The overall goal of this optimized mRNA interactome capture is to isolate and identify an mRNA-bound proteome from plant cells. This method is applied to arabidopsis thaliana leaf mesophil protoplasts, a cell type used as a versatile tool in various cellular assays. This method can help to answer key questions in the field of post-transcriptional gene regulation implants such as discovering and understanding the role of plant messengers RNA binding proteins.
The main advantage of this technique is that it can isolate and identify a plant messenger on a bound proteome directly from the physiological environment. So this method can provide insight into the messenger RNA-bound protein from aribidopsis leaf mesophil protoplasts. It also can be applied to other systems like other plant's protoplasts or tissues.
We first had the idea for this method when we noticed that it had been applied successfully to yeast and mammalian cell lines. Isolate arabidopsis leaf mesophil protoplasts for two different samples, as described in the text protocol. Keep the control sample that will not be UV crosslinked, or non-CL sample on ice.
Transfer the protoplast suspension to be crosslinked, or CL-sample to a large petri dish. Add 30 milliliters of ice-cold MMG solution to the dish to cover the surface and spread the cells by gentle pipetting. Cover the petri dish with aluminum foil when transported to the UV crosslinking apparatus.
Place the opened petri dish in a UV crosslinking apparatus immediately. Make sure that the distance between the dish and the UV lamp is eight centimeters and then irradiate the sample for one minute. After that, quickly aliquot the protoplast suspension from the petri dish into two new 50 milliliter round-bottom tubes.
Wash the bottom of the dish with an additional five milliliters of the ice-cold MMG solution to collect the remainder of the protoplasts. Divide the suspension between the two tubes. Next, centrifuge the non-CL and two CL sample tubes at 100 times g at four degrees celsius for five minutes.
After discarding the supernatant, place the tube with the non-CL pellet back on ice. Add 10 milliliters of ice-cold MMG solution to each CL sample tube and gently shake the tubes to re-suspend the pellets. After combining the two samples into one 50 milliliter round-bottom tube, centrifuge at 100 times g at four degrees celsius for five minutes.
After discarding the supernatant, keep the pellet on ice. Then add nine milliliters of ice-cold lysis binding buffer to the cell pellet of each sample. Lyse the protoplasts by pipetting up and down approximately twenty times, until the solution is a homogenous light green color.
For homogenization, pass the protoplast lysate two times through a 50 milliliter glass syringe with a narrow needle, and then incubate the lysate on ice for ten minutes. Freeze the samples in liquid nitrogen and store at minus 80 degrees celsius for up to three weeks. After thawing the tube with frozen protoplast lysate at room temperature, keep the tube on ice.
Aliquot 1.8 milliliters of oligo-d(T)magnetic beads into six new two milliliter round-bottom micro centrifuge tubes placed on ice. Wash the bead suspension in each tube with 600 microliters of lysis binding buffer by briefly pipetting up and down and gently rotate the beads on a rotator for two minutes. Keep the beads on ice.
Next, place all six tubes containing the beads into a magnetic rack for at least three minutes to allow enough time for the complete magnetic capture. When the beads are completely captured, the suspension will become clear. Then, discard the supernatant from all six tubes and immediately aliquot nine milliliters of protoplast lysate into them.
Mix the beads with lysate by pipetting until the suspension appears brown and homogenous. Incubate the tubes at four degrees celsius for one hour with gentle rotation. After incubation, place the tubes back into the magnetic rack for three minutes.
When all the beads are captured on the side of the tubes, collect the supernatant containing the protoplast lysate into six new two milliliter round-bottom micro centrifuge tubes. Next, add 1.5 milliliters of ice-cold wash buffer one to each tube containing beads, and resuspend the beads by pipetting. After gentle rotation for one minute, place the tubes back into the magnetic rack for three minutes.
Discard the supernatant and repeat this wash step once. Using 1.5 milliliters of ice-cold wash buffer two, repeat the same washing procedure twice. If the mRNP's have been efficiently isolated from the protoplast lysate, there should be a halo visible around the bead pellet in the CL sample.
Finally, repeat the washing procedure once with 1.5 milliliters of ice-cold low salt buffer. After washing, add 500 microliters of elution buffer to each tube, and gently re-suspend the beads by pipetting. Incubate at 50 degrees celsius for three minutes to release the poly-A tailed RNA's.
Gently re-suspend the beads by pipetting once and place the tubes back into the magnetic rack at four degrees celsius for five minutes. Transfer and combine eluents from all six tubes into a 15 milliliter conical-bottom tube placed on ice. This will yield approximately three millimeters of the eluent.
The quality and quantity of RNA can be immediately determined using a spectrophotometer. Prepare the oligo-d(T)beads to be re-used by washing them twice with one milliliter of ice-cold elution buffer. After that, wash them with one milliliter of ice-cold lysis binding buffer to adjust the salt concentration back to 500 millimolar.
Use these washed oligo-d(T)beads to repeat the binding, washing, and elution procedures for an additional two rounds. This will deplete the poly-A tailed RNA's from the protoplast lysate and yield a total of nine millimeters of eluent for each sample. Beads can be stored and used for another experiment for a maximum of three times.
To test the RNA for quality, digest the UV-crosslinked proteins with proteinase K solution according to the text protocol. Then, purify the RNA using the RNA purification kit to remove any residual contaminates. After purification, the samples are ready for the RNA quality test using qRT-PCR.
Begin by treating eight milliliters of each sample with 100 units of RNAse cocktail containing RNAse A and RNAse T1.After brief vortexing, incubate the samples at 37 degrees celsius for one hour. To concentrate mRNA binding proteins, or mRBP's, filter the samples using centrifugal filter units. Each sample will yield 75 microliters with two micrograms of protein.
For long-term storage, freeze the samples at minus 80 degrees celsius. To run an SDS-page gel, mix 25 microliters of each concentrated sample with 15 microliters of 2X loading dye and heat the samples at 95 degrees celsius for five minutes. Load the samples in a protein marker on an SDS-page gel with a five percent stacking gel and twelve percent resolving gel.
Next, wash the gel twice with ultrapure water for five minutes per wash. Use a commercial silver staining kit to stain the gel. If the sufficient number of starting protoplasts has been used, the protein bands will show within five minutes.
To further test the mRBP's, purify the peptides from a second 1D page gel as explained in the text protocol. These purified peptides can now be analyzed by nano reverse-phased liquid chromatography, mass spectrometry, and qualitative and quantitative proteomics. After oligo-d(T)bead capture, qRT-PCR demonstrated significantly higher levels of poly ubiquitin 10 reference mRNA versus 18S rRNA.
Since oligo-d(T)beads only bind to poly-A tailed RNA, this suggests that mRNA's are enriched in the eluent. Capture of mRBP's by oligo-d(T)beads is further evaluated by a silver-stained SDS-page gel. The gel shows a significantly higher protein amount in the UV-crosslinked CL sample versus the non-crosslinked non-CL sample.
The duration of UV irradiation can influence the efficiency of crosslinking. This silver-stained SDS-page gel confirms that one or three minutes of UV irradiation is the optimal condition, with the strongest band intensities. A total of 325 proteins were identified by quantitative proteomics analysis.
225 of these proteins were below the significance level due to low abundant peptides present for each protein, with high variability of peptide intensities. However, because all of them were qualitatively detected only in CL samples, they were all considered positive results. These 325 proteins were further classified into three categories, Ribosomal proteins, main RBP's, and candidate RBP's.
The candidate RBP's lack conventional RNA binding domains and most of their roles have not been validated, suggesting possible novel functions in RNA regulation. Once mastered, this technique can be done in one week if performed appropriately. After it's development, this technique paved the way for researchers in the field of molecular regulation pathways in plants to explore genome-wide messenger RNA-bound proteome in dicots and monocots cells and tissues.
