Methylated RNA Immunoprecipitation Assay to Study m⁵C Modification in Arabidopsis

Methylated RNA immunoprecipitation, also called MeRIP, is a method designed to identify methylated groups in whole transcriptomes. It can be adapted to RNA extracts of many model organisms. The method is simple, does not require any harsh chemical treatment, and can be used to map any RNA modification as long as specific variable.

To extract the RNA sample, first grind 200 milligrams of the plant tissue sample of interest to powder in liquid nitrogen, taking care that the tissue remains frozen throughout the procedure. Next, add one milliliter of RNA extraction reagent containing guanidine thiocyanate and acid phenol to the ground plant tissue and mix well by inversion, making sure that all of the tissue is wet. After 10 minutes at room temperature, centrifuge the samples and transfer the supernatant to a new 1.5 milliliter tube.

Add 200 microliters of 1-bromo-3-chloropropane to the supernatant and vigorously vortex the sample. Separate the phases by centrifugation and transfer approximately 500 microliters of the upper aqueous phase to a new 1.5 milliliter tube. Add 500 microliters of isopropanol and 50 microliters of three molar sodium acetate to the sample and mix well by inversion.

Incubate the sample for 10 minutes at minus 20 degrees Celsius before precipitating the RNA by centrifugation. After discarding the supernatant, wash the pellet two times with 500 microliters of 80%ethanol and one time with 500 microliters of 99%ethanol. After the last wash, dry the pellet for five to 10 minutes before dissolving in 30 microliters of RNase-free water.

Measure the RNA concentration on a spectrophotometer and digest 20 micrograms of the RNA sample in a DNase water and buffer solution. After a 30-minute incubation at 37 degrees Celsius, use an appropriate volume of DNase inactivation reagent to remove the enzyme and check the quality and purity of the isolated RNA by capillary electrophoresis according to standard protocols. If the RNA integrity number of the DNase digested sample is higher than seven, add at least 12 micrograms of each RNA sample to a new tube.

Spike in 0.1 nanogram of each previously prepared in vitro transcript, one methylated and one non-methylated, per three micrograms of RNA sample and bring the final volume in each tube to 80 microliters with RNase-free water. Then sonicate the entire volume of each RNA sample in individual sonication tubes and confirm the efficiency of sonication and the concentration of the RNA samples by capillary electrophoresis. The average size of the fragmented RNA should be around 100 nucleotides.

For methylated RNA immunoprecipitation, add nine micrograms of each sonicated RNA sample and up to 60 microliters of RNase-free water into new individual low binding tubes and heat the RNA to 70 degrees Celsius in a water bath for 10 minutes. At the end of the incubation, cool the samples for 10 minutes in an ice water bath and transfer 20 microliters of each sample into a new low binding tube for minus 80 degrees Celsius storage as input. Add RNase-free water to the remaining 40 microliters of each sample to a final volume of 860 microliters and split each sample into one low binding tube for immunoprecipitation and one low binding tube as a mock sample.

Add 50 microliters of 10X MeRIP buffer and 10 microliters of RNase inhibitor to each sample. Next, add 10 microliters of anti-M5C antibody to the immunoprecipitation tubes and 10 microliters of water to the mock tubes. Seal the tubes with parafilm and incubate the samples for 12 to 14 hours at four degrees Celsius with overhead rotation.

The next morning, add 40 microliters of beads to one 15 milliliter tube per sample. Wash the beads with three five-minute washes with 800 microliters of MeRIP buffer and overhead rotation per sample per wash, collecting the beads on a magnet after each wash to allow the buffer to be discarded. After the third wash, resuspend the beads in 40 microliters of fresh MeRIP buffer per sample and add 40 microliters of resuspended beads to the immunoprecipitation and mock sample tubes for a two-hour incubation at four degrees Celsius with overhead rotation.

At the end of the incubation, place the tubes on a magnetic rack for one minute before carefully discarding the supernatant without disturbing the beads. Wash the beads five times in 700 microliters of MeRIP buffer supplemented with 0.01%Tween-20 for 10 minutes per wash at room temperature with overhead rotation. After the last wash, resuspend the beads in 200 microliters of proteinase K digestion buffer and 3.5 microliters of proteinase K for a three-hour incubation at 50 degrees Celsius with shaking at 800 revolutions per minute.

At the end of the incubation, extract the RNA with 800 microliters of RNA extraction reagent and the acid guanidinium thiocyanate phenol chloroform extraction protocol as demonstrated. After the extraction, resuspend the extracted RNA pellet in exactly 20 microliters of RNase-free water. In this figure, a representative run of a good RNA sample is shown.

In the same sample after sonication, the presence of one uniform peak shifted to the left at a size of around 100 nucleotides can be observed. The lower concentration is caused by both the loss of RNA during fragmentation and the increased volume of the sample. The quality of the immunoprecipitation and mock samples can be evaluated by quantitative reverse transcriptase PCR.

In this representative analysis, around 80%of the methylated in vitro transcript was recovered in the immunoprecipitation sample while only approximately 2%was recovered in the mock sample. For the non-methylated control, the recovery was below 1%in both the immunoprecipitation and mock samples. After deep sequencing and aligning the reads to the genome, peak calling algorithms can be applied to identify the statistically significant windows which are enriched in the immunoprecipitation samples compared to the input.

Using the sequencing data, we can identify the motive at which the RNA methylation most often occurs and this motive can serve as bait to pull down specific binding proteins.