The overall goal of this experiment is to retrieve RNA-binding proteins of an RNA transcript from brown adipose tissue. This method can help answer key questions in this field, such as, what are the regulatory mechanisms of longlife coding RNA, and associated RNA proteins. The main advantage of this tactic is that it is easy to perform.
The RNA used to demonstrate this procedure is a fragment of the Antigen Receptor 3-Prime Untranslated Region, hereafter referred to as the AR 3-Prime UTR. Begin by amplifying the T7 AR oligo and the partial sequence of the firefly luciferase DNA from the psiCHECK-2 plasmid. The primers and parameters used are detailed in the accompanying document.
Following amplification, use the PCR producs as templates to synthesize biotinylated RNAs using an in vitro transcription kit, following the manufacturers instructions. Then, incubate the mixture at 37 degrees Celsius for four hours. After in vitro transcription, add one microliter of Dnase to the reaction and incubate it at 37 degrees Celsius for 15 minutes to degrade the template DNA.
Then, bring the total volume up to 60 microliters with water. Apply the biotinylated RNAs to purification columns to remove the salt and unincorporated nucleotides. Then, after measuring the RNA concentration, store the RNase at minus 80 degrees Celsius for subsequent pull-down assays.
On the day of the assay, thaw the frozen biotinylated RNA on ice. Then, to ensure that the RNA of interest has the proper secondary structure, heat 50 microliters of it at 90 degrees Celsius for two minutes. Chill on ice for two minutes.
Next, add 50 microliters of 2X RNA structure buffer and allow the RNA to fold at room temperature for 30 minutes. Next, resuspend streptavidin-coated magnetic beads in their original vial by briefly vortexing them. Then, transfer 150 microliters of the resuspended beads to a 1.5 milliliter tube.
Place the tube in a magnetic stand for one minute to separate the magnetic beads and the supernatant. Use a pipette to remove and discard the supernatant. Keep the bead pellet.
Wash the beads once by resuspending the bead pellet in one milliliter of the manufacturers recommended 1X binding and washing buffer, and then place the tube on a rotater for five minutes at room temperature. Wash with buffer A and buffer B twice. Following the incubation, discard the supernatant and keep the bead pellet.
Next, resuspend the bead pellet in 300 microliters of 2X washing and binding buffer. Split the beads equally into three 1.5-milliliter tubes with 100 microliters of beads per tube. Add 100 microliters of RNase-free water, 100 microliters of biotinylated firefly luciferase RNA and 100 microliters of biotinylated AR 3-Prime UTR RNA.
Incubate each bead mixture for one hour at room temperature with rotation. Then, place the tubes on the magnet for one minute. Remove and discard the supernatant.
Keep the bead pellets. Next, wash the beads twice, each time resuspending the bead pellets with 400 microliters of 1X washing and binding buffer, and then placing them on the rotater for five minutes at room temperature. Place the tubes on a magnet for one minute.
Then, remove and discard all of the supernatant. Resuspend each bead pellet in 50 microliters of nuclear isolation buffer. In preparation for the next part of the protocol, ensure that all buffers, as well as the dounce glass homogenizers are chilled on ice.
The cell lysate preparation should proceed on ice. For each RNA pull-down assay to be performed, prepare one 15-centimeter dish of differentiated preadipocytes from three-week old C57 Black Six wild type mice. Four to five days later, to harvest the cells, add 10 to 15 milliliters of PBF and then use a cell-scraper to remove the cells from the dish.
Transfer them to a 50-milliliter tube and then pellet them by centrifugation at 200 times G for five minutes at room temperature. After the spin, use one-milliliter pipette to aspirate and discard the supernatant. Then, resuspend the cell pellet in two milliliters of hypotonic buffer and incubate the cells on ice for fifteen minutes.
Transfer the cells to a 15-milliliter dounce glass homogenizer and shear the cells mechanically on ice with 20 strokes. Pellet the cell nuclei by centrifugation at 3300 times G for 15 minutes at four degrees Celsius. After the spin, transfer the supernatant to 1.5-milliliter tubes and keep the pellet on ice for later use.
To the supernatant, add 3-molar potassium chloride to obtain a final concentration of 150 millimolar. Spin at 20, 000 times G for 15 minutes at four degrees Celsius. Following centrifugation, there will be a lipid layer on the top.
Use a one-milliliter pipette to carefully remove it. Then, collect the citoplasmic cellular lysate supernatant. Next, using a one-milliliter pipette, resuspend the nuclei pellet in one milliliter of nuclear isolation buffer and transfer to a 15-milliliter dounce glass homogenizer.
Shear the nuclei mechanically on ice with 100 strokes. Pellet the nuclear debris by centrifugation at 20, 000 times G for 15 minutes at four degrees Celsius. Collect the supernatant as the nuclear cellular lysate.
These may be combined with the cytoplasmic lysate collected earlier or kept separate for later applications. Here, the samples are combined at a ratio of one-to-one. Add RNase inhibitor to the unfractionated cell lysate to obtain a final concentration of 0.5 units per microliter.
Then, set aside 100 microliters of the cell lysate on ice as an input control for western blotting. Split the cell lysate into three equal aliquots with one milliliter of cell lysate in each 1.5-milliliter tube and incubate with blank firefly luciferase RNA conjugated and AR 3-Prime UTR conjugated beads, respectively, for three hours at four degrees Celsius with rotation. After the incubation, place the tubes on the magnet for one minute.
Use a one-milliliter pipette to collect the supernatant, then isolate the RNA and assess the RNA intactness as described in the accompanying document. Keep the bead pellets on ice. Wash the magnetic beads six times.
Each time, resuspend the bead pellets in one milliliter of nuclear isolation buffer containing 40 units of RNase inhibitor and 0.25 percent igepal. Rotate the tubes for two minutes at room temperature. Place the tubes in a magnetic stand to separate the magnetic beads and supernatant.
After one minute, aspirate and discard the supernatant, then place the bead pellets on ice. To elute the RNA protein complexes from the beads for western blotting, first, resuspend each bead pellet in 75 microliters of nuclear isolation buffer. Then, add 25 microliters of 4X western blot-loading buffer.
containing SDS, to obtain a total volume of 100 microliters. Finally, boil beads in this 100-microliter solution at 95 degrees Celsius for five minutes. Perform western blotting for verification of RBP as described in the accompanying document.
In this experiment, an antigen receptor RNA fragment was used as the bait to capture its binding protein, HuR. Both a firefly luciferase RNA bait unrelated to the HuR protein and an aliquot of unconjugated blank streptavidin beads served as negative controls. RNA protein interactions can occur either in the nucleus or the cytoplasm, and this pull-down protocol can be applied to either total or fractionated nuclear or cytoplasmic cell lysates.
Analysis by western blotting showed HuR protein in the unfractionated cell lysate, or input, using the monoclonal antibody. The antigen receptor RNA elusion fraction also gave a positive result, indicating the presence of HuR protein in the adipocyte lysate using the antigen receptor RNA-conjugated beads for the RNA pull-down assay. Both the firefly luciferase RNA elution fraction and the blank bead sample gave negative results, indicating that non-specific interactions were not detectable using this approach.
Taken together, these results suggest that the interaction between the antigen-receptor RNA fragment and the HuR protein was specific. After watching this video, you should have a good understanding of how to retrieve RNA binding proteins off a RNA transcript from brown adipose tissue. Once mastered, this then can be done in 24 hours, if it is performed properly.
