There are more than 100 distinct modifications of RNA, two third of which are methylations. This protocol provides a method for sensitive and accurate analysis of RNA methylation writer activity. This easy-to-use technique allow us to quantify and to visualize methylated RNA without the use of antibodies, which are commonly prone to artifacts.
Demonstrating the procedure will be Samantha Shelton, a grad student from my lab. To begin this procedure, set up the 100-microliter RNA methyltransferase assay in a 1.5-milliliter tube on ice, as outlined in the text protocol. Vortex the tube to mix thoroughly, and centrifuge at 200 times g for five seconds to collect the solution at the bottom of the tube.
Incubate the tube at 37 degrees Celsius for two hours. Then, clean the reaction using column purification, as outlined in the text protocol. To perform the liquid scintillation count, set up the scintillation count rack with one vial per sample, one vial for a background measurement, and one vial for the swipe test.
Fill the vials with five milliliters of scintillation counting solution. Add 10 microliters of each eluted radioactive RNA sample into one vial. Tighten the lid, and mix gently.
To prepare the swipe test vials, thoroughly rub cotton swabs on all surfaces and equipment used during the procedure. Add these swabs to the vials filled with scintillation solution, and tighten the lid. To run the samples on the scintillation counter, open the counter hood, insert the rack into the machine, and close the hood.
Select Count Single Rack. Choose Select User Program. Select or create a program that measures tritium for 60 seconds, and hit Count Rack.
Repeat the scintillation count three times. After this, prepare and re-run a urea denaturing polyacrylamide gel, as outlined in the text protocol. Transfer 20 microliters of radioactive RNA material into a new 1.5-milliliter tube containing 20 microliters of 2X gel-loading buffer.
After preparing the ladder, incubate the samples at 70 degrees Celsius for 15 minutes. Wash the wells of the gel once more immediately before sample loading by pipetting buffer from the gel tanks down into the wells of the gel. Then, load 20 microliters of the prepared ladder, 20 microliters of the samples, and 20 microliters of 1X gel-loading buffer into the lanes of the gel.
Run the gel at 100 volts for 60 to 180 minutes, depending on the polyacrylamide percentage. Once the gel has finished running, remove the gel from the cassette and place it in a box containing 50 milliliters of 1X TBE buffer with five microliters of ultrasensitive nucleic acid gel stain. Incubate on a rocker for five minutes to stain the RNA.
After this, carefully take the gel out of the box, and place it on the UV transilluminator of the gel imaging system with the wells up and the ladder on the left. Focus the camera on the gel, turn on the UV light, and take an image of the gel. Then, turn off the UV exposure, and save the image as a TIFF file.
Place the gel back into the box, remove the TBE, and fix the gel with 50 milliliters of fixing solution on a rocker at room temperature for 30 minutes. Next, gently move the gel to a fresh black box containing 25 milliliters of the autoradiography enhancing solution. Incubate on the rocker at room temperature for 30 minutes.
Gently lift the gel, and place it face-down on a sheet of plastic wrap with the wells up and the ladder on the right-hand side. Place two sheets of chromatography paper on the back of the gel, and flip the entire stack. Pre-heat the gel dryer to 80 degrees Celsius.
Move the plastic cover on the gel dryer back. Insert the entire stack under the plastic cover, and move the plastic cover back down to create a seal. Dry the gel at 80 degrees Celsius for one hour.
Then, turn off the gel dryer, and gently remove the stack. Remove the wrap and second chromatography paper. Tape the remaining chromatography paper with the dried gel in an autoradiogram cassette.
In a dark room, add one sheet of autoradiography film. Store the cassette at negative 80 degrees Celsius, and develop the film at the appropriate time, depending on the signal intensity. Once the film is developed, place it on top of the cassette and use a lab marker to carefully mark the four edges of the gel, each well, and the position of the XC and BB dyes.
Scan the film at a resolution of either 300 or 600 pixels per inch, and save the image as a TIFF file. In this study, an antibody-free assay is demonstrated to analyze RNA methyltransferase activity. A typical run from an in vitro transcription reaction with the T7 RNA polymerase of the 7SK small nuclear RNA shows relatively short and highly structured RNA.
There are multiple undesired bands, both shorter and longer than 7SK, probably resulting from random transcriptional initiation or termination events. Because of this, gel purification following the in vitro transcription reaction is important to obtain a clean RNA sample. At this point, the RNA of interest can be identified by its location relative to the ladder and purified by gel purification.
A representative RNA methyltransferase assay using the lower limit of the recommended RNA and protein concentrations is shown here. This assay allows for both quantitative results from the scintillation counts, as well as qualitative results from the autoradiogram. Here, an RNA methyltransferase known to methylate 7SK is shown to be able to also methylate U6.Moreover, as recently shown for 7SK, binding of histone H4 to MePCE also inhibits U6 methylation.
This can be observed in both the scintillation count and the autoradiogram, showing the robustness of this protocol. RNA is susceptible to degradation, so please ensure to use RNase-free reagents and to thoroughly clean all surfaces and equipment. The radioactively labeled RNA obtained from this methyltransferase assay can be directly used to assess RNA demethylase activity or RNA binding activity by electrophoretic mobility shift assay, for example.
This method allows researchers to test the effect of different factors on RNA methyltransferase activity, including point mutations and small molecule inhibitor treatments. This method uses radioactive material, so be sure to wear the appropriate PPE, and be careful during handling and disposing of radioactive materials.
