Purification of Messenger Ribonucleoprotein Particles via a Tagged Nascent Polypeptide

The cytoplasmic fates of mRNAs are influenced by interactions between RNA-binding proteins and cis regulatory motifs. In the cytoplasm, mRNAs are present as messenger ribonucleoprotein particles, which include not only proteins that bind directly to the mRNA, but also additional proteins that are recruited via protein-protein interactions. Many labs have sought to purify such particles from cells, with limited success. We here describe a simple two-step procedure to purify actively translated mRNAs, with their associated proteins, from polysomes. We use a reporter mRNA that encodes a protein with three streptavidin binding peptides at the N-terminus. The polysomal reporter mRNA, with associated proteins, is purified via binding to a streptavidin matrix. The method takes four days, and can be applied in any cell that can be genetically manipulated. Using Trypanosoma brucei as a model system, we routinely purified 8% of the input reporter mRNA, with roughly 22-fold enrichment relative to un-tagged mRNAs, a final reporter-mRNA:total-mRNA ratio of about 1:10, and a protein purification factor of slightly over 1000-fold. Although the overall reporter mRNP composition is masked by the presence of proteins that are associated with many polysomal mRNAs, our method can be used to detect association of an RNA-binding protein that binds to specifically to a reporter mRNA.


Reagents
Reagents needed for procyclic cell culture • MEM-PROS mixture (Biochrom, cat. no • Laminar flow hoods to work with procyclic trypanosomes in the S1 cell culture and to extract RNA and perform Northern blots.

Reagent setup
Media for procyclic cells MEM-PROS mixture was mixed with MEM vitamins, MEM nonessential amino acid solution and 100mg phenol red (pH 7.4). The media was sterilized by filtration and stored at 4°C. One night before use the media was supplemented with heatinactivated FBS 10%(v/v), 7.5 mg/l hemin and 50U/ml penicillin/streptomycin. Cells Monomorphic Lister 427 procyclic trypanosomes were cultured in supplemented MEM-Pros medium at 27 o C to a density of 1 x 10 6 cells/ml. All work was done under sterile conditions in a S1 laboratory in a laminar flow hood. Zimmerman's Post Fusion Medium (ZPFM) 132mM NaCl, 8mM KCl, 8mM Na 2 HPO 4 ,1.5mM KH 2 PO 4 ,1.5mM MgAc x 4 H 2 O, 90 µM Ca(OAc) 2; pH 7.0, filter-sterilized and stored at 4°C Generation of stable cell lines in trypanosomes A total of 1x10 7 cells were used per transfection. The cells were washed once with PBS and resuspended in 0.5 ml of ZPFM. Then, approximately 8 µg of digested plasmid was mixed with the cells and transferred to a 2mm gap cuvette for electroporation. The settings used were 1.5 kV and resistance R2 in the BTX electroporator. On the next day, the selective antibiotic was added and the cells plated in serial dilution on a 24 well plate. Proliferating clones were picked later and checked for expression. Polysome buffer 20mM Hepes (pH 7.5), 25mM NaCl and 5mM MgCl 2 . (Schutz et al 1977). At the moment of the experiment add: 5 µg/ml leupeptin, 0.5 mg/ml heparin, 2mM DTT, 100 µg/ml cycloheximide, 40U/µl rRNasin, 200mM Sucrose, 1pill/10mL of Protease Inhibitors EDTA-free and 0.2% NP40 (the detergent was only added at the moment of lysis). Blocking buffer 20mM Hepes (pH 7.5), 25mM NaCl and 5mM MgCl 2 (Schutz et al 1977). At the moment of the experiment 0.1 mg/ml tRNA and 0.4 mg/ml heparin were added. Affinity Purification buffer 20mM Hepes (pH 7.5), 280mM NaCl, 5mM MgCl 2 . At the moment of the experiment add 0.4 mg/ml of heparin. Proteinase K buffer 0.2% SDS, 8mM EDTA and 0.8 µg of Proteinase K. Incubate 20 min at 42°C Sucrose gradients Gradients were prepared by adding the AP buffer supplemented with 5 µg/ml leupeptin, 0.5 mg/ml heparin and 2mM DTT to 15% and 50% sucrose mixtures. The sucrose layers were made manually, by loading first the 15% sucrose into the centrifuge tubes and later the 50% sucrose solution was loaded at the bottom of the tube, pushing the 15% solution up. The upper part of the centrifuge tubes were covered with parafilm and left in a 90° position for 2 h. Gradients were stored at -80°C.

Equipment setup
Sucrose density gradient fractionation The ultracentrifuge was pre-cooled before the centrifugation by starting the vacuum. The SW60 rotor and tubes were kept at 4°C overnight. 15-50% sucrose gradients were left to thaw overnight at 4°C in a place without external movement. ! CRITICAL STEP. Once the gradients were thaw, the clear lysate was loaded on top of the gradients at 4°C, without disturbing the sucrose layers and without making bubbles while loading. Also there must be 1-2 mm space left after loading in order to avoid the implosion of the tubes while ultracentrifugation ! CRITICAL STEP. The gradients were ultra centrifuged at 164,326 g for 2 h in the SW60 rotor. Fractionator setup The fractionator was assembled as indicated by the manufacturers' instructions (TELEDYNE ISCO. www.isco.com) and the tubing cleaned once with distilled water. Before the fractionation started, 60% sucrose was pumped into the tubes taking care that there were no bubbles left in the tubing. The needle and the equipment were adjusted properly to avoid leakage of the gradients and the UV-lamp turned on approximately 15 min in advance before its use. The polyribosome profile was recorded at 454 nm and takes approximately 12 min per gradient. For large-scale experiments, 6 gradients (a full rotor) were used. The profile can be recorded in paper or in the computer. At the end of the fractionation, the fractionator was washed twice with water and once with ethanol 70%; in order to avoid the formation of fungus or bacteria in the tubing, which can later contaminate the sample. Blocking step The streptavidin beads were blocked with 1ml of blocking buffer (see REAGENT SETUP) per 25 µl beads before the affinity purification. 25 µl is suitable for polysomes from 7 x 10 8 cells -see File S2. The beads were tumbled within the blocking buffer for 1 hour at 4°C, spun down at 400 g for 2 min and then washed twice with polysome buffer and once with affinity purification buffer (see REAGENT SETUP). Before the incubation with the polysomal fractions all buffer was removed from the beads.

Procedure
Induction of the 3SBPs-CAT mRNA TIMING Day1, 5 min 1. Add 200 ng/ml tetracycline to the culture in order to induce the expression of the reporters and leave to grow overnight. Depending on the expression of the vector the induction time can be reduced.

Collection of cells and cycloheximide treatment TIMING Day2, 30 min
2. Cells (3x10 8 procyclic trypanosomes per gradient) are collected by centrifugation at 500 g for 10 min at 4°C. (After heat shock double the number can be used because there are fewer polysomes.) From this step onwards everything must be done on ice or at 4°C if possible ! CRITICAL STEP. 3. Wash the pellet with 10 ml of media without serum per 8x10 6 cells and distributed in a Petri dish (145mm radius). 4. Cross-link once the RNA and protein complexes at 254nm (0.3 J/cm) using the UV StratalinkerTM 2400 5. Collect the cells in a 50ml falcon, add 100 µg/ml of cycloheximide and incubate for 5 min at room temperature. 6. Spin down the cells for 8 min at 500 g, resuspend the pellet in 1ml of polysome buffer without detergent and transferred to a 1.5ml LoBind eppendorf tube. 7. Spin the cells down for 3 min. In this step the cell pellets could be frozen in liquid nitrogen and store at -80°C.
Cell lysis TIMING Day2, 2 h + 20 min 8. Resuspend the cells in 200µl of polysome buffer with detergent. 9. Lyse by passing the cells 15 to 30 times through a 21G 11/2 needle (? TROUBLESHOOTING). CRITICAL STEP The lysis must be done at 4°C and can be supplemented with RNase inhibitors. Also adjust the salt concentration to 120 mM KCl. 10. Clear the lysate by centrifugation at 16,000 g for 10 min 11. Load the clear lysate carefully in the sucrose gradients and ultracentrifuge the gradients at 164,326 g for 2 h (? TROUBLESHOOTING). While the centrifuge is running clean and assemble the fractionator. Also wash the blocking buffer from the streptavidin beads (see EXPERIMENTAL SETUP).
Polyribosomal fractionation TIMING Day2, 20 min 12. Fractionate the samples by time (22 seconds each fraction) using the ISCO160 fractionator; this will give 400 µl fractions approximately. The lysate of 6-8x10 8 cells can be loaded into the sucrose gradients without saturation. Each gradient takes approximately 12 min to collect and detect (? TROUBLESHOOTING). CRITICAL STEP Make sure that the needle is assemble properly and that the gradient is tightly fixed to the fractionator in order to avoid the leakage of the sample. 13. Pool the polyribosomal fractions and take samples for Northern blot and/or Western blot (? TROUBLESHOOTING) If the fractionator is not in the cold room one can pre cool the tubes (at -20°C) and as soon as the sample is collected put it on ice.

Affinity Purification TIMING Day2, 2h
14. Add the polyribosomal fractions to the pre-blocked Streptavidin sepharose beads and tumble for 1 h. Perform all steps on ice or at 4°C ! CRITICAL STEP 15. Spin down the beads at 900 g for 3 min and leave on ice until the beads settle. Due to the high concentration of sucrose, this step can take approximately 20 min. 16. Take a sample of the unbound fraction for Northern and/or Western blot and wash the beads 3 times with affinity purification buffer. 17. Take samples of the wash for Northern and Western blot (? TROUBLESHOOTING) Preparation of samples for Mass spectrometry Day3, 30 min + overnight staining 18. Add 12.5µl of 4x sample buffer and boil the beads at 95°C for 10 min. 19. Load the samples in a pre-cast protein gels NuPAGE 4-12% Bis -Tris Gel (? TROUBLESHOOTING) and run the samples for 3 cm. 20. Stain with Coomassie brilliant blue and wash overnight.
Mass spectrometry, Day4, 5 min 21. Cut the samples as desired and send the bands to mass spectrometry (? TROUBLESHOOTING).

TIMING
Step 1 (day 1) approximately 5 min Steps 2-17 (day 2) approximately 5 h and 10 min Steps 18-20 (day 3) approximately 30 min and overnight incubation Step 21 (day 4) approximately 5 min See Figure 2 Troubleshooting Table1 Troubleshooting table   Problem Possible reasons Suggestions Cell lysis Not enough polyribosomes Incomplete cell lysis Use a 21G 11/2 needle for obtained the lysis, smaller needles will take more time for lysis and RNA degradation might occur. Check the efficiency of lysis by dilution 1:100 in the buffer (before and after the lysis) and observe/count the cells in a Newbauer chamber (load 10µl of the dilution into the chamber) Disturbed polysome profile Overloaded sucrose gradient Do not load more than 6x10 6 cells per gradient. In case of heat shock treatment 8x10 6 cells can be load Higher background Biotinylated proteins present in the lysate Block the lysate using Avidinagarose beads. Mix the lysate with 25µl of 50% Avidin slurry and tumble for 10 min at 4°C. Spin down the beads at 2400 g for 5 min and continue with the protocol in step 10 Cell fractionation Assembling the fractionator

Leakage of gradients
Fix the gradient to the fractionator using silicon paste and tightening the needle to the collector.

Disturbance of the gradients
Bubbles in the tubing Pass 60% sucrose trough the tubing before starting the fractionation Hit slightly the tubing to get rid of the bubbles while pumping 60% sucrose at full speed Leave sucrose pass through the needle in order to ensure that there are not bubbles inside the needle The 60% sucrose can be recover by pumping back and can remain at 4°C or even be frozen, if care is taken to avoid contamination. Affinity purification SBP-tag not binding to the streptavidin beads

Not enough SBPs
At least 3 SBPs must be present in order to be able to