This method should be of interest to researchers who are trying to isolate RNA protein complexes and identify their composition by mass spectrometry. We believe our protocol will be especially useful for purifying complexes that exist in cells in limited amounts and tend to dissociate during long multi-step purification procedures. The main advantage of this method is that it involves only a single purification step and could be used with RNA binding sites of any length and sequence.
Demonstrating some of the steps will be Xiao-cui Yang, who is a senior research technician in our group. For binding sites significantly longer than 65 nucleotides, obtain T7 generated RNA and an adaptor PCB oligonucleotide as outlined in the text protocol. Mix 20 picomoles of the RNA with 100 picomoles of the adaptor oligonucleotide in a 1.5 milliliter tube containing 100 microliters of binding buffer.
Place the tube in boiling water and let it incubate for 5 minutes. Then allow the water to cool down to room temperature. First supplement 1 milliliter of mouse nuclear extract with 80 millimolar EDTA at pH 8 to a final concentration of 10 millimolar.
Then add between 5 and 10 picomoles of the RNA substrate that has been tagged with the PCB moiety. Incubate on ice for 5 minutes, making sure to occasionally mix the sample. Next use a pre-cooled microcentrifuge at 4 degrees Celsius to centrifuge the mixture at 10, 000 times g for 10 minutes to remove any potential precipitates.
Carefully collect the supernatant into a new tube on ice, while being careful to avoid transferring the pellet. Transfer approximately 100 microliters of streptavidin agarose bead suspension to a 1.5 milliliter tube. Add approximately 1 milliliter of binding buffer to begin washing the beads.
Centrifuge the tube at 25 times g for 2 to 3 minutes and aspirate the supernatant. Repeat this washing and centrifugation process 2 to 3 times to equilibrate the beads with the binding buffer. Load the previously collected supernatant, which contains the assembled complex over the equilibrated beads.
Using a tube rotator, rotate the tube for 1 hour at 4 degrees Celsius to immobilize the RNA and bound complexes onto the beads. Next spin the tube at 25 times g for 2 to 3 minutes to collect the beads at the bottom of the tube. Aspirate the supernatant.
And rinse the beads twice with binding buffer, using the previously described washing process. After removing the supernatant of the second wash, add 1 milliliter of binding buffer and rotate the sample for 1 hour at 4 degrees Celsius. Spin down the sample at 25 times g for 2 to 3 minutes.
Next add 1 milliliter of binding buffer and transfer the suspension to a new tube. Rotate the sample at 4 degrees Celsius for up to 1 hour. Centrifuge the immobilized complexes at 25 times g for 2 to 3 minutes.
Then aspirate supernatant, and add 200 microliters of binding buffer. Transfer the beads to a 500 microliter tube. Turn on a high intensity UV lamp that emits UV light at 365 nanometers and allow it to reach full brilliance.
Fill up the bottom of a Petri dish with tightly packed ice stacking it over the dish's lid. Briefly vortex the tube containing the immobilized complexes. Then place it horizontally on the ice and cover it with the pre-warmed lamp, making sure that the sample is 2 to 3 centimeters from the surface of the bulb.
Irradiate the sample for a total of 30 minutes while frequently inverting and vortexing the tube to ensure uniform exposure and to prevent overheating. After this, spin down the beads at 25 times g for 2 to 3 minutes and collect the supernatant. Centrifuge this supernatant once more using the same conditions.
Collect the resulting supernatant making sure to leave a small amount at the bottom of the tube to avoid transferring any residual beads. Using SDS-PAGE electrophoresis, separate a fraction of the UV eluted supernatant and the corresponding fraction from the material left on the beads after UV elution. Next use a commercially available silver staining kit to stain the gel.
Evaluate the efficiency of UV elution by comparing the intensities of the proteins present in the UV eluted supernatant and those left on the beads following UV irradiation. Excise the protein bands of interest and determine their identities by mass spectrometry. After this, directly analyze a fraction of the UV eluted supernatant by mass spectrometry to determine the entire proteome of the purified material in an unbiased manner.
In this study, stem loop RNA tagged with photo-cleavable biotin is incubated with 1 milliliter of a cytoplasmic S100 extract obtained from mice myeloma cells. And the effect and efficiency of UV elution is analyzed by silver staining. A number of proteins are detected on the streptavidin beads before UV elution.
Irradiation with long wave UV releases only some of these proteins into the supernatant leaving a non-specific background on the beads. This emphasizes the importance of the UV elution step. Mass spectrometry identifies the major UV eluted proteins as 3'hExo and SLBP with SLBP being represented by both the full length protein and a number of shorter degradation products.
Smaller amounts of other proteins identified as various RNA binding proteins also selectively released into solution by the UV irradiation. UV elution of immobilized Drosophila histone pre-mRNA tagged with photo-cleavable biotin incubated with a nuclear extract resulted in a selective release of only a small number of proteins into the supernatant with an intense background of non-specific proteins remaining on the beads. Mass spectrometry identifies these proteins to be components of the U7 snRNP.
They are not detected in the presence of processing competitors that block binding of U7 snRNP to histone pre-mRNA. It is important to use high intensity UV lamp and place it a close distance to the irradiated sample while also making sure that it doesn't overheat. The UV elution method yields native RNA protein complexes that lack major contaminants and are directly suitable for additional purification steps and functional assays.
Avoid eye and skin exposure during UV irradiation.
