An Oligonucleotide-based Tandem RNA Isolation Procedure to Recover Eukaryotic mRNA-Protein Complexes

This method can help answer key questions in the field of gene expression regulation such as how RNA-binding proteins assemble on particular RNAs in vivo and thereby impose post-transcriptional gene regulation. The main advantage of this technique is that it does not need any cloning or genetic manipulation. It can be adapted to any model organism or subtype.

To design oligonucleotides, analyze the secondary structure of the mRNA or a fragment thereof using online tools. First, enter the nucleotide sequence in the empty box, then in the Basic Options box select minimum free energy and avoid isolated base pairs. In the Output Options box, select interactive RNA secondary structure plot, and finally click the Proceed button.

A new window will pop up that displays the secondary structure of the mRNA of interest. Select at least three different 21 to 24-nucleotide-long sequences within the mRNA of interest, preferentially in regions lacking extensive secondary structures and located in 3-prime untranslated regions. Select regions with a guanidine/cytosine ratio close to 50%and lacking nucleotide tandem repeats to avoid potential formation of hairpins or self-annealing.

Manually design 2-prime-methoxy modified RNA oligonucleotides bearing a biotin moiety at the 3-prime that are fully complementary to the selected regions within the desired target mRNA. Use a suitable online tool to adjust the melting temperature of the RNA hybrids to between 60 and 65 degrees and having a good linguistic sequence complexity. Use the Basic Local Alignment Search Tool to search for potential ASO cross-hybridization with other mRNAs in the transcriptome.

Select Nucleotide BLAST, insert the sequence in the empty box, and select the organism of interest. Keep the remaining parameters as default and click BLAST. Transfect HEK293 cells at 70%confluency in a 10-centimeter tissue culture dish by mixing two micrograms of the reporter gene with 20 microliters of transfection reagent and adding to the cells.

Place the cells in an incubator at 37 degrees Celsius for 48 hours before harvesting. On the day of harvest, remove the medium with a serological pipette and quickly wash the cells twice with 10 milliliters of PBS pre-warmed at 37 degrees Celsius. Next, add six milliliters of PBS to the dish and place on ice.

Then expose the cells to UV light at 100 millijoules per square centimeter in a UV-crosslinker. After UV exposure, scrape off the cells and PBS and transfer to a 15-milliliter tube. Then spin down the cells at 250 times g for 10 minutes at four degrees Celsius.

After removing the supernatant with a pipette, resuspend the cells in two milliliters of pre-chilled lysis buffer by pipetting up and down five or six times while keeping the tube on ice. Transfer the lysate with a pipette to a five-milliliter tube placed on ice and sonicate for three rounds consisting of 20-second bursts at 10-micron amplitude with 30 seconds of cooling periods on ice. After transferring the lysate to two-milliliter tubes, centrifuge at 15, 000 times g for 10 minutes at four degrees Celsius.

Collect the supernatant and transfer to a new tube. To begin RNA isolation, equilibrate one milligram of oligo(dT)coupled magnetic beads in 500 microliters of lysis buffer. Remove the tube from the rotator, place it on a magnetic rack, and remove the lysis buffer.

Combine four milligrams of HEK293 protein extract with the oligo(dT)25-coupled magnetic beads. Mix the samples vigorously, then incubate for 10 minutes at 25 degrees Celsius. Place the tubes on a magnetic stand for 10 seconds, then remove the supernatant.

Keep the supernatant on ice for subsequent rounds of recovery. Add 500 microliters of wash buffer A to the beads and vortex for five seconds. Collect the beads with a magnet and then wash the beads twice with 500 microliters of wash buffer B.To elute the RNA, add 30 microliters of 10-millimolar Tris-HCl and incubate the tube at 80 degrees Celsius for two minutes with continuous shaking at 1, 000 RPM.

Transfer the tube directly to the magnetic stand and collect the eluate as quickly as possible after 10 seconds or so to prevent potential rebinding of mRNAs to the beads at lower temperatures. The eluates from repeated rounds are then combined and can be stored at minus 80 degrees Celsius. To capture specific mRNAs, add 30 microliters of streptavidin-coupled magnetic beads to one milliliter of binding and wash buffer containing 0.1 milligram per milliliter E.coli transfer RNA.

Equilibrate on a rotator for one hour at room temperature. After an hour, wash the beads three times with 750 microliters of binding and wash buffer. Vortex the tube, then remove the B&W buffer, then resuspend in 30 microliters of buffer and keep on ice until use.

Dilute approximately 35 micrograms of total protein from the previously poly(A)isolation in 100 microliters of binding and wash buffer, then add 200 picomoles of the appropriate antisense oligonucleotide and incubate at 70 degrees Celsius for five minutes to facilitate annealing. Remove the entire heat block from the device and place it at room temperature for 10 minutes to cool down slowly. Next, add the 30 microliters of equilibrated streptavidin-coupled magnetic beads to the sample, then incubate the mixture for 30 minutes at 25 degrees Celsius with constant shaking at 950 rpm in a mixer.

Place the tube on the magnetic stand, remove the supernatant, and wash the beads three times with 750 microliters of pre-warmed binding and wash buffer at 55 degrees Celsius. Add 20 microliters of 10-millimolar Tris-HCl and place at 90 degrees Celsius with constant shaking at 950 rpm for 10 minutes to elute the RNA. After 10 minutes, place the tube in the magnetic stand and immediately collect the eluate.

This is an agarose gel used for the detection of mRNAs with RT-PCR using specific primers upon capture with oligo(dT)and an antisense oligonucleotide to p27 mRNAs from HEK293 cell extract. A control without antisense oligonucleotides is also shown. The input lanes show the total RNA from the cross-linked cells.

In addition, results from competition with poly(A)are also shown. Shown here is an immunoblot analysis of mRNA-bound proteins with antibodies detecting human antigen R, unknown RNA-binding protein, and beta-Actin, which is a negative control protein. The blot shows that human antigen R was successfully detected in poly(A)RNA isolates upon capture of p27 mRNAs with antisense oligonucleotides.

Human antigen R was not detected in control isolates without antisense oligonucleotide. The input lanes show the proteins in the extract from cross-linked cells and results from competition with poly(A)are also shown. Following this procedure, other method like mass spectrometry can be performed to systematically analyze the entire sample of protein interacting with a particular RNA in vivo.

Importantly, TRIP is a versatile approach that can be adapted to all types of polyadenylated RNAs across organisms to understand the dynamic arrangement of RNA protein interactions. Therefore, it can be used to investigate developmentally-controlled or disease-related RNAs and may eventually lead to new targets for therapeutic intervention.