7 macrophages. 10 7 Cells were either untreated or treated O / N with 100 µM hemin or desferrioxamine. Cytoplasmic lysates were prepared by EMSA with a 32 P-labeled probe IRE in the absence (above) or in the presence of 2% 2-ME (prepared and analyzed below). The positions of the IRE / IRP1 and IRE / irp2 complexes and the free IRE probe are indicated by arrows. The star shows a non-specific band. Shorter and longer exposure times of the autoradiograms are shown in the left and right windows. Please click here to view a larger version of this image.


Figure 2. Analysis of the IRE binding activities in the liver of wild type (wt), IRP1 - / - and irp2 - / -. Mice 5 week old mice (n = 2 for each genotype), all in C57BL / 6 background 29, were placed on a standard or high iron diet (containing 2% carbonyl iron). After one week the animals were euthanized. Liver protein extracts were available from EMSA with a 32 P-labeled probe IRE processed and analyzed in the absence (top) or in the presence of 2% 2-ME (bottom). The positions of the IRE / IRP1 and IRE / irp2 complexes and the free IRE probe are indicated by arrows. The star shows a non-specific band. Shorter and longer exposure times of the autoradiograms are shown in the left and right windows. Please click here to view a larger version of this image.


Figure 3. Analysis of IRE binding activities in liver and spleen of wild type (wt) and hjv - / -. Mice 10 week old mice (n = 2 for each genotype), all in C57BL / 6 background 30 were put to sleep. Liver and spleen protein extracts were prepared by EMSA with a 32 P-labeled probe IRE in the absence (top) or in the presence of prepared and analyzed 2% 2-ME (bottom). The positions of the IRE / IRP1 and IRE / irp2 complexes and the free IRE probe are indicated by arrows. The star shows a non-specific band. Shorter and longer exposure times of the autoradiograms are shown in the left and right windows. Please click here to view a larger version of this image.

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Discussion

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Here we describe a protocol designed to study the IRE-binding activities of IRP1 and irp2 and we show representative data. By using different probes, this protocol can also be used for the investigation of other RNA-binding proteins. A critical step is the size of the probe. Use of long probes, which is common when the exact binding site is not known, 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 RNAse T1 (Step 6.6) to remove. The quality of the probe is also very important. Best results are obtained with freshly prepared radiolabeled RNA. Gel cleansing 10 is not necessary, however, it can change the appearance of the EMSA if the in vitro transcription reaction Probe preparation yields premature products to improve (and when no RNAse T1 digestion step is included). Running native gel electrophoresis too quickly may lead to overheating, which could lead to dissociation of RNA / protein complexes and into fuzzy bands.

Quantitative results can be obtained by analyzing the intensity of the retarded bands with phosphor imaging. Quantifications are only valid when the RNA probe is present in excess and are not restrictive for RNA binding. The affinity of the RNA / protein interaction, expressed by the dissociation constant K d, associated with the free energy? G O linked can be shown under equilibrium conditions by titration of the RNA binding activity with decreasing amounts of radioactively labeled probe 3 be rated. The IRE / IRP1 interaction has been extensively physicochemical 9,31 marked. EMSA titration experiments with different amounts of an RNA binding protein can be performed to determine the stoichiometry 3 to calculate. Specificity for binding can be validated by competition experiments with an excess of unlabeled "cold" competitor RNAs 3. Only certain competitors should reduce the intensity of the delayed radioactive band. Specificity can also be determined by the inclusion of an antibody against the RNA binding protein in the EMSA reaction, which is a "supershift" 7 revealed are monitored. This can also be used to determine the identity of the RNA-binding protein. No "supershift" should be noted with non-specific antibodies or pre-immune serum.

In summary, the EMSA is a powerful method for the analysis of RNA / protein interactions. It's relatively easy to do and requires minimal infrastructure. It is not as simple as the filter binding assay, but will obtain more informative and accurate data when multiple RNA binding proteins interact specifically with the probe (such as IRP1 and irp2). It should be noted that, in contrast to murine, human IRE / IRP1 and IRE / irp2 complexes, co-migration into EMSA. However, they can be separated with "supershifts̶1; with specific antibodies 7. The EMSA is not valuable for detailed mapping of RNA / protein interactions; other techniques, such as the RNAse protection assay, toeprinting, or crosslinking 32 are more suitable. Still, the EMSA is an excellent choice for discovering new RNA / protein interactions, as has been the case with IRPs so far 8, but also for the matching data obtained with arithmetic or high-through experimental approaches. In addition, the EMSA is superior for analyzing specificity and physicochemical properties of RNA / protein interactions.

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Materials

SurnameCompanyCatalog NumberComments
leupeptinSIGMAL2884
PMSFSIGMA78830
BioRad Protein AssayBIORAD500-0006
T7 RNA polymeraseThermoscientificEPO111
RNase inhibitorInvitrogen15518-012
UTP [alpha-32P]Perkin-ElmerNEG507H
Scintillation liquidBeckman Coulter141349
heparinSIGMAH0777
Rnase T1ThermoscientificEN0541
Name of the equipment
Tissue ruptorQiagen9001271
Scintillation counterBeckman CoulterLS6500
Protean II xi CellBIORAD165-1834
20 wells combsBIORAD165-18681.5 mm thick
1.5 mm spacersBIORAD165-1849
PowerPacBIORAD164-5070

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