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

The overall goal of the following experiment is to analyze RNA protein interactions from cell extracts using an electrophoretic mobility shift assay. This is achieved by first preparing a protein extract from cells or tissues in a separate step, a gene specific radio labeled RNA probe is synthesized and purified. The protein extract is then mixed with the labeled RNA probe to allow formation of specific RNA protein complexes.

Finally, the reaction product is loaded onto a non denaturing poly acrylamide gel and analyzed by electrophoresis free RNA probe is separated from the RNA protein complexes by virtue of its faster mobility. The electrophoretic mobility shift assay is widely used to analyze RNA protein interactions. We could analyze the binding of iron regulatory proteins, IRP one and IRP two to their target RNA element.

But the method can also be applied for the study of other RNA binding proteins demonstrating this procedure will be done by Dr.Kain Philippine research associate in my laboratory and by Dr.Nicole Wilkinson, a postdoctoral fellow For adhere cells grown in dishes. Wash the cells twice with ice cold PBS, then add one milliliter of ice cold PBS and harvest the cells with a plastic cell scraper. Transfer the loose cell suspension into a fresh 1.5 milliliter centrifuge tube.

Place the tube in a pre chilled four degree Celsius micro centrifuge. Spin down at a low speed for five minutes and aspirate the sup natant. The cells will appear as a white pellet at the bottom of the tube.

For every 10 million cells harvested, add 100 microliters of ice cold cytoplasmic lysis buffer. Loosen the cell pellet by pipetting up and down several times and then incubate the suspension on ice for 20 minutes. This will solubilize the cellular membrane and release all cytosolic contents into the buffer.

To isolate the solubilized cytosolic fraction, spin the tube down at full speed for 10 minutes in a four degree Celsius centrifuge, transfer the clarified snat into a new 1.5 milliliter tube on ice and discard the pellet. Next, determine the total protein concentration of the resulting cytoplasmic extract using the Bradford assay. Aliquot the resulting cytoplasmic extract into smaller tubes and store at negative 80 degrees Celsius until use to produce a cytoplasmic extracts from fresh tissues.

Begin the harvesting process by laying the euthanized animal on a clean pad over a dissecting board. Open the abdomen with scissors. Remove the liver and the spleen by using scissors and forceps.

Rinse each tissue in approximately 50 milliliters ice cold PBS to prevent tissue degradation. Immediately cut tissues into small pieces approximately one to two cubic millimeters with a scalpel without delay. Place the tissues in a fresh cryo tube and snap.

Freeze a sample in liquid nitrogen. Store the frozen tissues at minus 80 degrees Celsius until they are needed. Begin making the cytoplasmic extract by transferring the previously frozen tissue sample into a two milliliter tube with a flat bottom containing 250 to 500 microliters of ice cold lysis buffer.

Place a tissue homogenizer tip into the tube and turn on the apparatus at medium power. After 10 seconds of homogenization immediately place the tube on ice for 20 minutes to prevent protein denaturation. To isolate the cytosolic fraction, spin the tube down at full speed for 10 minutes in a four degrees Celsius centrifuge, transfer the clarified supernatant into a new 1.5 milliliter tube on ice and discard the pellet.

Determine the total protein concentration of the resulting cytoplasmic extract using the Bradford assay. Aqua the resulting cytoplasmic extract into smaller tubes and store at negative 80 degrees Celsius until it is needed. Before continuing with the protocol, set up a radiation safe workbench complete with a plexiglass shield Geiger counter.

Do cytometry monitor tags on lab coats and appropriate radiation specific sharps and non sharpp waste containers. Starting with a de linearized DNA plasmid containing a cloned iron response element. As a template, set up a 20 microliter in vitro RNA transcription reaction by mixing the template reaction buffer, partially radioactive nucleotides and the RNA polymerase with a pipette at room temperature.

To initiate RNA synthesis, incubate the sample at 40 degrees Celsius for one hour. Terminate the in vitro transcription reaction by adding one microliter of 0.5 molar EDTA pH 8.0. Mix in the EDTA by pipetting up and down.

Now use a standard alcohol precipitation protocol to purify the RNA product. To begin, add 10 microliters of TRNA and 82.5 microliters of three molar sodium acetate at pH 5.2 to the post synthesis mixture and vortex. To mix the TRNA will act as a precipitation carrier and will increase the final RNA yield to precipitate the RNA.

Add 273 microliters of 95 to 100%ethanol and mix by vortexing. Allow the precipitation reaction to proceed by leaving the tube on the bench for 10 minutes at room temperature to isolate the RNA, spin the tube down in a room temperature centrifuge at full speed for 10 minutes. With a pipette, carefully discard the supernatant and do not disturb the pellet.

The precipitated RNA may exist either as a small white pellet near the bottom of the tube or it may be invisible to the naked eye. Wash the pellet by adding 100 to 500 microliters of 70%ethanol. Spin down the sample at full speed for 10 minutes at room temperature and then decant the snat with the lid.

Open dry the RNA pellet by leaving the tube open on the bench for 10 to 15 minutes. Reconstitute the solid radio labeled RNA by adding 100 microliters of nuclease free water. Quantify the extent of radioactive nucleotide incorporation by placing the RNA solution in a liquid sation Counter Eloqua.

The radio labeled RNA Probe into smaller tubes and store at minus 80 degrees Celsius until it is needed. Frozen aliquots can be used for up to three weeks before starting the electrophoretic mobility shift assay, prepare a standard 6%non denaturing acrylamide gel of at least 16 by 16 centimeters in size to facilitate larger sample volumes per lane. And to enhance the mechanical stability of a gel this size.

Use glass spacers and combs of at least one millimeter thick. To begin the electrophoretic mobility shift assay thaw the previously frozen cytoplasmic lysate and the radio labeled RNA probes on eyes for the protein component of the experiment. Dilute 25 micrograms of the cytoplasmic extract with the cytoplasmic lysis buffer to a final total volume of 10 microliters.

When needed, add one microliter of two me stock solution to the mixture and keep the protein sample on ice. For the RNA component, dilute the radio labeled RNA probe in nuclease free water down to 200, 000 counts per minute per microliter heat to nature the RNA at 95 Celsius for one minute and cool the solution down at room temperature for at least five minutes. To initiate the protein RNA binding reaction, add one microliter of radio labeled RNA probe to the protein extract allow binding to occur for 20 minutes at room temperature.

To increase specificity of the electrophoretic mobility shift assay, add one microliter of heparin stock to the reaction. If radiolabeled RNA probes are longer than 60 nucleotides, add an additional one microliter of RNAs T one. Allow the binding reaction to continue for another 10 minutes at room temperature to further enhance specificity and allow better separation of the RNA protein complex.

Add three microliters of loading buffer and pipette up and down to mix. Then load the entire reaction onto the 6%acrylamide gel and run the gel at five volts per centimeter for 60 minutes. Be sure to cover the apparatus with proper radiation shielding.

Disassemble the gel apparatus and transfer the gel onto a large filter paper. Dry the gel using a gel drying vacuum apparatus in a dark room. Place the gel and filter paper combination on top of a piece.

Film after exposure, develop and air dry the film. The optimum exposure time can vary from one hour to overnight. One can assess the iron dependent binding capacity of I RRP one and I RRP two to the radioactive IRE probes in tissue culture cells with variable iron content.

Thus, IRE binding activity is induced in iron depleted cells previously treated with the K later deferoxamine. Conversely, IRE binding activity is suppressed in iron loaded cells previously treated with heman in iron loaded cells. The IRE binding activity of I RRP one is lost following assembly of an iron sulfur cluster while I RRP two undergoes iron dependent degradation.

The iron cell cluster of IRP one can be destroyed by treatment of cell extracts with 2%two me, which allows monitoring its dormant IRE binding activity. The activity of I RRP two is not recovered by two me. In this next experiment, the IRE binding activities of I RRP one and IRP two as a function of cellular iron concentration are assessed in the context of fresh liver tissues of wild type I RRP one knockout and I RRP two knockout mice.

Here the data show that feeding mice with a high iron diet known to increase hepatic iron load promotes a reduction in the IRE binding activities of I RRP one and IRP two in liver extracts from wild type mice. I rrp two IRE complexes are skewed by the presence of I RRP one. They are readily visualized in liver extracts of I RRP one knockout mice.

Here the high iron diet leads to complete inactivation of IRP two. After watching this video, you should have a good understanding of how to analyze RNA protein interactions by the electrophoretic mobility shift assay. Remember that the quality of the RNA probe is critical for success.