mRNA Interactome Capture from Plant Protoplasts

1. Arabidopsis Leaf Mesophyll Protoplast Isolation

NOTE: Arabidopsis leaf mesophyll protoplasts are essentially isolated as described by Yoo et al., 2007, with several modifications⁴⁰.

1. Growth of plants
   1. Soak approximately 200 Arabidopsis thaliana Col-0 ecotype seeds in sterilized water for 2 days at 4 °C in darkness for stratification.
      NOTE: This number of seeds is enough for one non-CL sample and one CL sample (see step 1.3).
   2. Prepare pots with a 50% (v/v) mixture of soil and vermiculite and soak the pots with distilled water in a tray to wet the soil.
   3. Sow and distribute the stratified seeds on wet soil. Do not cover the seeds with additional soil because Arabidopsis seeds need light for germination.
      NOTE: Plant growth conditions are a 12 h light/12-h dark cycle at 23 °C, with a light intensity of 100 µmol m^-2 s^-1, in a growth room for 5 weeks before flowering. 4-week-old plants can also be used⁴⁰.
      NOTE: All described materials and reagents from step 1.2.1 to step 3.2.3 are for two samples (i.e., one non-CL sample (the control sample that is not subjected to UV-C irradiation) and one CL sample (the research sample that is subjected to UV-C irradiation)).
2. Preparation of isotonic enzyme solution
   1. Make 80 mL of primary isotonic enzyme solution (400 mM mannitol, 20 mM KCl, and 20 mM MES buffer (pH 5.7)) and immediately heat the solution in a 60 °C incubator for 10 min.
   2. Add 1.2 g of Cellulase R10 (w/v 1.5%) and 0.32 g of Macerozyme R10 (w/v 0.4%) powder to the warm primary isotonic enzyme solution.
   3. Carefully bring the enzyme powder into solution by applying gentle stirring. Rapidly heat the solution in a 60 °C incubator for 10 min until that the enzyme solution is clear light brown.
   4. Cool the solution on ice for 3 min and add 800 µL of 1 M CaCl₂ and 800 µL of 10% (w/v) BSA to the solution.
   5. Homogenize the solution by pipetting and filtering through a 0.22-µm filter. Aliquot this final isotonic enzyme solution into two large-scale Petri dishes (150 mm x 20 mm).
3. Preparation of Arabidopsis rosette leaf strips
   NOTE: 150 leaves for each Petri dish is recommended for one non-CL sample or one CL sample.
   1. Choose and cut a total of 300 fully expanded 2^nd– or 3^rd-pair true leaves (average: 3 – 4 per rosette) into 0.5- to 1-mm leaf strips using a new and sharp razorblade.
   2. Transfer and submerge the strips immediately into the isotonic enzyme solution.
      NOTE: To avoid any contact with light, always cover the Petri dishes with aluminum foil. Do not crush the leaves. Confirm that all strips are submerged in the solution and are not floating on the surface.
4. Isolation of Arabidopsis leaf mesophyll protoplasts
   1. Place the aluminum foil-covered Petri dishes into vacuum desiccators and infiltrate the leaf strips under vacuum for 30 min. Subsequently, incubate the Petri dishes in darkness without shaking at room temperature for 2.5 h.
   2. Gently and briefly shake the dishes horizontally by hand, attempting to release the protoplasts entirely into the enzyme solution.
5. Harvesting and counting the protoplasts
   1. Filter the protoplast suspension through a 35 to 75 µm nylon mesh. Aliquot the filtrate of each sample into two 50 mL round-bottom tubes (approximately 20 mL of filtrate in each tube).
   2. Rinse each Petri dish with 10 mL of W5 buffer (154 mM NaCl, 125 mM CaCl₂, 5 mM KCl, and 2 mM MES buffer (pH 5.7)) to transfer the rest of the protoplasts to the enzyme solution. Add this 10-mL filtrate of each sample to the tubes. Cover the tubes with aluminum foil and keep them on ice.
   3. Spin all four tubes for 5 min at 100 x g to pellet the protoplasts. Discard the supernatant and gently resuspend the protoplast pellets in each tube with 10 mL of W5 buffer.
      NOTE: When resuspending the protoplast pellets, gently reverse the tube upside down and gently spin the tube by hand until the pellets have disappeared. Do not pipette the pellet directly to avoid damaging the cells.
   4. Repeat step 1.5.3 once and combine the protoplast suspension from two tubes of each sample into one 50-mL round-bottom tube. Keep the tubes on ice.
   5. Count the protoplasts using a hemocytometer.
      NOTE: Each 20 cells within 25 cubes equals 2 x 10⁵ protoplasts/mL. 150 leaves should yield approximately 1 x 10⁷ cells. Modulate to have an equal total number of protoplasts in the non-CL and CL samples.
   6. Keep the protoplasts on ice for 30 min. Afterwards, spin the tubes for 5 min at 100 x g. Discard the supernatant and resuspend the protoplasts in each tube with 20 mL of MMg solution (400 mM Mannitol, 15 mM MgCl₂, and 4 mM MES buffer (pH 5.7)).

2. In Vivo mRNA-protein Crosslinking by UV Irradiation

NOTE: Keep the non-CL sample tube on ice. The CL sample must be immediately UV irradiated.

1. Transfer the protoplast suspension containing 1 x 10⁷ cells from the CL sample tube to a new large-scale Petri dish (150 mm x 20 mm) and add 30 mL of ice-cold MMg solution to cover the plate surface. Spread the cells in the whole MMg buffer by gentle pipetting. Immediately place the Petri dish without the top cover in a UV crosslinking apparatus.
   NOTE: The height between the UV lamp and the Petri dish is 8 cm.
2. Irradiate the sample with 254 nm wavelength UV light and 0.00875 W/cm² UV intensity for 1 min, 3 min, or 5 min. After irradiation, rapidly aliquot the protoplast suspension into two new 50 mL round-bottom tubes. Wash the bottom of the dish with an additional 5 mL of ice-cold MMg solution to collect the remainder of the protoplasts and add this cell suspension to these two tubes.
   NOTE: 1 min was chosen as the optimal condition. The explanation can be found in the Representative Results.

3. Protoplast Lysis under Denaturing Conditions

1. Preparation of protoplast pellets for lysis
   1. Spin the non-CL sample tube together with the two CL sample tubes from step 2 at 100 x g for 5 min and discard the supernatant. Place the tube of the non-CL sample protoplast pellet back on ice.
   2. Add 10 mL of ice-cold MMg solution to each CL sample tube, gently resuspend the pellets, and combine them back into one 50 mL round-bottom tube. Spin the tube at 100 x g for 5 min. After removing the supernatant, keep the CL sample tube protoplast pellet on ice.
2. Protoplast lysis and homogenizing protoplast lysate
   1. Add 9 mL of ice-cold lysis/binding buffer (500 mM LiCl, 0.5% (w/v) Lithium Dodecyl Sulphate (LiDS), 5 mM Dithiothreitol (DTT), 20 mM Tris-HCl (pH 7.5), and 1 mM EDTA (pH 8.0)) to the cell pellet of each sample. Roughly lyse the protoplasts by pipetting up and down approximately 20 times until the suspension is homogenously light green.
   2. Pass the protoplast lysate two times through a 50-mL glass syringe with a narrow needle (0.9 x 25 mm²) for homogenization. Incubate the lysate on ice for 10 min. Freeze the lysates in liquid nitrogen and store at -80 °C for up to 3 weeks.

4. mRNP Pull-down and Purification by Oligo-d(T)₂₅ Beads

NOTE: All the following described materials and reagents, from step 4.1 to step 4.4, are only for one sample (i.e., one non-CL sample or one CL sample). The protoplast lysate must be added immediately to the tubes containing beads after the bead supernatant lysis/binding buffer is discarded.

1. Preparation of oligo-d(T)₂₅ magnetic beads for oligo(dT) capture
   1. Thaw the frozen protoplast lysate at room temperature. When the lysate is fully thawed, keep the tube of lysate on ice.
   2. Aliquot 1.8 mL of oligo-d(T)₂₅ magnetic beads (5 mg/mL) into 6 new 2-mL round-bottom microcentrifuge tubes on ice. In each tube, wash the bead suspension homogenously with 600 µL of lysis/binding buffer by briefly pipetting up and down and gently rotating at 4 °C for 2 min. Keep the beads on ice.
2. Binding
   1. Place all 6 tubes containing the beads into a magnetic rack at 4 °C for 3 min, which will result in the magnetic capture of the beads and the clearing of the suspension.
      NOTE: When the tubes are placed into the magnetic rack, wait until the beads are completely captured, at least 3 min.
   2. Discard the supernatant and immediately aliquot 9 mL of protoplast lysate into these 6 tubes. Mix well by pipetting until a homogeneous brown suspension appears and incubate the beads in protoplast lysate at 4 °C for 1 h by applying gentle rotation.
      NOTE: An incubation time from 30 min to 1 h is recommended.
3. Washing
   1. Place the tubes back into the magnetic rack at 4 °C for 3 min. When all beads are captured on the side of the tubes, collect the protoplast lysate into 6 new 2-mL round-bottom microcentrifuge tubes and temporarily store them at 4 °C. DO NOT discard the protoplast lysate immediately; keep the lysate at 4 °C.
   2. Add 1.5 mL of ice-cold wash buffer 1 (500 mM LiCl, 0.1% (w/v) LiDS, 5 mM DTT, 20 mM Tris-HCl (pH 7.5), and 1 mM EDTA (pH 8.0)) to the beads in each tube. Resuspend the beads and apply gentle rotation for 1 min. Place the tubes back into the magnetic rack at 4 °C for 3 min and discard the supernatant. Repeat this wash step with wash buffer once.
   3. Repeat the same procedure of this wash step twice using 1.5 mL of ice-cold wash buffer 2 (500 mM LiCl, 5 mM DTT, 20 mM Tris-HCl (pH 7.5), and 1 mM EDTA (pH 8.0)) and once using 1.5 mL of ice-cold low-salt buffer (200 mM LiCl, 20 mM Tris-HCl (pH 7.5), and 1 mM EDTA (pH 8.0)).
      NOTE: If the mRNPs have been efficiently isolated from the protoplast lysate, there should be a "halo" visible around the bead pellet in the CL sample during the wash step with wash buffer 2 (Figure 1B).
4. Elution
   1. Add 500 µL of elution buffer (20 mM Tris-HCl (pH 7.5) and 1 mM EDTA (pH 8.0)) to the beads in each tube. Gently resuspend the beads and incubate at 50 °C for 3 min to release the poly(A)-tailed RNAs. Gently resuspend the beads once by pipetting. Place the tubes back into the magnetic rack at 4 °C for 5 min.
   2. Transfer and combine all eluents (approximately 3 mL in total) to a clean, sterile RNase-free, 15-mL conical-bottom tube on ice.
      NOTE: The quality and quantity of RNA can be immediately determined using a spectrophotometer device.
      NOTE: The RNA concentration of each sample is approximately 10 ng/µL, with an A₂₆₀/A₂₈₀ ratio of 1.9. The A₂₆₀/A₂₈₀ ratio should be between 1.6 and 2.0 because the isolated poly(A)-tailed RNAs are usually greater than 70% pure. If the RNA concentration is too low, increase the number of starting protoplasts to a maximum of 10⁹. Samples can be frozen in liquid nitrogen and kept at 80 °C for long-term storage.
   3. Repeat step 4.2 to step 4.4 for an additional two rounds and re-use the oligo-d(T)₂₅ beads to deplete the poly(A)-tailed RNAs from the protoplast lysate.
      NOTE: After three rounds of pull-down procedure, there should be a total of 9 mL of eluent for each non-CL or CL sample. Perform all work at 4 °C to avoid the degradation of RNA. Follow step 4.5 or step 4.6 to re-use or regenerate the beads.
5. Preparation of re-used oligo-d(T)₂₅ beads
   1. Wash the beads twice with 1 mL of ice-cold elution buffer and once with 1 mL of ice-cold lysis/binding buffer to adjust the salt LiCl concentration back to 500 mM.
   2. Follow step 4.1, discard the supernatant, transfer the stored protoplast lysate in each tube, and repeat the whole procedure from step 4.2 to step 4.4 for an additional two rounds.
6. Regeneration of oligo-d(T)₂₅ beads
   NOTE: Beads can be stored and used for another experiment (maximum re-use is three times).
   1. Follow step 4.4, add 1 mL of 0.1 M NaOH to the beads, and incubate by rotating at room temperature for 5 min. For each tube, wash two times with 1 mL of wash buffer 1 and three times with 1x PBS (pH 7.4) containing 0.1% Tween-20 for equilibration. Keep the beads from each tube in 300 µL of sterile RNase-free 1x PBS (pH 7.4) containing 0.1% Tween-20 at 4 °C for long-term storage.
      NOTE: Beads used for Arabidopsis CL samples in this experiment can only be used for the same plant samples the next time.

5. Proteinase K Treatment and mRNA Purification

1. Proteinase K treatment
   1. Add 8 µL of Proteinase K solution (2 µg/µL) to 1 mL of the eluent from each sample to digest the UV-crosslinked proteins. Briefly vortex.
2. mRNA purification
   1. Incubate the eluent at 37 °C for 1 h. Purify the RNA using the RNA purification kit to remove residual contaminants.
      NOTE: After purification, the RNAs are ready for the RNA quality test using the qRT-PCR assay.
      NOTE: Other kits, such as RNA mini kit and TRIzol reagent, can also be used¹⁴.

6. qRT-PCR Assay

1. Reverse transcription
   1. Achieve efficient synthesis of first-strand cDNA using the reverse transcription system, with 1 µg of RNA as the template. Dilute the sample to 5 ng/µL with nuclease-free water.
2. cDNA quantification using qRT-PCR
   1. Amplify and quantify the cDNA using the qPCR master mix and the real-time PCR cycler, with 10 ng of cDNA as the template. Use the following PCR program: 95 °C for 10 min (stage 1, one cycle) and 95 °C for 15 s and 60 °C for 1 min (stage 2, 40 cycles).
      NOTE: The reference gene used here was an endogenous internal control gene, UBQ10 (AT4G05320). qRT-PCR primers for UBQ10 and 18S rRNA were described in Li et al., 2014 and Durut et al., 2014, respectively^45,46. Primer sequences are listed in the Material Table.

7. RNase Treatment and mRBP Concentration

1. RNase treatment
   1. Add approximately 100 U of RNase cocktail containing RNase A and RNase T1 into 8 mL of eluent. Include a negative-control sample with RNase cocktail in which the eluent is replaced by nuclease-free water. Briefly vortex and incubate at 37 °C for 1 h.
2. mRBP concentration
   1. After RNase digestion, concentrate the eluent using centrifugal filter units. The end volume is 75 µL, with a total protein yield of approximately 2 µg of each sample after concentration. Place the samples at -80 °C for long-term storage.

8. SDS-PAGE and Silver Staining

1. SDS-PAGE
   1. Mix 25 µL of the concentrated eluent (CL sample) or the control sample (non-CL sample) with 15 µL of 2x loading dye. Heat the sample at 95 °C for 5 min and load it on an SDS-PAGE gel containing 5% stacking gel and 12% resolving gel, including a protein marker.
   2. Condense the proteins at 60 V for 40 min and separate at 160 V for approximately 1 h until the loading dye reaches the end of the resolving gel.
2. Silver-staining assay
   1. Wash the SDS-PAGE gel twice with ultrapure water for 5 min each time. Perform the silver-staining of the proteins using a commercial silver stain kit.
      NOTE: The protein bands must be shown within 5 min. If the bands are visible beyond 5 min with very light color, it is suggested to increase the number of starting protoplasts up to the maximum of 10⁹.

9. Trypsin Digest of Protein Bands and Peptide Purification

1. Trypsin digest
   NOTE: Run a second 1D-PAGE gel by taking another 25 µL of concentrated mRBP solution from each sample for mRBP identification; silver-stained gels cannot be used since they are not compatible with MS analysis.
   1. After running the 1D-PAGE gel, fix the gel in fixation solution (50% (v/v) methanol and 10% (v/v) acetic acid) for 1 h. Stain the gel in stain solution (0.1% (w/v) Coomassie Brilliant Blue R-250, 50% (v/v) methanol, and 10% (v/v) acetic acid) for 20 min with gentle shaking. Destain the gel in destain solution (40% (v/v) methanol and 10% (v/v) acetic acid) for 1 h. After destaining, keep the gel in 5% (v/v) acetic acid.
      NOTE: During destaining, new destain solution can be replaced until the background is completely destained.
   2. Cut the bands of interest out of the gel. Cut the bands further into pieces of approximately 1 mm³ for the subsequent optimal trypsin digestion and extraction of peptides. Hydrate the gel pieces with 50 µL of 100 mM ammonium bicarbonate (NH₄HCO₃) for 10 min. Cover the gel pieces completely.
      1. Remove the hydrating solution carefully and dehydrate the gel pieces with acetonitrile (CH₃CN) for 10 min. Carefully remove the acetonitrile solution.
      2. Repeat the process from hydration to dehydration twice. Finally, remove the acetonitrile solution.
         NOTE: The gel run time depends on the purpose of the experiment. A long run times allows a good separation of proteins, so the stained specific gel bands can be cut out for further analysis. If the purpose is to keep the proteins and remove contaminants, a short run time is sufficient.
   3. Add 500 µL of 6.6 mM DTT solution and incubate the gel pieces for 10 min. Remove the DTT solution and wash the gel pieces twice with 500 µL of 95% (v/v) acetonitrile solution.
      1. After washing, remove the acetonitrile solution and incubate the gel pieces with 500 µL of 55 mM iodoacetic acid (IAA) solution for 10 min in the darkness.
      2. After incubation, remove the solution and wash the gel pieces again with 500 µL of 95% (v/v) acetonitrile solution. Dry the gel pieces.
   4. Embed the gel pieces completely with 25 µL of digestion buffer (50 mM NH₄HCO₃, 5 mM CaCl₂, and 6 ng/µL trypsin) and keep on ice for 45 min to let the trypsin permeate into the gel. Incubate the gel pieces overnight at 37 °C for enzymatic digestion.
2. Peptide purification
   1. Add 80 µL of 50 mM NH₄HCO₃ to the gel pieces in the digestion buffer and vortex repeatedly to extract the tryptic peptides. Collect the extract. Repeat this extraction step twice with 80 µL of 50% (v/v) CH₃CN and 5% (v/v) formic acid (FA).
      NOTE: The total final extract should be approximately 240 µL.
   2. Pool and concentrate the extracts into approximately 10 µL by vacuum concentration or lyophilization and add 25 µL of 0.1% (v/v) aqueous trifluoroacetic acid (TFA) solution. Desalt the samples with µ-C18 columns, following the manufacturer's protocol.
   3. Elute the peptides with 4 – 10 µL of 60% (w/v) CH₃CN and 0.1% (v/v) FA. Lyophilize (freeze-dry) the peptides and store them at -20 °C until analysis.

10. Nano-LC-MS

1. Nano reverse-phase liquid chromatography
   NOTE: Perform the LC-MS analysis with a mass spectrometer that is online-coupled with a liquid chromatography instrument. This instrument should be equipped with a C18 column (2 µm particle, 100 Å pore size, and 50 µm x 15 cm in dimension).
   1. Resuspend the lyophilized peptides in 16 µL of solution (2% (v/v) CH₃CN and 0.1% (v/v) FA). Inject and load 5 µL of peptide solution at a flow rate of 5 µL/min on a C18 precolumn (3 µm particle size, 100 Å pore size, nanoviper, and 75 µm x 2 cm in dimension).
   2. 10.1.2. Separate the peptides using a 95-min gradient with a flow rate of 300 nL/min. During this separation gradient, increase the mobile phase B from 4% to 10%, 10% to 25%, and 25% to 45% in 5 min, 50 min, and 18 min, respectively. Finally, steeply increase the mobile phase B to 95% in 1 min. After each 95-min separation gradient, apply an inherent rinse step containing a 10-min gradient from 4% to 95% in 5 min.
      NOTE: Mobile phase A is 99.9% H₂O and 0.1% (v/v) FA, and mobile phase B is 19.92% H₂O, 80% (w/v) CH₃CN, and 0.08% (v/v) FA.
2. Mass spectrometry assay
   1. Operate the mass spectrometer in data-dependent mode with a mass range from 400 to 1,600 m/z.
   2. Select up to ten of the most intense ions in MS1 to generate the fragmentation spectra. Set the resolution to 70,000 full width at half maximum (FWHM) for MS1 and 17,000 for MS2, with an automatic gain control (AGC) target of 3,000,000 and 1,000,000 ions and a maximum ion injection time (IT) of 256 and 64 ms for MS1 and MS2, respectively.
   3. Set the dynamic exclusion to 10 s to avoid repeated fragmentation of the most abundant ions.
      NOTE: Here, ions with one or more than six charges are excluded for MS2.
3. Qualitative proteomics: peptide and protein identification
   1. Analyze fragmentation spectra using software such as Peaks studio software^47,48,49.
      NOTE: Other software packages are also available for peptide identification, such as NovoHMM³⁰ and PepNovo³⁷ for de novo sequencing and SEQUEST⁵⁴ and Mascot⁵⁵ for database searching.
   2. Combine spectra with the same mass (< 2 ppm difference) and retention times (< 2 min) and only retain spectra with a quality threshold higher than 0.65 to search the Swiss-Prot database (version: December 2013) for the taxonomy set to model plant Arabidopsis thaliana. Use the following search parameters: a precursor mass tolerance of 10 ppm through the use of the monoisotopic mass; a fragment mass tolerance of 20 mmu; trypsin as the digestion enzyme, with a maximum of 2 missed cleavages; cysteine carbamidomethylation as the fixed modification; methionine oxidation as the variable modification; and a maximum of 3 variable post-translational modifications per peptide. After identification, manually set the score thresholds for reliable peptide and protein identification so that a false discovery rate (FDR) <5% is acquired.
4. Quantitative proteomics: statistical analysis of mass spectrometry data
   1. Use LC-MS (e.g. Progenesis) for the label-free quantitation of proteomics data.
      NOTE: MS1 peptide peak areas (or intensity) are linked to the peptides identified by the studio software according to their mass, with the tolerated error at a maximum of 10 ppm.
      NOTE: This software calculates the abundance of a peptide by integrating the peak areas of all the charge states between 2⁺ and 8⁺. The quantitative data is linked to the PEAKS peptide identification by an inhouse script (mass matching with the tolerated error at maximum of 10 ppm).
   2. Calculate the average log2-fold changes (CL/non-CL) for each peptide. Group the fold-changes of the unique peptides by protein.
      NOTE: In each group, the final fold-change of a protein equals the average fold-changes of its peptides.
   3. Calculate the p-value using Student's t-test for each group to evaluate the statistical significance. Apply R software package (version 3.3.0) to correct the p-values for the FDR of all groups using the Benjamini-Hochberg (BH) method.
   4. Draw the volcano plot using the "calibrate" function package (version 1.7.2) compatible with the R software package (version 3.3.0).
      NOTE: Here, the -log10-adjusted p-values (-log10 (adj. p-value)) are a function of the log2-fold changes of all identified proteins. Proteins are treated as positive hits in the mRNA-bound proteome if their log2-fold changes are greater than 2, with or without significance.

11. Catalog of the Identified mRNA-bound Proteome

1. Classify the identified proteins from step 10 into three categories based on the items of "molecular functions and biological process" via the Gene Ontology (GO) database and "family and domain" via the InterPro database.
2. As category I, or "Ribosomal proteins," contain all detected ribosomal proteins, define proteins from category II, or "Main RBPs," as containing annotated protein domains that interact with RNAs or link to RNA binding with known or unknown functions in RNA biology.
3. As proteins with known or unknown functions in RNA biology without annotated RNA-binding domains are placed into category III, or "Candidate RBPs," define enzymes from category III based on annotations from the IntEnz database.