Novel RNA-Binding Proteins Isolation by the RaPID Methodology

The overall goal of this procedure is to efficiently isolate specific RNA with proteins that are associated with the RNA in vivo. This method can help answer key questions in RNA metabolism and regulation, such as what is a distinct set of fau-bee-pees bound to a particular transcript. The main advantage of this technique is the high efficiency of isolation, which allows follow up identification of noble mRNA associated proteins.

The rapid methodology utilizes a yeast strain that co-expresses MS2-tagged MRNA and a fusion protein of an MS2 binding protein and a streptavadin binding protein. Grow 500 milliliters of yeast cells at 30 degrees Celsius in synthetic dextrose or SD selective medium to an OD600 of 0.8 to 1.0. When the cells are ready, centrifuge at 3000 times G for 4 minutes at room temperature and discard the supernatant.

Wash the cells in PBS and centrifuge again. Discard the supernatant and re-suspend the cells in an equal volume of SD without methionine. Incubate for 45 to 60 minutes at 30 degrees celcius to induce expression of the fusion protein.

After 45 to 60 minutes, set aside 10 milliliters of the cells for RNA extraction and use the remaining cells for rapid purification. To begin this procedure, add to the cells 1.35 milliliters of 37%formaldehyde, to a final concentration of 0.1%to cross-link the RNA-protein complexes. Continue incubating at 30 degrees Celsius for 10 minutes.

To stop cross-linking, add 26.5 milliliters of 2.5 molar glycine, to a final concentration of 0.125 molar, and incubate at 30 degrees Celsius for 3 minutes. From here on, keep everything on ice and work quickly to minimize RNA degradation. Centrifuge the cells at 3000 times G for 4 minutes at 4 degrees Celsius.

Discard the supernatant, add 45 milliliters of cold PBS, and centrifuge again. Remove the supernatant and re-suspend the cells in 5 milliliters of rapid lysis buffer that contains a high concentration of Rnase inhibitors. Divide the cell suspension into aliquots of 500 microliters in screw-capped microfuge tubes and add approximately 400 milligrams of chilled glass beads to each aliquot.

Lyse the cells in a bead-beater for three minutes. Immediately put the lysate on ice. Transfer the lysate to new microfuge tubes.

Cut the top off a 5 milliliter syringe and put it on an empty 15 milliliter tube to serve as an adapter for screw-cap tubes. Use a hot needle to pierce a small hole in the bottom of each tube and place them on top of the adapted 15 milliliter tubes. Centrifuge the tub assemblies at 3000 times G for one minute at 4 degrees Celsius to elute the lysate to the 15 milliliter tubes.

Transfer the flowthrough from each 15 milliliter tube to a new 1.5 milliliter tube and centrifuge at 10, 000 times G for 10 minutes at 4 degrees Celsius to clear the cell debris. Pool the supernatant from all 1.5 milliliter tubes into a single 15 milliliter tube. Set aside 1/50th and 1/100th volume of the lysate for RNA and protein isolation respectively.

Begin the rapid procedure for isolating RNA protein complexes by adding 300 micrograms of Avidin to the cell lysate. Incubate for 30 minutes at 4 degrees Celsius under constant rolling. While the cell lysate is being incubated with Avidin, pre-wash the streptavidin beads, transfer about 300 microliters of the streptavadin bead slurry to weigh 1.5 milliliters microcentriuge tube, centrifuge the preparation, and then remove the upper supernatant which will result in 250 microliters of beads.

Add 1 milliliter of rapid lysis buffer to the beads and centrifuge at 660 times G for 2 minutes at 4 degrees Celsius. Remove the supernatant and wash again with rapid lysis buffer. To block the beads, add 0.5 milliliters of rapid lysis buffer, 0.5 milliliters of BSA, and 10 microliters of yeast TRNA and incubate for 1 hour at 4 degrees Celsius with constant rotation.

Wash the beads twice with 1 milliliter of rapid lysis buffer. Add 250 microliters of the pre-washed streptavidin beads to the avidin containing lysate and incubate for 1 hour at 4 degrees Celsius with constant rotation. This incubation time is a compromise between providing sufficient binding time and minimizing the chances for RNA degradation.

After one hour, centrifuge at 660 times G for 2 minutes at 4 degrees Celsius to clear the streptavidin beads. Transfer the supernatant to a 15 milliliter tube. Set aside 1/50th and 1/100th volume of the lysate for RNA and protein isolation respectively.

Add 1 milliliter of rapid lysis buffer to the beads. Mix by pipetting, transfer to a new 1.5 milliliter microcentrifuge tube and centrifuge at 660 times G for 2 minutes at 4 degrees Celsius. Wash three more times with rapid lysis buffer.

After the final wash with rapid lysis buffer, remove the supernatant, add 1 milliliter of rapid wash buffer, and rotate for 5 minutes at 4 degrees Celsius. Centrifuge and wash 2 more times with rapid wash buffer. Wash the beads with 1 milliliter of PBS and centrifuge as before.

Remove the supernatant, add to the beads 120 microliters of PBS, and rotate for 5 minutes in a roller. Centrifuge as before and collect all the supernatant for RNA and protein extraction as the wash sample. To elute RNA and RNA-associated proteins, add 120 microliters of 6 millimolar biotin in PBS to the beads, and incubate for 45 minutes at 4 degrees Celsius with constant rotation.

Centrifuge the beads and transfer the eluded material to a new 1.5 milliliter tube. Spin the eluted sample again and transfer the upper phase to a new 1.5 milliliter tube to ensure no carry-over of beads. Take samples for RNA or protein extraction.

To determine the isolation efficiency and quality of RNA, Northern Analysis was performed for two tagged RNAs. PMP1 and FPR1, from cast cell lysis, rapid cell lysis, and after elution with biotin. Ribosomal RNAs were detected by ethidium bromide staining, and the lack of ribosomal RNAs in the elution sample indicates the stringency of purification.

The stringency and specificity of the purification are further demonstrated by the lack of a signal of an untagged MRNA in the elution samples. The apparent signal in the FPR1 lane, which is not of the size of ACT1, is a leftover from previous hybridization with the MS2L probe. Although the input RNA is somewhat more degraded compared to the RNA that was purified by the Hot Phenol protocol, a significant amount of full length tagged RNA is isolated specifically, as revealed by the strong signal in the elution fractions.

Protein samples can be analyzed by SDS-PAGE and silverstaining prior to proteomics analysis. The MS2-CP-GFP-SBP fusion protein, indicated by the arrow, demonstrates equal protein loading. The asterisks indicate bands with differential intensities that were later cut out of the gel for mass-spectrometry analysis.

While attempting this procedure, it's important to wear gloves, ensure a clean work-area, prepare solution with RNA free water, and work quickly and efficiently to reduce protocol time. Don't forget that working with reagents like phenol and formaldehyde can be extremely hazardous, and precautions such as working in a chemical hood should always be taken while performing this procedure. After watching this video, you should have a good understanding of how to efficiently and effectively purify RNA of fayn-tress and its associated body.

Following this procedure, other methods like RNA-Seq can be performed in order to detect RNAs that bind the transcript of interest.