This protocol provides a method to identify RNA interactors of an RNA binding protein PKR in a genome-wide manner. It helps us to better understand post-transcriptional regulation of gene expression by RNA binding protein. The main advantage of this technique is that it utilizes efficient crosslinking of formaldehyde to fix RNase bound to RNA-binding proteins and enrich them through immunoprecipitation.
Activated PKR is observed in patients with neurodegenerative disease, such as Parkinson's and Alzheimer's disease. Identifying PKR-interacting double-stranded RNase will help us to better understand the pathogenesis of these degenerative diseases. This method can be used to study RNA interactors of any RNA-binding proteins.
In particular, we believe that this method is useful to study proteins that recognize the structured RNase, such as double-stranded RNase. Check the homogeneity of the cycle-arrested sample before harvesting cells. In addition, it is critical to optimize the immunoprecipitation efficiency using the best antibody available.
To begin, seed 750, 000 or one million HeLa cells for S-or M-phase-arrested samples, respectively. Grow cells at 37 degrees Celsius and 5%carbon dioxide for 24 hours. Treat the cells with two-millimolar thymidine, and incubate at 37 degrees Celsius for 18 hours.
Wash the cells two times with PBS. Then, add fresh media, and incubate at 37 degrees Celsius for nine hours. For S-phase-arrested cells, treat cells with two-millimolar thymidine.
For M-phase-arrested cells, treat cells with 100 nanograms per milliliter of nocodazole. Incubate at 37 degrees Celsius for 15 hours. For an S-phase sample, collect cells with a cell scraper, and transfer into a 15-milliliter conical tube.
For an M-phase sample, tap the side of the cell culture dish to detach M-phase-arrested cells, and transfer them into a 15-milliliter conical tube. Next, centrifuge the cells at 380 g at room temperature for three minutes. Remove the supernatant, and resuspend the cells with one milliliter of cold PBS.
Transfer cells into 1.5-milliliter microcentrifuge tube. Centrifuge the cells at 10, 000 g at four degrees Celsius for 30 seconds, and remove the supernatants completely. To perform formaldehyde crosslinking, first add 750 microliters of 0.1%paraformaldehyde to the cells, and incubate at room temperature for 10 minutes to fix the cells.
Then, add 250 microliters of one-molar glycine and incubate at room temperature for an additional 10 minutes to quench the reaction. Centrifuge the cells at 10, 000 g at four degree Celsius for 30 seconds. Remove the supernatants completely.
Next, resuspend with one milliliter of PBS. Centrifuge the cells at 10, 000 g at four degrees Celsius for 30 seconds, and remove the supernatant. Resuspend with 400 microliters of fCLIP lysis buffer supplemented with 0.2 volume percent protease inhibitor and two volume percent RNase inhibitor.
Incubate on ice for 10 minutes, and sonicate using an ultrasonicator. Next, add 11 microliters of five-molar sodium chloride to adjust sodium chloride concentration to 150 millimolar, and vortex briefly. Then, centrifuge at 21, 130 g at four degree Celsius for 15 minutes.
Transfer the supernatants to a pre-chilled 1.5 milliliter microcentrifuge tube. To perform immunoprecipitation, first add 20 microliters of protein A beads into a 1.5-milliliter microcentrifuge tube. Then, wash the beads with 400 microliters of fCLIP lysis buffer.
Centrifuge the beads at 1, 000 g at four degrees Celsius for one minute. Next, remove the supernatant, add 400 microliters of fCLIP lysis buffer carefully, and gently resuspend the beads by inverting three to four times. Repeat the wash step three times.
After the final wash, remove the supernatants completely. Resuspend the beads in 300 microliters of fCLIP lysis buffer. Add 8.3 microliters of five-molar sodium chloride to adjust sodium chloride concentration to 150 millimolar.
Add 10 microliters of protein kinase RNA-activated antibody, and incubate on a rotator at four degrees Celsius for three hours. Next, centrifuge the beads at 1, 000 g at four degrees Celsius for one minute. Remove the supernatant, add 400 microliters of fCLIP wash buffer, and gently resuspend the beads by inverting three to four times.
Repeat the wash step two times. After the final wash, remove the supernatants completely. Next, add 300 microliters of lysate, and incubate at four degrees Celsius on a rotator for three hours.
Then, centrifuge the beads at 1, 000 g at four degrees Celsius for one minute. Remove the supernatant, add 400 microliters of fCLIP wash buffer, and gently resuspend the beads by inverting three to four times. Repeat the wash step three times.
After the final wash, remove the supernatant completely. Next, add 300 microliters of fCLIP elution buffer. Incubate in a ThermoMixer at 25 degrees Celsius for three hours to elute protein kinase RNA-activated from the beads.
Centrifuge at 1, 000 g at room temperature, and transfer the supernatants to a clean siliconized tube. Finally, add 300 microliters of Proteinase K and incubate overnight at 65 degrees Celsius. The efficiency of the cell cycle arrest of the HeLa cells at the S or M phase were examined using FACS.
D7F7 antibody showed a superior ability to immuno precipitate protein kinase RNA activated. Western blot analysis of PKR immuno precipitated or total HeLa lysate showed only one strong band corresponding to the size of PKR, indicating that the D7F7 antibody is highly specific in recognizing PKR. Using different PKR antibodies with different immuno precipitation efficiency, resulted in a different class distribution of map sequencing reads.
Decrease in radioisotope signal for PKR co-immunoprecipitated RNase was observed upon successful rRNA removal. PKR co-immunoprecipitated RNA sample, showed strong enrichment of mitochondrial RNase compared to the negative controls. Strand specific reverse transcription was used to distinguish the heavy and light strand mitochondrial RNase that were co-immunoprecipitated with PKR for S or M phase arrested cells.
The most critical step in preparing cell cycle arrest sample are where you must washing the cells two times with PBS and where you must collect only cells pastel for homogeneity or would be impaired sample. For ethically process, the most critical steps are the formaldehyde crosslinking and quantity. After this procedure the eluted RNAs can be analyzed using northern blotting, qRT PCR or high throughput sequencing.
These experiments can identify the type and relative amount of RNase bound to PKR. This technique can be applied to study and identify RNA interactors of RNA binding proteins which will enhance our understanding of post transcriptional regulation of gene expression, mediated by RNA binding proteins. Formaldehyde is hazardous so the fixation, solution and the initial wash buffer need to be handled with caution.
