Novel RNA-Binding Proteins Isolation by the RaPID Methodology

Note: Insert a sequence consisting of 12 MS2-binding sites (MS2 loops; MS2L) into the desired genomic locus, usually between the open reading frame (ORF) and the 3' UTR. A detailed protocol for this integration is provided elsewhere²². Verify proper insertion and expression by PCR, northern analysis or RT-PCR^20,23. It is important to verify that the integration did not intervene with the synthesis of the 3'UTR. In addition, a plasmid-expressing MS2-CP fused to SBP under the expression of an inducible promoter (methionine depletion) should also be introduced into the cells²⁴. An identical strain, excluding the introduced MS12 loops, should be used as a control for the detection of non-specific signals.

1. RaPID Purification

1. Grow 500 ml of yeast cells (use 250-1,000 ml of yeast cells depending on the expression level of the gene of interest) expressing MS2-tagged mRNA²⁰ and MS2-CP-GFP-SBP fusion protein²⁴ to OD₆₀₀ 0.8 – 1.0 at 30 ^oC in the appropriate growth medium (e.g., Synthetic Dextrose (SD) selective medium).
2. Centrifuge cells at 3,000 x g for 4 min at RT and discard the supernatant.
3. Wash cells in 1x phosphate buffered saline (PBS), centrifuge as before (step 1.2), resuspend in an equal volume (step 1.1) of SD without methionine, and incubate for 45 – 60 min at 30 ^oC to induce expression of the MS2-CP-GFP-SBP fusion protein.
   Note: Longer induction time may cause aggregation of GFP.
4. Set aside 10 ml of cells and extract RNA from this sample using the simple "hot phenol" method²⁵. Caution: Phenol is highly toxic and should be handled according to its safety instructions.
   Note: This RNA is a good reference for the quality of isolated RNA (Figure 1A).
5. For 500 ml cells, add 1.35 ml 37% formaldehyde to a final concentration of 0.1% to crosslink the RNA-protein complexes. Continue incubation at 30 ^oC for 10 min. Caution: Formaldehyde is highly toxic and should be handled according to its safety instructions.
6. Add 26.5 ml of 2.5 M glycine to a final concentration of 0.125 M and incubate for 3 min to stop crosslinking. From this step on, put everything on ice to minimize RNA degradation.
7. Centrifuge cells at 3,000 x g for 4 min at 4^oC and wash with 45 ml cold 1x PBS.
8. Resuspend cells in 1 ml RaPID Lysis buffer per 100 ml of initial cell culture.
9. Split into aliquots of 500 µl in screw-capped microfuge tubes and add ~ 400 mg of chilled glass beads to each aliquot (roughly a full 0.2 ml PCR tube).
10. Lyse cells in a bead beater for 3 min. Immediately put the lysate on ice.
    Note: Cell lysis releases cellular RNases, which are the major cause of RNA degradation. RaPID Lysis buffer contains a high concentration of RNase inhibitors, such as the nonspecific RNase inhibitor heparin and specific inhibitors. Working quickly and keeping everything cold is also recommended.
11. Transfer the lysate to new tubes. Pierce a small hole in the bottom of each microfuge tube with a hot needle (0.8 mm x 40 mm) and place the microfuge tubes on top of the 15 ml tubes. Use the head of a 5 ml syringe as an adaptor between the microfuge tubes and the 15 ml tubes. Centrifuge the tubes assembly at 3,000 x g for 1 min at 4 ^oC.
12. Transfer the flow-through from the 15 ml tube to a new 1.5 ml tube and centrifuge at 10,000 x g for 10 min at 4 ^oC to clear cell debris.
13. Pool supernatants from all 1.5 ml tubes into a single 15 ml tube, and set aside 1/50 and 1/100 volume of the lysate for RNA and protein isolation, respectively.
    Note: These will serve as the "Input" samples for subsequent analysis (Figure 1).
14. Add 300 µg of avidin per 500 ml of initial yeast culture and incubate for 30 min (this can be reduced to 10 min) at 4 ^oC under constant rolling (10 rpm).
    Note: Avidin blocks biotin and biotinylated proteins from binding to the streptavidin beads.
15. Pre-wash the streptavidin beads before incubation with the cell lysate.
    1. Transfer about 300 µl of the streptavidin beads slurry to a 1.5 ml microcentrifuge tube and remove the upper supernatant. This will result in 250 µl of beads. Wash the beads twice with 1 ml of RaPID lysis buffer. Centrifuge the streptavidin beads at 660 x g for 2 min at 4^oC between each step.
    2. Block the beads by incubating for 1 hr with 0.5 ml of RaPID lysis buffer, 0.5 ml of 10 mg/ml BSA and 10 µl of 10 mg/ml yeast tRNA. Beads can be left O/N in blocking solution at 4^oC with rotation if necessary.
    3. Wash twice with 1 ml of RaPID Lysis buffer.
       Note: Magnetic beads can also be used to reduce time and background. These, however, have lower capacity than sepharose beads.
16. Add 250 µl of the pre-washed streptavidin beads to the avidin-containing lysate (from step 1.14) and incubate 1 hr at 4 °C with constant rotation (10 rpm).
    Note: This incubation time is a compromise between providing sufficient binding time and minimizing chances for RNA degradation.
17. Centrifuge at 660 x g for 2 min at 4 ^oC to clear the streptavidin beads and transfer the supernatant to a new 15 ml tube. Set aside 1/50 and 1/100 volume of the lysate for RNA and protein isolation, respectively.
    Note: These will be the "Unbound" samples.
18. Wash the beads with 1 ml of RaPID Lysis buffer, shake gently, transfer to a new 1.5-ml microcentrifuge tube, and centrifuge as in step 1.17. Repeat this step three times.
19. Wash the beads with 1 ml of RaPID Wash buffer, and this time rotate (10 rpm) for 5 min at 4 °C. Repeat this step twice.
20. Wash the beads with 1 ml of 1x PBS and centrifuge as above.
21. Wash the beads again with 120 µl of 1x PBS. Centrifuge as above, and take the entire supernatant for RNA and protein extraction as the "Wash" sample.
    Note: This last wash sample will indicate the stringency of the washes and should be of the same volume as the Elution sample. This will simplify processing and loading in subsequent assays.
22. To elute RNA and RNA-associated proteins from the column, add 120 µl of 6 mM biotin in 1x PBS and incubate for 45 min at 4 °C with constant rotation (10 rpm).
23. Centrifuge the beads as above and transfer the eluted material to a new 1.5 ml tube. Spin the eluted sample again and transfer the upper phase to a new 1.5 ml tube to ensure no carryover of beads.
24. Take samples for RNA or protein extraction.
    Note: The volume taken should depend on the following assay and expression level of the RNA or protein of interest. As a guideline, for northern analysis²⁶, take 80% of the sample, for Western blot^23,27 or RT-PCR²⁸, take 20%, and for mass spectrometry²⁹ to discover new proteins, take the entire sample.

2. RNA Extraction

1. Add an equal volume of 2x Crosslinking Reversal Buffer and incubate for 45 min at 65 °C. Continue directly for RNA extraction.
   Note: The Crosslinking Reversal Buffer contains ethylenediaminetetraacetic acid (EDTA), which significantly improves RNA extraction²⁰.
2. Use the standard phenol:chloroform method²⁶ to extract RNA from the "input" and "unbound" samples (steps 1.13 and 1.17).
   Note: These samples contain a large amount of the RNase inhibitor heparin, which can inhibit subsequent Reverse Transcriptase-(RT)utilizing assays (e.g., RT-PCR), therefore, LiCl precipitation³⁰ is advised to remove it. The "wash" and "elution" samples (steps 1.21 and 1.24) contain low amounts of RNA and are precipitated through the following steps.
3. Add an equal volume of 8M Guanidinium-HCl (GuHCl) and two volumes of 100% ethanol to the "wash" and "elution" samples. Incubate at -80 °C for at least 2 hr.
   Note: Guanidinium will efficiently denature proteins and thereby inhibit RNases and proteases in the sample.
4. Centrifuge at 20,000 x g, 4 °C for 30 min and discard the supernatant. Wash the pellet with cold 80% ethanol and centrifuge again at 20,000 x g, 4 °C for 15 min.
5. Dissolve the RNA pellet in 400 µl of RNase free water and precipitate again to remove any leftover GuHCl or other contaminants. Add 40 µl of 3 M sodium acetate, pH 5.2 and 800 µl of 100% ethanol, incubate at -20 °C for at least 1 hr.
6. Centrifuge and wash as in step 2.4 and remove all ethanol with a 10 µl tip. Resuspend the RNA pellet in 10 µl RNase free water. Take extra care as the RNA pellet is usually not visible.
   Note: Samples can be used for northern analysis as described in²³ (Figure 1).

3. Protein Preparation for Western Blot or Mass Spectrometry Analysis

1. Add 1/3 volume of 4x Laemmli Sample Buffer (LSB) to protein samples and incubate 45 min at 65 °C to reverse the crosslinking of RNA and proteins.
   Note: Samples can be stored at -20 °C.
2. Run proteins on a SDS polyacrylamide gel (SDS-PAGE)³¹. Adjust the percentage of the gel according to the size of proteins to be analyzed.
   Note: 10% acrylamide gel gives a wide range of separation and is good as a first approximation.
3. Transfer proteins to the membrane as in the standard immunoblot analysis²⁷ for control of isolation efficiency (Figure 1C). Stain with either Coomassie Blue R250 or silver stain before mass spectrometry analysis.
   Note: Silver stain is the preferred choice because it is more sensitive and allows better detection (Figure 1B). Staining can be done with either manually prepared solutions or commercial kits. The incubation time of the gel in the final dye solution should be determined experimentally. Long incubation may reveal low abundant proteins, but can cause highly expressed proteins such as MS2-CP-SBP-GFP to be over stained and may mask surrounding proteins in their vicinity. Additionally, a high background might occur. Follow the reaction carefully, and add Stop Solution at the appropriate time.
4. Cut out bands from the gel and transfer them to 1.5 ml tubes. Store samples at -20 °C, or send for protein extraction and trypsin digestion followed by LC-MS/MS analysis³².
5. As an alternative proteomic approach to exclude the gel separation step, digest eluted material from step 1.24 in solution using trypsin and subject to LC-MS/MS analysis³³.
   Note: Omitting the SDS-PAGE separation simplifies the protocol and increases yield. However, samples might contain traces of the detergent NP-40, which inhibits mass spectrometry analysis. To overcome this problem, perform the following steps:
   1. Run protein on SDS-PAGE³¹ for 10 – 15 min.
   2. Cut out the entire portion of the lane (i.e., where the proteins are located).
      Note: The protein sample might include a significant amount of the MS2-CP-SBP protein that may obscure signals of low abundance proteins.