An Optimized Quantitative Pull-Down Analysis of RNA-Binding Proteins Using Short Biotinylated RNA

This protocol enables the identification of protein binding partners of a known RNA sequence by pulling them down from cellular extract using biotinylated RNA oligonucleotides. Compared to methods that require detergents or high temperatures to detach the proteins from the RNA, these protocols instead uses very mild conditions that allows also to preserve potential protein complexes. Demonstrating the procedure will be Jakob Rupert, a PhD student under my provision.

Begin total protein harvest by removing the medium from the wells of the HEK 293T cell plate, and wash the wells of the six well plates with one milliliter of PBS. Discard PBS and transfer the plates to ice, before adding 200 microliters of lysis buffer to each well. Detach and break the cells using a cell scraper before transferring the cell extract derived from two wells into the same 1.5 milliliter tube.

After 30 minutes of incubation on ice and centrifugation, transfer the supernatant into a pre-cooled tube. Next, calculate the volume of protein extract corresponding to 1.5 milligrams of proteins. Then bring all the samples to a final volume of 600 microliters using a lysis buffer.

Keep the samples on ice until use. To prepare the beads, mix them in the storage buffer by flicking the tube. After calculating the 100 microliters of slurry medium per sample, place the calculated volume of the slurry into a magnetic rack.

To wash the beads, remove the storage solution, and wash the beads by adding one milliliter of lysis buffer and inverting it manually. Then remove the buffer using the magnetic rack and repeat the washing step. To the beads, add a volume of lysis buffer equal to the initial volume of slurry medium, and mix it by flicking the tube before dispensing the medium uniformly into as many 1.5 milliliter tubes as samples.

For bead blocking, remove the buffer using the magnetic rack and add 600 microliters of 0.25 milligrams per milliliter of yeast TRNA solution prepared in lysis buffer. Incubate it for one hour at room temperature on a rotating wheel. Remove the TRNA solution using the magnetic rack before adding 600 microliters of lysis buffer, and wash it by mixing manually.

Repeat the washing step and discard the buffer. For each tube containing the initial 100 microliters of the slurry medium, prepare 200 micrograms of RNA oligonucleotide in 600 microliters of lysis buffer. Add the oligo to the beads and incubate for one hour at room temperature while rotating.

Remove the solution from the beads and wash twice with 600 microliters of lysis buffer by rotating the tubes for five minutes each at room temperature. Discard the buffer. For protein binding on beads, separate 5%or 30 microliters of volume from the 600 microliters protein solution and keep it as input or IN for further analysis.

Add the remaining protein mix to each tube of loaded beads, and incubate with slow rotations overnight at four degrees Celsius. Remove the unbound protein fraction from the beads using the magnetic rack. Save 5%or 30 microliters of its volume and label it flow through or FT.Add one milliliter of wash buffer 1 to the beads, and rotate it for five minutes at four degrees Celsius.

Discard the buffer and repeat this wash one time. Add one milliliter of wash buffer 2 to the beads. And after incubating at four degrees Celsius on a rotator for five minutes, discard the supernatant.

For eluting the specific binders, add 100 microliters of elution buffer 1 to the beads. After mixing manually by flicking, incubate it for five minutes at room temperature. Place the tubes in a thermal mixer and shake vigorously for five minutes at 95 degrees Celsius.

Then place the tube into the magnetic rack and collect the eluted fraction into a clean tube. Once done, quickly spin the beads with a bench centrifuge to maximize the recovery of the eluate. And save 5%or five microliters of the total eluate or EL volume for further analyses.

The eluted proteins were identified by mass spectrometry. Plotting the significantly enriched proteins in a volcano plot revealed that the total protein content and the enriched proteins eluted from RNA was significantly higher than RNA, suggesting that RNA can establish a higher number of specific interactions. As examples of the agreement between predicted and obtained results, the interaction scores for plus RNA and minus RNA with proteins from human proteome showed that HNRNPH3 was predicted to bind plus RNA selectively, and PCBP2 to interact specifically with RNA.

In addition, the protein RBM41 was predicted to be promiscuous for both RNA oligonucleotides. The mass spectroscopy analysis of protein pull-down assay samples confirmed the presence of HNRNPH3 in RNA and PCBP2 in minus RNA. While RBM41 was found to interact with both.

Western blot was used to detect the presence of TDP-43 in the results and protocol optimization steps. In RNA, TDP-43 was observed in the input sample and the eluate, indicating its presence from the start. TDP-43 was retained by RNA during the washing steps and was eluted at the end with a high salt buffer.

By mapping protein RNA interactions, this method can reveal macromolecular networks behind many physiological pathways. For example, transcriptional regulation or pathological mechanisms including cancer and neurodegeneration.