Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing (RIPiT-Seq)

RNA immunoprecipitation in tandem followed by sequencing, or RIPiT-Seq, is a method for identifying what regions of RNA are bound by a particular pair of RNA binding proteins. The most unique advantage of RIPiT is its ability to target two different proteins in two purifications. This provides a powerful way to enrich for a compositionally distinct RNA protein complex from other complexes.

Also, RIPiT-Seq is UV-crosslinking independent, and can be applied to many proteins that act on RNA without directly binding it. While this method does not directly extend towards therapy or diagnosis, RNA binding proteins and RNA processing have been associated with several diseases like neuropathies and cancer. RIPiT-Seq provides a tool for studying functions of disease-linked RNA binding proteins.

RIPiT-Seq requires a system where an epitope-tagged protein can be expressed. It has only been tested in HEK293 cells, and is most amenable to cell culture. However, now with our ability to tag proteins using CRISPR, RIPiT-Seq can be applicable to diverse modern systems.

The success of RIPiT depends upon two efficient immunoprecipitations, so it will be critical to optimize immunoprecipitation conditions for the antibody being used. Also, working with RNA requires proper precautions, so RNase-free reagents should be used during and after RNA extraction. Demonstrating the procedure will be Lauren Woodward, a graduate student in my lab.

Start by washing monolayer cells gently with 15 milliliters of chilled PBS per 15 centimeter plate. Then scrape off cells into 30 milliliters of PBS and collect them in a 50 milliliter conical tube. Pellet the cells by centrifuging at 400 g for 10 minutes at 4 degrees Celsius, and discard the supernatant.

Add four milliliters of ice cold hypotonic lysis buffer and use a P1000 pipette to re-suspend the cells. Transfer the lysate to a five milliliter tube and incubate on ice for 10 minutes. Place the lysate in an ice bath and sonicate according to manuscript directions.

Then add 108 microliters of five molar sodium chloride to adjust the salt concentration to 150 millimolar. Then, centrifuge the lysate at 12, 000 g for 10 minutes at four degrees Celsius, and collect 20 microliters of the supernatant for western blot analysis. Pre-wash FLAG agarose beads according to manuscript directions and apply remaining supernatant to 750 microliters of beads in a five milliliter tube.

Incubate the FLAG agarose beads with the cell extract for one to three hours at four degrees Celsius with gentle mixing. After incubation, pellet the beads by centrifugation at 400 g for one minute at four degrees Celsius, and collect 20 microliters of the supernatant for western blot analysis. Discard the remaining supernatant and wash the beads by resuspending them in four milliliters of iso wash buffer.

Pellet the beads again and remove the supernatant. Dilute RNase I in 750 microliters of iso wash buffer according to manuscript directions, and add it to the FLAG agarose beads. Incubate the mixture at four degrees Celsius with gentle mixing, and then pellet the beads by centrifugation.

Collect 20 microliters of supernatant for western blot analysis, and discard the rest. Then, wash the beads with iso wash buffer as previously described. Next, perform affinity elution by adding 375 microliters of elution buffer to the beads, and shaking the mixture gently at four degrees Celsius for one to two hours.

After incubation, pellet the beads and collect a 15 microliter aliquot of the elution for western blot analysis. While affinity elution is underway, perform magnetic bead antibody conjugation. Begin by washing 50 microliters of magnetic beads in one milliliter of iso wash buffer.

Resuspend the beads in 100 microliters of conjugation buffer and add the appropriate amount of antibody. Then, incubate the beads for at least 10 minutes at room temperature. Wash the magnetic beads twice with conjugation buffer and resuspend the beads in 375 microliters of RIPiT dilution buffer.

Store the beads on ice until immunoprecipitation. Apply the FLAG affinity elution to the antibody-coupled magnetic beads and incubate with gentle mixing for one to two hours at four degrees Celsius. Capture the magnetic beads with a magnet and collect 15 microliters of supernatant for western blot analysis.

Then, wash the beads seven times with iso wash buffer. Proceed with denaturing elution by resuspending magnetic beads in 100 microliters of clear sample buffer and incubating the suspension on ice for 10 minutes. During incubation, periodically flick the beads to resuspend them.

Capture the magnetic beads on a magnet and collect 15 microliters of elution for western blot analysis. Transfer the remaining elution to a new 1.5 milliliter tube. To extract the RNA, add 320 microliters of RNase-free water and 400 microliters of phenol-chloroform isoamyl alcohol to the RIPiT elution.

Vortex the mixture and then centrifuge it at 12, 000 g for five minutes. Collect 350 microliters of the aqueous phase into a separate tube. Add sodium acetate, magnesium chloride, glycogen, and ethanol to the collected sample and incubate the mixture overnight at negative 20 degrees Celsius.

On the next day, pellet the RNA by centrifugation at 12, 000 g for 30 minutes at four degrees Celsius and wash it in 70%ethanol. After the ethanol wash, treat the RNA with T4 polynucleotide kinase, or PNK, without any ATP, and perform phenol-chloroform purification according to manuscript directions. Resuspend the clean RNA in 4.5 microliters of RNase-free water.

To quantify RNA yield, radiolabel the RNA with PNK and 32P labeled ATPs according to manuscript directions. Use a low range DNA ladder and a 20 to 40 nucleotide synthetic oligo for size and quantity standards. Resolve the labeled RNA on a 26%urea-polyacrylamide gel.

Dry the gel according to the manuscript directions and expose the gel to a phosphoscreen until adequate signal is detected. This technique has been used to investigate the interactions of the exon junction complex or EJC. Western blot analysis of proteins purified from each major step in the RIPiT procedure show that purified EJC contains both EIF4A3 and MAGOH.

The negative control, HNRNPA1, is not detected in the elution. The strength of the RNA footprint signal correlates with the amount of RNA protein interaction. A stronger footprint is observed when RIPiT was performed on cells treated with puromycin, which increases EJC occupancy on RNA.

To ensure that the RIPiT has been effective, it's important to test the efficiency of the IP by western blot. It's also important to assess the amount and the quality of the RNA prior to proceeding with deep sequencing. In 2018, the Singh lab successfully used this technique to purify two compositionally distinct variations of the exon junction complex and identify RNA binding patterns.

If researchers choose to detect RNA footprints with autoradiography, all proper radiation safety procedures and protocols should be followed.