This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Korean government Ministry of Science and ICT (NRF-2016R1C1B2009886).

1. Solution and cell preparation
<ol>
	<li>Solution preparation
	<ol>
		<li>For the cell culture medium, prepare medium for HeLa cell culture by adding 50 mL of fetal bovine serum (FBS) to 500 mL of Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM). 
		NOTE: Antibiotics can be added to the cell culture medium, but we do not use antibiotics.</li>
		<li>For the 0.1% paraformaldehyde, dissolve 4% (w/v) paraformaldehyde in 1x Phosphate-Buffered Saline (PBS) with heating on a hot plate and dilute to make 30 mL of 0.1% (v/v) paraformaldehyde by adding 1x PBS. 
		CAUTION: Perform all steps in a fume hood and be careful not to boil the paraformaldehyde solution. Protect the 0.1% solution from light and store at 4 &#176;C. Use the solution within one month. 
		NOTE: The pH of the final 0.1% (v/v) paraformaldehyde solution should be around 7.</li>
		<li>Prepare fCLIP lysis buffer: 20 mM Tris-HCl (pH 7.5), 15 mM NaCl, 10 mM EDTA, 0.5% (v/v) nonidet-p40 (NP-40), 0.1% (v/v) Triton X-100, 0.1% (v/v) sodium dodecyl sulfate (SDS), and 0.1% (w/v) sodium deoxycholate. Add triple distilled water (TDW) to 40 mL. Store at 4 &#176;C.</li>
		<li>Prepare fCLIP wash buffer: 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10 mM EDTA, 0.1% (v/v) NP-40, 0.1% (v/v) Triton X-100, 0.1% (v/v) SDS, and 0.1% (w/v) sodium deoxycholate. Add TDW to 40 mL. Store at 4 &#176;C.</li>
		<li>Prepare 4x PK buffer: 400 mM Tris-HCl (pH 7.5), 200 mM NaCl, 40 mM EDTA, and 4% (v/v) SDS. Add TDW to 40 mL. Store at room temperature.</li>
		<li>Prepare fCLIP elution buffer: 20 mL of 4x PK buffer, 21 g of urea, and 8.5 mL of TDW. Prepare fresh.</li>
		<li>Prepare RNA elution buffer: 0.3 M NaOAc and 2% (w/v) SDS. Store at room temperature.</li>
		<li>Prepare 2x RNA loading dye: 0.025% (w/v) bromophenol blue, 0.025% (w/v) xylene cyanol, 5 mM EDTA, 0.05% (w/v) SDS, and 95% (v/v) formamide. Store at -20 &#176;C.</li>
		<li>Prepare 1x TBE buffer: 0.089 M Tris-Borate and 0.002 M EDTA. Prepare 500 mL of TBE buffer.</li>
	</ol>
	</li>
	<li>Preparation of S or M phase-arrested cells
	<ol>
		<li>Seed ~750,000 or ~1,000,000 HeLa cells for S or M phase-arrested samples, respectively. Grow cells at 37 &#176;C and 5% CO2 for 24 h.</li>
		<li>Treat the cells with 2 mM thymidine and incubate for 18 h at 37 &#176;C.</li>
		<li>Wash the cells two times with PBS. Add fresh media and incubate for 9 h at 37 &#176;C.</li>
		<li>For S phase-arrested cells, treat cells with 2 mM thymidine. For M phase-arrested cells, treat cells with 100 ng/mL nocodazole. Incubate for 15 h at 37 &#176;C and harvest cells. 
		NOTE: The homogeneity of the cell cycle samples can be checked using FACS.</li>
	</ol>
	</li>
</ol>
2. Formaldehyde cross-linking and immunoprecipitation
<ol>
	<li>Cell harvest
	<ol>
		<li>For an S phase sample, collect cells with a cell scraper and transfer into a 15 mL 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 mL conical tube. 
		NOTE: To increase the homogeneity of the M phase-arrested cells, do not use a cell scraper.</li>
		<li>Centrifuge the cells at 380 x g at room temperature for 3 min. Remove the supernatant and re-suspend with 1 mL of cold PBS and transfer into a 1.5 mL microcentrifuge tube.</li>
		<li>Centrifuge the cells at 10,000 x g at 4 &#176;C for 30 s. Remove the supernatant completely.</li>
	</ol>
	</li>
	<li>Formaldehyde crosslinking
	<ol>
		<li>Add 750 &#956;L of 0.1% paraformaldehyde for 10 min at room temperature to fix the cells.</li>
		<li>Add 250 &#956;L of 1 M glycine and incubate an additional 10 min at room temperature to quench the reaction. Centrifuge the cells at 10,000 x g at 4 &#176;C for 30 s. Remove the supernatant completely.</li>
		<li>Re-suspend with 1 mL of PBS. Centrifuge the cells at 10,000 x g at 4 &#176;C for 30 s and remove the supernatant.</li>
		<li>Re-suspend with 400 &#956;L of fCLIP lysis buffer supplemented with 0.2% (v/v) protease inhibitor and 2% (v/v) RNase inhibitor. Incubate on ice for 10 min and sonicate using an ultrasonicator.</li>
		<li>Add 11 &#956;L of 5 M NaCl to adjust NaCl concentration to ~150 mM. Vortex briefly and centrifuge at 21,130 x g at 4 &#176;C for 15 min. Transfer the supernatant to a pre-chilled 1.5 mL microcentrifuge tube. 
		NOTE: Retain 40 &#956;L of supernatant in a new centrifuge tube as the input sample. Store the input sample at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Immunoprecipitation
	<ol>
		<li>Add 20 &#956;L of Protein A beads into a 1.5 mL microcentrifuge tube.</li>
		<li>Wash the beads with 400 &#956;L of fCLIP lysis buffer. Centrifuge the beads at 1,000 x g at 4 &#176;C for 1 min. Remove the supernatant and add 400 &#956;L of fCLIP lysis buffer carefully. Gently re-suspend the beads by inverting 3~4 times.</li>
		<li>Repeat the wash step three times. After the final wash, remove the supernatant completely.</li>
		<li>Re-suspend the beads in 300 &#956;L of fCLIP lysis buffer. Add 8.3 &#956;L of 5 M NaCl to adjust NaCl concentration to ~150 mM. Add 10 &#956;L of PKR antibody and incubate for 3 h on a rotator at 4 &#176;C.</li>
		<li>Centrifuge the beads at 1,000 x g at 4 &#176;C for 1 min. Remove the supernatant and add 400 &#956;L of fCLIP wash buffer. Gently re-suspend the beads by inverting 3~4 times.</li>
		<li>Repeat the wash step two times. After the final wash, remove the supernatant completely. 
		NOTE: Never use a vortex mixer. Invert the tube gently with hands.</li>
		<li>Add 300 &#956;L of lysate and incubate for 3 h at 4 &#176;C on a rotator. 
		NOTE: Longer incubation time may result in increased background binding.</li>
		<li>Centrifuge the beads at 1,000 x g at 4 &#176;C for 1 min. Remove the supernatant and add 400 &#956;L of fCLIP wash buffer. Gently re-suspend the beads by inverting 3~4 times.</li>
		<li>Repeat the wash step three times. After the final wash, remove the supernatant completely.</li>
		<li>Add 300 &#956;L of fCLIP elution buffer. Incubate in a thermomixer for 3 h at 25 &#176;C to elute PKR from the beads. 
		NOTE: Prepare the fCLIP elution buffer fresh.</li>
		<li>Centrifuge at 1,000 x g at room temperature and transfer the supernatant to a clean siliconized tube. 
		NOTE: A microcentrifuge tube is also ok, but siliconized tube is preferred to prevent evaporation during the de-crosslinking step (step 2.3.12).</li>
		<li>Add 300 &#956;L of proteinase K (20 mg/mL) and incubate overnight at 65 &#176;C. 
		NOTE: Use a thermomixer with heated cover to avoid evaporation.</li>
	</ol>
	</li>
	<li>RNA extraction
	<ol>
		<li>Add 580 &#956;L of acid-phenol chloroform and vortex for 30 s. Incubate at 37 &#176;C for 1 h.</li>
		<li>Vortex for 30 s and centrifuge at 12,000 x g at room temperature for 10 min.</li>
		<li>Transfer the top layer into a clean 1.5 mL microcentrifuge tube. Add 1/10 volume of 3 M NaOAc, 0.5 &#956;L of coprecipitant (e.g., Glycoblue), and an equal volume of isopropanol. Incubate overnight at -20 &#176;C.</li>
		<li>Precipitate pellets by centrifuging at maximum speed at 4 &#176;C for 1 h. 
		NOTE: If RNA/DNA pellets were not observed, add an additional 0.5 &#956;L of coprecipitant and centrifuge for an additional 1 h. Continue this step until RNA/DNA pellets are observed.</li>
		<li>Remove the supernatant and add 1 mL of 75% ethyl alcohol. Centrifuge at 12,000 x g at 4 &#176;C for 5 min. Repeat the wash step one more time. Remove the supernatant completely and dry the pellet at room temperature.</li>
		<li>Add 42 &#956;L of TDW, 5 &#956;L of DNase I buffer, 1 &#956;L of RNase Inhibitor, and 2 &#956;L of DNase I. Incubate at 37 &#176;C for 1 h.</li>
		<li>Add 150 &#956;L of TDW and 200 &#956;L of acid-phenol chloroform. Vortex for 30 s. Centrifuge at 12,000 x g at room temperature for 10 min.</li>
		<li>Transfer the top layer into a clean 1.5 mL microcentrifuge tube. Add 20 &#956;L of 3 M NaOAc, 0.5 &#956;L of coprecipitant, and 1 mL of 100% ethyl alcohol. Incubate overnight at -80 &#176;C.</li>
		<li>Precipitate pellets and wash with 75% ethyl alcohol as described in steps 2.4.4 to 2.4.5.</li>
		<li>Re-suspend in the appropriate amount of TDW.</li>
	</ol>
	</li>
</ol>
3. Sequencing Library Preparation
<ol>
	<li>rRNA removal
	<ol>
		<li>Re-suspend the RNA pellets from step 2.4.10 with 28 &#956;L of TDW. 
		NOTE: The maximum amount of total RNA should be less than 5 &#956;g.</li>
		<li>Follow the procedures provided by the rRNA removal kit&#8217;s reference guide to remove rRNA.</li>
		<li>Clean up rRNA depleted RNAs by adding 160 &#956;L of magnetic beads.
		<ol>
			<li>Mix by pipetting ~15 times and incubate at room temperature for 15 min.</li>
			<li>Attach the tube on a magnetic bar and remove the supernatant.</li>
			<li>Add 300 &#956;L of 80% ethyl alcohol while the beads are still attached on the magnetic bar and incubate for 30 s.</li>
			<li>Replace ethyl alcohol with a fresh 300 &#956;L solution. Air dry the beads on the magnetic bar for 12 min at room temperature.</li>
			<li>Re-suspend the beads in 12 &#956;L of TDW and mix by pipetting 15 times.</li>
			<li>Attach the beads on the magnetic bar and move the supernatant to a clean PCR tube.</li>
		</ol>
		</li>
		<li>Deplete the fragmented ribosomal RNAs (rRNAs) following the procedures provided by rRNA Depletion Kit.</li>
		<li>Clean up the RNAs by adding 110 &#956;L of magnetic beads and repeating steps 3.1.3.1 to 3.1.3.6.</li>
	</ol>
	</li>
	<li>RNA labeling and adaptor ligation
	<ol>
		<li>On rRNA-depleted RNAs, add 2 &#956;L of 10x CIAP buffer, 1 &#956;L of RNase inhibitor, and 2 &#956;L of antarctic alkaline phosphatase. Incubate at 37 &#176;C for 1 h and then at 65 &#176;C for 5 min to inactivate the phosphatase.</li>
		<li>Add 3 &#956;L of 10x PNK buffer, 1.5 &#956;L of RNase inhibitor, 1.5 &#956;L of T4 PNK enzyme, 0.8 &#956;L of r-ATP, and 1.7 &#956;L of TDW. Incubate at 37 &#176;C for 50 min.</li>
		<li>Add 1.5 &#956;L of 10 mM ATP and incubate at 37 &#176;C for additional 40 min.</li>
		<li>Stop the reaction by adding 170 &#956;L of the RNA elution buffer.</li>
		<li>Add 200 &#956;L of acid-phenol chloroform and vortex for 30 s. Centrifuge at 12,000 x g at room temperature for 10 min.</li>
		<li>Transfer the top layer into a clean 1.5 mL microcentrifuge tube. Add 20 &#956;L of 3 M NaOAc, 0.5 &#956;L of coprecipitant, and 1 mL of 100% ethyl alcohol. Incubate overnight at -80 &#176;C.</li>
		<li>Precipitate RNA pellets and wash with 75% ethyl alcohol as described in steps 2.4.4 to 2.4.5. Re-suspend in 4.5 &#956;L of TDW and add 9 &#956;L of 2x RNA loading dye.</li>
		<li>Heat the sample at 95 &#176;C for 5 min and load on a 10% Urea-PAGE gel.</li>
		<li>Run the gel at 370 V for 40 min. 
		NOTE: Pre-run the gel at 370 V for 90 min before loading the sample.</li>
		<li>Cut the gel at the ~100 &#8211; 500 nucleotide region and break the gel.</li>
		<li>Add 700 &#956;L of 0.3 M NaCl and incubate overnight at 4 &#176;C on a rotator.</li>
		<li>Transfer the solution to a column and centrifuge at maximum speed at room temperature for 5 min.</li>
		<li>Transfer the eluate into a clean 1.5 mL microcentrifuge tube. Add 1/10 volume of 3 M NaOAc, 0.5 &#956;L of coprecipitant, and an equal volume of isopropanol. Incubate overnight at -20 &#176;C.</li>
	</ol>
	</li>
	<li>3&#39; adaptor ligation
	<ol>
		<li>Precipitate RNA pellets and wash with 75% ethyl alcohol as described in steps 2.4.4 to 2.4.5.</li>
		<li>Re-suspend the RNA pellets in 6.5 &#956;L of TDW and add 1 &#956;L of 10 &#956;M 3&#39; adaptor.</li>
		<li>Transfer the solution to a PCR tube and incubate at 70 &#176;C for 2 min.</li>
		<li>Add 1 &#956;L of 10x ligation buffer, 0.5 &#956;L of RNase inhibitor, and 1 &#956;L of T4 RNA ligase 2. Incubate at 28 &#176;C for 1 h, 25 &#176;C for 6 h, and 22 &#176;C for 6 h.</li>
		<li>Add 12 &#956;L of 2x RNA loading dye and heat at 95 &#176;C for 5 min.</li>
		<li>Gel purify the 3&#39; adaptor ligated RNAs as described in 3.2.9 to 3.2.13.</li>
	</ol>
	</li>
	<li>5&#39; adaptor ligation
	<ol>
		<li>Precipitate RNA pellets and wash with 75% ethyl alcohol as described in steps 2.4.4 to 2.4.5.</li>
		<li>Re-suspend the RNA pellets in 4.2 &#956;L of TDW and add 1 &#956;L of 5 &#956;M 5&#39; adaptor.</li>
		<li>Transfer the solution to a PCR tube and incubate at 70 &#176;C for 2 min.</li>
		<li>Add 0.8 &#956;L of 10x ligase buffer, 0.4 &#956;L of RNase inhibitor, 0.8 &#956;L of 10 mM ATP, 0.8 &#956;L of T4 RNA ligase 1. Incubate at 28 &#176;C for 1 h, 25 &#176;C for 6 h, and 22 &#176;C for 6 h.</li>
	</ol>
	</li>
	<li>Reverse transcription and PCR amplification
	<ol>
		<li>On 5&#8217; adaptor ligated RNAs, add 1 &#956;L of 4 &#956;M reverse transcription primer. Incubate at 70 &#176;C for 2 min and immediately cool to 4 &#176;C.</li>
		<li>Add 4 &#956;L of 5x FS buffer, 4 &#956;L of 2.5 mM dNTP, 1 &#956;L of 0.1 M DTT, 1 &#956;L of RNase inhibitor, and 1 &#956;L of reverse transcriptase. Incubate at 50 &#176;C for 1 h and 70 &#176;C for 15 min.</li>
		<li>Prepare PCR amplification
		<ol>
			<li>Mix 1 &#956;L of RT product, 1 &#956;L of 25 &#956;M RPI primer, 1 &#956;L of 25 &#956;M RP1 primer, 10 &#956;L of 5x HF buffer, 4 &#956;L of 2.5 mM dNTP, 32.5 &#956;L of TDW, and 0.5 &#956;L of high-fidelity polymerase.</li>
			<li>Run PCR program for 11~13 cycles. 
			NOTE: The program for the PCR is: 98 &#176;C for 30 s for a hot start, 98 &#176;C for 10 s, 60 &#176;C for 30 s and 72 &#176;C for 45 s for amplification, and 72 &#176;C for 5 min. The amplification step is repeated for 11-13 cycles.</li>
		</ol>
		</li>
		<li>Clean up the PCR products using 40 &#956;L of the magnetic beads and following steps 3.1.3.1 to 3.1.3.6 but using 10 &#956;L of TDW to elute the DNA.</li>
		<li>Add 2 &#956;L of 10x DNA loading dye. Load the sample on a 6% acrylamide gel and run at 200 V for 30 min.</li>
		<li>Stain with Sybr gold (0.01% (v/v) in 1x TBE buffer) for 5 min at room temperature.</li>
		<li>De-stain in 1x TBE buffer for 5 min at room temperature.</li>
		<li>Cut the gel at the ~200 &#8211; 700 nucleotide region and break the gel.</li>
		<li>Add 700 &#956;L of 0.3 M NaCl and incubate overnight at room temperature to elute the DNA.</li>
		<li>Load everything onto a column and centrifuge at maximum speed at room temperature for 5 min.</li>
		<li>Transfer the eluate into a clean 1.5 mL microcentrifuge tube. Add 1/10 volume of 3 M NaOAc, 0.5 &#956;L of coprecipitant, and an equal volume of isopropanol. Incubate overnight at -20 &#176;C.</li>
		<li>Precipitate DNA pellets and wash with 75% ethyl alcohol as described in steps 2.4.4 to 2.4.5. Re-suspend in 20 &#956;L of TDW.</li>
		<li>Analyze the sample using a sequencer.</li>
	</ol>
	</li>
</ol>
4. Analysis of PKR-interacting RNAs using qRT-PCR
<ol>
	<li>Method 1: Random hexamer reverse transcription
	<ol>
		<li>Re-suspend RNA pellets from step 2.4.10 with 8.5 &#956;L of TDW and transfer the solution to a PCR tube.</li>
		<li>Add 0.5 &#956;L of 100 &#956;M random hexamers and 4 &#956;L of 2.5 mM dNTP mix.</li>
		<li>Heat the solution at 65 &#176;C for 5 min and immediately incubate on ice for at least 1 min.</li>
		<li>Make the reaction mix: 4 &#956;L of 5x SSIV buffer, 1 &#956;L of 100 mM DTT, 1 &#956;L of RNase Inhibitor, and 1 &#956;L of reverse transcriptase (200 U/&#956;L). Add the reaction mix to the RNA solution.</li>
		<li>Incubate the mixture at 23 &#176;C for 10 min, 50 &#176;C for 10 min, and 80 &#176;C for 10 min.</li>
		<li>Analyze the cDNA using a real-time PCR system. 
		NOTE: The program for the real-time PCR is: 95 &#176;C for 3 min for hot start, 95 &#176;C for 5 s and 60 &#176;C for 10 s for amplification, and 95 &#176;C for 30 s, 65 &#176;C for 30 s, and 95 &#176;C for 30 s for melt. The amplification step is repeated for 40 cycles.</li>
	</ol>
	</li>
	<li>Method 2: Strand-specific reverse transcription
	<ol>
		<li>Prepare a master mix of reverse primers: Mix equal amounts of gene specific reverse transcription primers containing CMV promoter sequence followed by ~20 nucleotides of gene specific sequences. 
		NOTE: The combined concentration of all gene specific RT primers is 4 &#956;M.</li>
		<li>Re-suspend RNA pellets from step 2.4.10 with 8.5 &#956;L of TDW and transfer the solution to a PCR tube.</li>
		<li>Add 0.5 &#956;L of 4 &#956;M master mix of reverse transcription primers and 4 &#956;L of 2.5 mM dNTP mix.</li>
		<li>Heat the solution at 65 &#176;C for 5 min and immediately incubate on ice for at least 1 min.</li>
		<li>Prepare the reaction solution by mixing 4 &#956;L of 5x SSIV buffer, 1 &#956;L of 100 mM DTT, 1 &#956;L of RNase Inhibitor, and 1 &#956;L of reverse transcriptase (200 U/&#956;L). Add the reaction solution to the RNA-primer mix.</li>
		<li>Incubate the solution at 50 &#176;C for 10 min and 80 &#176;C for 10 min.</li>
		<li>Analyze the cDNA using the real-time PCR system. 
		NOTE: The program for the real-time PCR is same as the one described in Method 1.</li>
	</ol>
	</li>
</ol>

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<li>Onuki, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+RNA-dependent+protein+kinase+is+involved+in+tunicamycin-induced+apoptosis+and+Alzheimer%27s+disease%5BTitle%5D)+AND+%22The+EMBO+Journal%22%5BJournal%5D)">An RNA-dependent protein kinase is involved in tunicamycin-induced apoptosis and Alzheimer's disease.</a> The EMBO Journal. 23 (4), 959-968 (2004).</li>
<li>Peel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Activation+of+the+cell+stress+kinase+PKR+in+Alzheimer%27s+disease+and+human+amyloid+precursor+protein+transgenic+mice%5BTitle%5D)+AND+%22Neurobiology+of+Disease%22%5BJournal%5D)">Activation of the cell stress kinase PKR in Alzheimer's disease and human amyloid precursor protein transgenic mice.</a> Neurobiology of Disease. 14 (1), 52-62 (2003).</li>
<li>Peel, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Double-stranded+RNA-dependent+protein+kinase%2C+PKR%2C+binds+preferentially+to+Huntington%27s+disease+%28HD%29+transcripts+and+is+activated+in+HD+tissue%5BTitle%5D)+AND+%22Human+Molecular+Genetics%22%5BJournal%5D)">Double-stranded RNA-dependent protein kinase, PKR, binds preferentially to Huntington's disease (HD) transcripts and is activated in HD tissue.</a> Human Molecular Genetics. 10 (15), 1531-1538 (2001).</li>
<li>Suen, K. C., Yu, M. S., So, K. F., Chang, R. C., Hugon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Upstream+signaling+pathways+leading+to+the+activation+of+double-stranded+RNA-dependent+serine%2Fthreonine+protein+kinase+in+beta-amyloid+peptide+neurotoxicity%5BTitle%5D)+AND+%22Journal+of+biological+chemistry%22%5BJournal%5D)">Upstream signaling pathways leading to the activation of double-stranded RNA-dependent serine/threonine protein kinase in beta-amyloid peptide neurotoxicity.</a> Journal of biological chemistry. 278 (50), 49819-49827 (2003).</li>
<li>Nakamura, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+critical+role+for+PKR+complexes+with+TRBP+in+Immunometabolic+regulation+and+eIF2alpha+phosphorylation+in+obesity%5BTitle%5D)+AND+%22Cell+Reports%22%5BJournal%5D)">A critical role for PKR complexes with TRBP in Immunometabolic regulation and eIF2alpha phosphorylation in obesity.</a> Cell Reports. 11 (2), 295-307 (2015).</li>
<li>Saito, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enhancement+of+the+interferon-induced+double-stranded+RNA-dependent+protein+kinase+activity+by+Sindbis+virus+infection+and+heat-shock+stress%5BTitle%5D)+AND+%22Microbiology+and+Immunology%22%5BJournal%5D)">Enhancement of the interferon-induced double-stranded RNA-dependent protein kinase activity by Sindbis virus infection and heat-shock stress.</a> Microbiology and Immunology. 34 (10), 859-870 (1990).</li>
<li>Youssef, O. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Potential+role+for+snoRNAs+in+PKR+activation+during+metabolic+stress%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences%22%5BJournal%5D)">Potential role for snoRNAs in PKR activation during metabolic stress.</a> Proceedings of the National Academy of Sciences. 112 (16), 5023-5028 (2015).</li>
<li>Murtha-Riel, P., Davies, M. V., Choi, S. Y., Hershey, J. W., Kaufman, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expression+of+a+Phosphorylation-resistant+Eukaryotic+Initiation+Factor+2+a-Subunit+Mitigates+Heat+Shock+Inhibition+of+Protein+Synthesis%5BTitle%5D)+AND+%22The+Journal+of+Biological+Chemistry%22%5BJournal%5D)">Expression of a Phosphorylation-resistant Eukaryotic Initiation Factor 2 a-Subunit Mitigates Heat Shock Inhibition of Protein Synthesis.</a> The Journal of Biological Chemistry. 268, 12946-12951 (1993).</li>
<li>Benkirane, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oncogenic+potential+of+TAR+RNA+binding+protein+TRBP+and+its+regulatory+interaction+with+RNA-dependent+protein+kinase+PKR%5BTitle%5D)+AND+%22The+EMBO+Journal%22%5BJournal%5D)">Oncogenic potential of TAR RNA binding protein TRBP and its regulatory interaction with RNA-dependent protein kinase PKR.</a> The EMBO Journal. 16 (3), 611-624 (1997).</li>
<li>Koromilas, A., Roy, S., Barber, G., Katze, M., Sonenberg, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Malignant+transformation+by+a+mutant+of+the+IFN-inducible+dsRNA-dependent+protein+kinase%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Malignant transformation by a mutant of the IFN-inducible dsRNA-dependent protein kinase.</a> Science. 257 (5077), 1685-1689 (1992).</li>
<li>Kim, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((PKR+is+activated+by+cellular+dsRNAs+during+mitosis+and+acts+as+a+mitotic+regulator%5BTitle%5D)+AND+%22Genes+%26+Development%22%5BJournal%5D)">PKR is activated by cellular dsRNAs during mitosis and acts as a mitotic regulator.</a> Genes & Development. 28 (12), 1310-1322 (2014).</li>
<li>Kim, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Deletion+of+human+tarbp2+reveals+cellular+microRNA+targets+and+cell-cycle+function+of+TRBP%5BTitle%5D)+AND+%22Cell+Reports%22%5BJournal%5D)">Deletion of human tarbp2 reveals cellular microRNA targets and cell-cycle function of TRBP.</a> Cell Reports. 9 (3), 1061-1074 (2014).</li>
<li>Zhu, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Suppression+of+PKR+promotes+network+excitability+and+enhanced+cognition+by+interferon-gamma-mediated+disinhibition%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Suppression of PKR promotes network excitability and enhanced cognition by interferon-gamma-mediated disinhibition.</a> Cell. 147 (6), 1384-1396 (2011).</li>
<li>Dagon, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Double-stranded+RNA-dependent+protein+kinase%2C+PKR%2C+down-regulates+CDC2%2Fcyclin+B1+and+induces+apoptosis+in+non-transformed+but+not+in+v-mos+transformed+cells%5BTitle%5D)+AND+%22Oncogene%22%5BJournal%5D)">Double-stranded RNA-dependent protein kinase, PKR, down-regulates CDC2/cyclin B1 and induces apoptosis in non-transformed but not in v-mos transformed cells.</a> Oncogene. 20 (56), 8045-8056 (2001).</li>
<li>Elbarbary, R. A., Li, W., Tian, B., Maquat, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((STAU1+binding+3%27+UTR+IRAlus+complements+nuclear+retention+to+protect+cells+from+PKR-mediated+translational+shutdown%5BTitle%5D)+AND+%22Genes+%26+Development%22%5BJournal%5D)">STAU1 binding 3' UTR IRAlus complements nuclear retention to protect cells from PKR-mediated translational shutdown.</a> Genes & Development. 27 (13), 1495-1510 (2013).</li>
<li>Cho, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((LIN28A+is+a+suppressor+of+ER-associated+translation+in+embryonic+stem+cells%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">LIN28A is a suppressor of ER-associated translation in embryonic stem cells.</a> Cell. 151 (4), 765-777 (2012).</li>
<li>Licatalosi, D. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((HITS-CLIP+yields+genome-wide+insights+into+brain+alternative+RNA+processing%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">HITS-CLIP yields genome-wide insights into brain alternative RNA processing.</a> Nature. 456 (7221), 464-469 (2008).</li>
<li>Van Nostrand, E. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Robust+transcriptome-wide+discovery+of+RNA-binding+protein+binding+sites+with+enhanced+CLIP+%28eCLIP%29%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">Robust transcriptome-wide discovery of RNA-binding protein binding sites with enhanced CLIP (eCLIP).</a> Nature Methods. 13 (6), 508-514 (2016).</li>
<li>Kim, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((PKR+Senses+Nuclear+and+Mitochondrial+Signals+by+Interacting+with+Endogenous+Double-Stranded+RNAs%5BTitle%5D)+AND+%22Molecular+Cell%22%5BJournal%5D)">PKR Senses Nuclear and Mitochondrial Signals by Interacting with Endogenous Double-Stranded RNAs.</a> Molecular Cell. 71 (6), 1051-1063 (2018).</li>
<li>Kim, B., Jeong, K., Kim, V. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genome-wide+Mapping+of+DROSHA+Cleavage+Sites+on+Primary+MicroRNAs+and+Noncanonical+Substrates%5BTitle%5D)+AND+%22Molecular+Cell%22%5BJournal%5D)">Genome-wide Mapping of DROSHA Cleavage Sites on Primary MicroRNAs and Noncanonical Substrates.</a> Molecular Cell. 66 (2), 258-269 (2017).</li>
<li>Ricci, E. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Staufen1+senses+overall+transcript+secondary+structure+to+regulate+translation%5BTitle%5D)+AND+%22Nature+Structural+%26+Molecular+Biology%22%5BJournal%5D)">Staufen1 senses overall transcript secondary structure to regulate translation.</a> Nature Structural & Molecular Biology. 21 (1), 26-35 (2014).</li>
<li>Kim, B., Kim, V. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((fCLIP-seq+for+transcriptomic+footprinting+of+dsRNA-binding+proteins%3A+Lessons+from+DROSHA%5BTitle%5D)+AND+%22Methods%22%5BJournal%5D)">fCLIP-seq for transcriptomic footprinting of dsRNA-binding proteins: Lessons from DROSHA.</a> Methods. , (2018).</li>
<li>Dhir, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitochondrial+double-stranded+RNA+triggers+antiviral+signalling+in+humans%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Mitochondrial double-stranded RNA triggers antiviral signalling in humans.</a> Nature. 560 (7717), 238-242 (2018).</li>
</ol>

The authors have nothing to disclose.

The process to prepare S or M phase-arrested samples is illustrated in Figure 1. To arrest cells at the S phase, we used a thymidine double block method where we treated cells with thymidine two times with a 9 h release in between to ensure high arrest efficiency (Figure 1A). For M phase arrest, we treated cells once with thymidine followed by a 9 h release and then applied nocodazole to block cells at prometaphase (Figure 1B). One ...

Protein kinase RNA-activated (PKR), also known as eukaryotic initiation factor 2-alpha kinase 2 (EIF2AK2), is a well-characterized protein kinase that transmits information provided by RNAs. It belongs to the eukaryotic translation initiation 2 subunit alpha (eIF2&#945;) kinase family and phosphorylates eIF2&#945; at serine 51 in response to infection to suppress global translation1. In this context, PKR is activated by viral double-stranded RNAs (dsRNAs), which provide a platform for PKR dimerization and autophosphorylation2. In addition to eIF2&#945;, PKR can also phosphorylate p53, insulin receptor substrate 1, inhibitor &#954;B, and c-Jun N-terminal kinase (JNK) to regulate activity of numerous signal transduction pathways3,4,5,6.
PKR was originally identified as a kinase that phosphorylated eIF2&#945; during poliovirus infection by recognizing poliovirus&#8217; dsRNAs7,8. PKR is increasingly found to play multifaceted roles beyond immune response, and its aberrant activation or malfunction is implied in numerous human diseases. Activated/Phosphorylated PKR (pPKR) is frequently observed during apoptosis and is a common characteristic of patients with degenerative diseases, particularly neurodegenerative diseases such as Huntington&#8217;s, Parkinson&#8217;s, and Alzheimer&#8217;s disease9,10,11,12,13. In addition, PKR is activated under various stress conditions such as metabolic stress and heat shock14,15,16,17. On the other hand, inhibition of PKR results in increased cell proliferation and even malignant transformation18,19. PKR function is also important in normal brain function and during the cell cycle as the level of pPKR is elevated during the M phase20,21,22. In this context, pPKR suppresses global translation and provides cues to key mitotic signaling systems that are required for proper cell division20. Moreover, prolonged activation of PKR resulted in G2/M phase cell cycle arrest in Chinese hamster ovary cells23. Consequently, PKR phosphorylation is regulated by the negative feedback loop to ensure rapid deactivation during M/G1 transition21.
Despite the wide range of PKR function, our understanding of PKR activation is limited due to the lack of a standardized high-throughput experimental approach to capture and identify dsRNAs that can activate PKR. Previous studies have shown that PKR can interact with dsRNAs formed by two inverted Alu repeats (IRAlus)20,24, but the possibility of the existence of additional cellular dsRNAs that can activate PKR during the cell cycle or under stress conditions in human cells was unexplored. The conventional approach in identifying RNA-interactors of an RNA binding protein (RBP) uses UV light to crosslink RNA-RBP complexes25,26,27. A recent study applied this UV crosslinking approach in a mouse system and identified that small nucleolar RNAs can regulate PKR activation during metabolic stress16. By utilizing high crosslinking efficiency of formaldehyde, &#160;we presented an alternative method to identify PKR-interacting RNAs during the cell cycle in HeLa cells28. A similar approach has been applied to study other dsRBPs such as Staufen and Drosha29,30,31. We found that PKR can interact with various types of noncoding RNAs such as short interspersed nuclear element (SINE), long interspersed nuclear element (LINE), endogenous retrovirus element (ERV), and even alpha-satellite RNAs. In addition, we showed that PKR can interact with mitochondrial RNAs (mtRNAs), which form intermolecular dsRNAs through complementary interaction between the heavy-strand and the light strand RNAs28. A recent publication further supported our data that some mtRNAs exist in a duplex form and can activate dsRNA sensors such as melanoma differentiation-associated protein 5 to induce interferons32. More importantly, the expression and subcellular localization of mtRNAs are modulated during the cell cycle and by various stressors, which may be important in their ability to regulate PKR activation28.
In this article, we present a detailed protocol for a recently developed formaldehyde crosslinking and immunoprecipitation (fCLIP) method to capture and analyze PKR-interacting RNAs during the cell cycle. We demonstrate the method to prepare cell cycle arrest samples using thymidine and nocodazole. We then present the fCLIP process to isolate PKR-bound RNAs and a method to prepare high-throughput sequencing library to identify these RNAs. Furthermore, we delineate detailed procedures to analyze PKR-bound RNAs using qRT-PCR. Specifically, we present a strand-specific reverse transcription procedure to analyze the strandedness of mtRNAs. The described protocol is optimized for HeLa cells and PKR, but key steps such as the preparation of cell cycle sample, fCLIP, and strand-specific qRT-PCR analysis can be easily modified to study cellular dsRNAs or to identify RNA interactors of other dsRBPs.

<table><tbody><tr><td>0.5 M EDTA, pH 8.0</td><td>Thermo Fisher Scientific</td><td>AM9260G</td><td></td></tr><tr><td>1 M Tris, pH 7.0</td><td>Thermo Fisher Scientific</td><td>AM9855G</td><td></td></tr><tr><td>1 M Tris, pH 8.0</td><td>Thermo Fisher Scientific</td><td>AM9855G</td><td></td></tr><tr><td>1.7 mL microcentrifuge tube</td><td>Axygen</td><td>MCT-175-C</td><td></td></tr><tr><td>10% Nonidet-p40 (NP-40)</td><td>Biosolution</td><td>BN015</td><td></td></tr><tr><td>10X DNA loading buffer</td><td>TaKaRa</td><td>9157</td><td></td></tr><tr><td>15 mL conical tube</td><td>SPL</td><td>50015</td><td></td></tr><tr><td>3&#039; adaptor</td><td></td><td></td><td>5&#039;-rApp NN NNT GGA ATT CTC GGG TGC CAA GG/3ddC/-3&#039;</td></tr><tr><td>3 M Sodium Acetate pH 5.5</td><td>Thermo Fisher Scientific</td><td>AM9740</td><td></td></tr><tr><td>5&#039; adaptor</td><td></td><td></td><td>5&#039;-GUU CAG AGU UCU ACA GUC CGA CGA UCN NNN-3&#039;</td></tr><tr><td>5 M NaCl</td><td>Thermo Fisher Scientific</td><td>AM9760G</td><td></td></tr><tr><td>50 mL conical tube</td><td>SPL</td><td>50050</td><td></td></tr><tr><td>Acid-phenol chloroform, pH 4.5</td><td>Thermo Fisher Scientific</td><td>AM9722</td><td></td></tr><tr><td>Agencourt AMPure XP</td><td>Beckman Coulter</td><td>A63881</td><td>Magnetic beads DNA/RNA clean up</td></tr><tr><td>Antarctic alkaline phosphatase</td><td>New England Biolabs</td><td>M0289S</td><td></td></tr><tr><td>Anti-DGCR8</td><td></td><td></td><td>Made in house</td></tr><tr><td>Anti-PKR (D7F7)</td><td>Cell signaling technology</td><td>12297S</td><td></td></tr><tr><td>Anti-PKR (Milli)</td><td>Millipore EMD</td><td>07-151</td><td></td></tr><tr><td>ATP (100 mM)</td><td>GE Healthcare</td><td>GE27-2056-01</td><td></td></tr><tr><td>Bromophenol blue sodium salt</td><td>Sigma-aldrich</td><td>B5525</td><td></td></tr><tr><td>Calf intestinal alkaline phosphatase</td><td>TaKaRa</td><td>2250A</td><td></td></tr><tr><td>Cell scraper 25 cm 2-position</td><td>Sarstedt</td><td>83.183</td><td></td></tr><tr><td>CMV promoter sequence</td><td></td><td></td><td>5&#039;-CGCAAATGGGCGGTAGGCGTG-3&#039;</td></tr><tr><td>Dulbecco&#039;s modified eagle medium</td><td>Welgene</td><td>LM001-05</td><td></td></tr><tr><td>dNTP mixture (2.5 mM)</td><td>TaKaRa</td><td>4030</td><td></td></tr><tr><td>Ethanol, Absolute, ACS Grade</td><td>Alfa-Aesar</td><td>A9951</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Merck</td><td>M-TMS-013-BKR</td><td></td></tr><tr><td>Formamide</td><td>Merck</td><td>104008</td><td></td></tr><tr><td>Glycine</td><td>Bio-basic</td><td>GB0235</td><td></td></tr><tr><td>GlycoBlue coprecipitant (15 mg/mL)</td><td>Thermo Fisher Scientific</td><td>AM9516</td><td></td></tr><tr><td>Isopropanol</td><td>Merck</td><td>8.18766.1000</td><td></td></tr><tr><td>NEBNext rRNA Depletion Kit</td><td>New England Biolabs</td><td>E6318</td><td>rRNA Depletion Kit</td></tr><tr><td>Nocodazole</td><td>Sigma-Aldrich</td><td>M1404</td><td></td></tr><tr><td>Normal rabbit IgG</td><td>Cell signaling technology</td><td>2729S</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>6148</td><td></td></tr><tr><td>PCR forward primer (RP1)</td><td></td><td></td><td>5&#039;-AAT GAT ACG GCG ACC ACC GCG ATC TAC ACG TTC AGA GTT CTA CAG TCC GA-3&#039;</td></tr><tr><td>PCR index reverse primer (RPI)</td><td></td><td></td><td>5&#039;-CAA GCA GAA GAC GGC ATA CGA GAT NNN NNN GTG ACT GGA GTT CCT TGG CAC CCG AGA ATT CCA-3&#039;</td></tr><tr><td>PCR tubes with flat cap, 0.2 mL</td><td>Axygen</td><td>PCR-02-C</td><td></td></tr><tr><td>Phosphate bufered saline (PBS) Tablet</td><td>TaKaRa</td><td>T9181</td><td></td></tr><tr><td>Phusion high-fidelity DNA polymerase</td><td>New England Biolabs</td><td>M0530</td><td>High-fidelity polymerase</td></tr><tr><td>PlateFuge microcentrifuge with swing-out rotor</td><td>Benchmark</td><td>c2000</td><td></td></tr><tr><td>Polynucleotide kinase (PNK)</td><td>TaKaRa</td><td>2021A</td><td></td></tr><tr><td>Proteinase K, recombinant, PCR Grade</td><td>Sigma-Aldrich</td><td>3115879001</td><td></td></tr><tr><td>qPCR primer sequence: CO1 Heavy</td><td></td><td></td><td>Forward/Reverse: 5&amp;prime;-GCCATAACCCAATACCAAACG-3&amp;prime;/5&amp;prime;-CGCAAATGGGCGGTAGGCGTG-3&amp;prime;</td></tr><tr><td>qPCR primer sequence: CO1 Light</td><td></td><td></td><td>Forward/Reverse: 5&amp;prime;-TTGAGGTTGCGGTCTGTTAG-3&amp;prime;/5&amp;prime;-CGCAAATGGGCGGTAGGCGTG-3&amp;prime;</td></tr><tr><td>qPCR primer sequence: CO2 Heavy</td><td></td><td></td><td>Forward/Reverse: 5&amp;prime;-CTAGTCCTGTATGCCCTTTTCC-3&amp;prime;/5&amp;prime;-CGCAAATGGGCGGTAGGCGTG-3&amp;prime;</td></tr><tr><td>qPCR primer sequence: CO2 Light</td><td></td><td></td><td>Forward/Reverse: 5&amp;prime;-GTAAAGGATGCGTAGGGATGG-3&amp;prime;/5&amp;prime;-CGCAAATGGGCGGTAGGCGTG-3&amp;prime;</td></tr><tr><td>qPCR primer sequence: CO3 Heavy</td><td></td><td></td><td>Forward/Reverse: 5&amp;prime;-CCTTTTACCACTCCAGCCTAG-3&amp;prime;/5&amp;prime;-CGCAAATGGGCGGTAGGCGTG-3&amp;prime;</td></tr><tr><td>qPCR primer sequence: CO3 Light</td><td></td><td></td><td>Forward/Reverse: 5&amp;prime;-CTCCTGATGCGAGTAATACGG-3&amp;prime;/5&amp;prime;-CGCAAATGGGCGGTAGGCGTG-3&amp;prime;</td></tr><tr><td>qPCR primer sequence: CYTB Heavy</td><td></td><td></td><td>Forward/Reverse: 5&amp;prime;-CAATTATACCCTAGCCAACCCC-3&amp;prime;/5&amp;prime;-CGCAAATGGGCGGTAGGCGTG-3&amp;prime;</td></tr><tr><td>qPCR primer sequence: CYTB Light</td><td></td><td></td><td>Forward/Reverse: 5&amp;prime;-GGATAGTAATAGGGCAAGGACG -3&amp;prime;/5&amp;prime;-CGCAAATGGGCGGTAGGCGTG-3&amp;prime;</td></tr><tr><td>qPCR primer sequence: GAPDH</td><td></td><td></td><td>Forward/Reverse: 5&amp;prime;-CAACGACCACTTTGTCAAGC-3&amp;prime;/5&amp;prime;-CGCAAATGGGCGGTAGGCGTG-3&amp;prime;</td></tr><tr><td>qPCR primer sequence: ND1 Heavy</td><td></td><td></td><td>Forward/Reverse: 5&amp;prime;-TCAAACTCAAACTACGCCCTG-3&amp;prime;/5&amp;prime;-CGCAAATGGGCGGTAGGCGTG-3&amp;prime;</td></tr><tr><td>qPCR primer sequence: ND1 Light</td><td></td><td></td><td>Forward/Reverse: 5&amp;prime;-GTTGTGATAAGGGTGGAGAGG-3&amp;prime;/5&amp;prime;-CGCAAATGGGCGGTAGGCGTG-3&amp;prime;</td></tr><tr><td>qPCR primer sequence: ND4 Heavy</td><td></td><td></td><td>Forward/Reverse: 5&amp;prime;-CTCACACTCATTCTCAACCCC-3&amp;prime;/5&amp;prime;-CGCAAATGGGCGGTAGGCGTG-3&amp;prime;</td></tr><tr><td>qPCR primer sequence: ND4 Light</td><td></td><td></td><td>Forward/Reverse: 5&amp;prime;-TGTTTGTCGTAGGCAGATGG-3&amp;prime;/5&amp;prime;-CGCAAATGGGCGGTAGGCGTG-3&amp;prime;</td></tr><tr><td>qPCR primer sequence: ND5 Heavy</td><td></td><td></td><td>Forward/Reverse: 5&amp;prime;-CTAGGCCTTCTTACGAGCC-3&amp;prime;/5&amp;prime;-CGCAAATGGGCGGTAGGCGTG-3&amp;prime;</td></tr><tr><td>qPCR primer sequence: ND5 Light</td><td></td><td></td><td>Forward/Reverse: 5&amp;prime;-TAGGGAGAGCTGGGTTGTTT-3&amp;prime;/5&amp;prime;-CGCAAATGGGCGGTAGGCGTG-3&amp;prime;</td></tr><tr><td>qPCR primer sequence: ND6 Heavy</td><td></td><td></td><td>Forward/Reverse: 5&amp;prime;-TCATACTCTTTCACCCACAGC-3&amp;prime;/5&amp;prime;-CGCAAATGGGCGGTAGGCGTG-3&amp;prime;</td></tr><tr><td>qPCR primer sequence: ND6 Light</td><td></td><td></td><td>Forward/Reverse: 5&amp;prime;-TGCTGTGGGTGAAAGAGTATG-3&amp;prime;/5&amp;prime;-CGCAAATGGGCGGTAGGCGTG-3&amp;prime;</td></tr><tr><td>Random hexamer</td><td>Thermo Fisher Scientific</td><td>SO142</td><td></td></tr><tr><td>Recombinant Dnase I (Rnase-free) (5 U/&amp;mu;L)</td><td>TaKaRa</td><td>2270A</td><td></td></tr><tr><td>Recombinant Rnase inhibitor (40 U/&amp;mu;L)</td><td>TaKaRa</td><td>2313A</td><td></td></tr><tr><td>Ribo-Zero rRNA Removal Kit</td><td>Illumina</td><td>MRZH116</td><td>rRNA Removal Kit</td></tr><tr><td>Rotator</td><td>FINEPCR, ROTATOR AG</td><td>D1.5-32</td><td></td></tr><tr><td>RT primer sequence: CO1 Heavy</td><td></td><td></td><td>5&amp;prime;-CGCAAATGGGCGGTAGGCGTGTTGAGGTTGCGGTCTGTTAG-3&amp;prime;</td></tr><tr><td>RT primer sequence: CO1 Light</td><td></td><td></td><td>5&amp;prime;-CGCAAATGGGCGGTAGGCGTGGCCATAACCCAATACCAAACG-3&amp;prime;</td></tr><tr><td>RT primer sequence: CO2 Heavy</td><td></td><td></td><td>5&amp;prime;-CGCAAATGGGCGGTAGGCGTGGTAAAGGATGCGTAGGGATGG-3&amp;prime;</td></tr><tr><td>RT primer sequence: CO2 Light</td><td></td><td></td><td>5&amp;prime;-CGCAAATGGGCGGTAGGCGTGCTAGTCCTGTATGCCCTTTTCC-3&amp;prime;</td></tr><tr><td>RT primer sequence: CO3 Heavy</td><td></td><td></td><td>5&amp;prime;-CGCAAATGGGCGGTAGGCGTGCTCCTGATGCGAGTAATACGG-3&amp;prime;</td></tr><tr><td>RT primer sequence: CO3 Light</td><td></td><td></td><td>5&amp;prime;-CGCAAATGGGCGGTAGGCGTGCCTTTTACCACTCCAGCCTAG-3&amp;prime;</td></tr><tr><td>RT primer sequence: CYTB Heavy</td><td></td><td></td><td>5&amp;prime;-CGCAAATGGGCGGTAGGCGTGGGATAGTAATAGGGCAAGGACG-3&amp;prime;</td></tr><tr><td>RT primer sequence: CYTB Light</td><td></td><td></td><td>5&amp;prime;-CGCAAATGGGCGGTAGGCGTGCAATTATACCCTAGCCAACCCC-3&amp;prime;</td></tr><tr><td>RT primer sequence: GAPDH</td><td></td><td></td><td>5&amp;prime;-CGCAAATGGGCGGTAGGCGTGTGAGCGATGTGGCTCGGCT-3&amp;prime;</td></tr><tr><td>RT primer sequence: ND1 Heavy</td><td></td><td></td><td>5&amp;prime;-CGCAAATGGGCGGTAGGCGTGGTTGTGATAAGGGTGGAGAGG-3&amp;prime;</td></tr><tr><td>RT primer sequence: ND1 Light</td><td></td><td></td><td>5&amp;prime;-CGCAAATGGGCGGTAGGCGTGTCAAACTCAAACTACGCCCTG-3&amp;prime;</td></tr><tr><td>RT primer sequence: ND4 Heavy</td><td></td><td></td><td>5&amp;prime;-CGCAAATGGGCGGTAGGCGTGTGTTTGTCGTAGGCAGATGG-3&amp;prime;</td></tr><tr><td>RT primer sequence: ND4 Light</td><td></td><td></td><td>5&amp;prime;-CGCAAATGGGCGGTAGGCGTGCCTCACACTCATTCTCAACCC-3&amp;prime;</td></tr><tr><td>RT primer sequence: ND5 Heavy</td><td></td><td></td><td>5&amp;prime;-CGCAAATGGGCGGTAGGCGTGTTTGGGTTGAGGTGATGATG-3&amp;prime;</td></tr><tr><td>RT primer sequence: ND5 Light</td><td></td><td></td><td>5&amp;prime;-CGCAAATGGGCGGTAGGCGTGCATTGTCGCATCCACCTTTA-3&amp;prime;</td></tr><tr><td>RT primer sequence: ND6 Heavy</td><td></td><td></td><td>5&amp;prime;-CGCAAATGGGCGGTAGGCGTGGGTTGAGGTCTTGGTGAGTG-3&amp;prime;</td></tr><tr><td>RT primer sequence: ND6 Light</td><td></td><td></td><td>5&amp;prime;-CGCAAATGGGCGGTAGGCGTGCCCATAATCATACAAAGCCCC-3&amp;prime;</td></tr><tr><td>Siliconized polypropylene 1.5 mL G-tube</td><td>Bio Plas</td><td>4167SLS50</td><td></td></tr><tr><td>Sodium dedecyl sulfate</td><td>Biosesang</td><td>S1010</td><td></td></tr><tr><td>Sodium deoxycholate</td><td>Sigma-Aldrich</td><td>D6750</td><td></td></tr><tr><td>SUPERase In Rnase inhibitor</td><td>Thermo Fisher Scientific</td><td>AM2694</td><td></td></tr><tr><td>SuperScript III reverse transcriptase</td><td>Thermo Fisher Scientific</td><td>18080093</td><td>Reverse transcriptase for library preparation</td></tr><tr><td>SuperScript IV reverse transcriptase</td><td>Thermo Fisher Scientific</td><td>18090010</td><td>Reverse transcriptase for qRT-PCR</td></tr><tr><td>SYBR gold nucleic acid gl stain</td><td>Thermo Fisher Scientific</td><td>S11494</td><td></td></tr><tr><td>T4 polynucleotide kinase</td><td>New England Biolabs</td><td>M0201S</td><td></td></tr><tr><td>T4 RNA ligase 1 (ssRNA Ligase)</td><td>New England Biolabs</td><td>M0204</td><td></td></tr><tr><td>T4 RNA ligase 2, truncated KQ</td><td>New England Biolabs</td><td>M0373</td><td></td></tr><tr><td>Thermomixer</td><td>Eppendorf ThermoMixer C with ThermoTop</td><td></td><td></td></tr><tr><td>Thymidine</td><td>Sigma-Aldrich</td><td>T9250</td><td></td></tr><tr><td>Tris-borate-EDTA buffer (TBE)</td><td>TaKara</td><td>T9122</td><td></td></tr><tr><td>Triton X-100</td><td>Promega</td><td>H5142</td><td></td></tr><tr><td>Ultralink Protein A sepharose beads</td><td>Thermo Fisher Scientific</td><td>22810</td><td>Protein A beads</td></tr><tr><td>Ultrasonicator</td><td>Bioruptor</td><td></td><td></td></tr><tr><td>Urea</td><td>Bio-basic</td><td>UB0148</td><td></td></tr><tr><td>Vortex mixer</td><td>DAIHAN Scientific</td><td>VM-10</td><td></td></tr><tr><td>Xylene cyanol</td><td>Sigma-Aldrich</td><td>X4126</td><td></td></tr><tr><td>&amp;gamma;-&lt;sup&gt;32&lt;/sup&gt;P-ATP (10 &amp;mu;Ci/&amp;mu;L, 3.3 &amp;mu;M)</td><td>PerkinElmer</td><td>BLU502A100UC</td><td></td></tr></tbody></table>

studying rna interactors of protein kinase rna-activated during the mammalian cell cycle

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral RNAs. When bound to viral double-stranded RNAs (dsRNAs), PKR undergoes dimerization and subsequent autophosphorylation. Phosphorylated PKR (pPKR) becomes active and induces phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2&#945;) to suppress global translation. Increasing evidence suggests that PKR can be activated under physiological conditions such as during the cell cycle or under various stress conditions without infection. However, our understanding of the RNA activators of PKR is limited due to the lack of a standardized experimental method to capture and analyze PKR-interacting dsRNAs. Here, we present an experimental protocol to specifically enrich and analyze PKR bound RNAs during the cell cycle using HeLa cells. We utilize the efficient crosslinking activity of formaldehyde to fix PKR-RNA complexes and isolate them via immunoprecipitation. PKR co-immunoprecipitated RNAs can then be further processed to generate a high-throughput sequencing library. One major class of PKR-interacting cellular dsRNAs is mitochondrial RNAs (mtRNAs), which can exist as intermolecular dsRNAs through complementary interaction between the heavy-strand and the light-strand RNAs. To study the strandedness of these duplex mtRNAs, we also present a protocol for strand-specific qRT-PCR. Our protocol is optimized for the analysis of PKR-bound RNAs, but it can be easily modified to study cellular dsRNAs or RNA-interactors of other dsRNA binding proteins.

A schematic for the process to arrest HeLa cells at the S or M phase of the cell cycle is shown in Figure 1. For an M phase-arrested sample, we can clearly visualize round shaped cells under the microscope (Figure 2A). To examine the efficiency of the cell cycle arrest, the nuclear content of the cell can be analyzed using FACS (Figure 2B). Figure 3 shows representative data for immunoprecipitation efficiency test, where ...

Watch this Scientific Journal Video about Studying RNA Interactors of Protein Kinase RNA-Activated during the Mammalian Cell Cycle at JoVE.com

Studying RNA Interactors of Protein Kinase RNA-Activated during the Mammalian Cell Cycle

We present experimental approaches for studying RNA-interactors of double-stranded RNA binding protein kinase RNA-activated (PKR) during the mammalian cell cycle using HeLa cells. This method utilizes formaldehyde to crosslink RNA-PKR complexes and immunoprecipitation to enrich PKR-bound RNAs. These RNAs can be further analyzed through high-throughput sequencing or qRT-PCR.

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral ...

studying-rna-interactors-protein-kinase-rna-activated-during

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

6.4K Views.  Korea Advanced Institute of Science and Technology (KAIST). Dit protocol biedt een methode om RNA interactors van een RNA bindend eiwit PKR op een genoombrede manier te identificeren. Het helpt ons om de post-transcriptional regulatie van genexpressie door RNA-bindende eiwitten beter te begrijpen. Het belangrijkste voordeel van deze techniek is dat het gebruik maakt van efficiënte crosslinking van formaldehyde vast te stellen RNase gebonden aan RNA-bindende eiwitten en verrijken door middel van immunoprecipitatie.Geactiveerde PKR wordt waargen...

Video: Studying RNA Interactors of Protein Kinase RNA-Activated during the Mammalian Cell Cycle

Isolation of Cognate RNA-protein Complexes from Cells Using Oligonucleotide-directed Elution

Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. They are composed of position-dependent, cis-acting RNA elements and unique combinations of RNA binding proteins. Defining the composition of a specific RNP is essential to achieving a fundamental understanding of gene regulation. The isolation of a select RNP is akin to finding a needle in a haystack. Here, we demonstrate an approach to isolate RNPs associated at the 5' untranslated region of a select mRNA in asynchronous, transfected cells. This cognate RNP has been demonstrated to be necessary for the translation of select viruses and cellular stress-response genes.
The demonstrated RNA-protein co-precipitation protocol is suitable for the downstream analysis of protein components through proteomic analyses, immunoblots, or suitable biochemical identification assays. This experimental protocol demonstrates that DHX9/RNA helicase A is enriched at the 5' terminus of cognate retroviral RNA and provides preliminary information for the identification of its association with cell stress-associated huR and junD cognate mRNAs.

This manuscript describes an approach to isolate select cognate RNPs formed in eukaryotic cells via a specific oligonucleotide-directed enrichment. We demonstrate the applicability of this approach by isolating a cognate RNP bound to the retroviral 5' untranslated region that is composed of DHX9/RNA helicase A.

Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. ...

Studying Cell Cycle-regulated Gene Expression by Two Complementary Cell Synchronization Protocols

The gene expression program of the cell cycle represents a critical step for understanding cell cycle-dependent processes and their role in diseases such as cancer. Cell cycle-regulated gene expression analysis depends on cell synchronization into specific phases. Here we describe a method utilizing two complementary synchronization protocols that is commonly used for studying periodic variation of gene expression during the cell cycle. Both procedures are based on transiently blocking the cell cycle in one defined point. The synchronization protocol by hydroxyurea (HU) treatment leads to cellular arrest in late G1/early S phase, and release from HU-mediated arrest provides a cellular population uniformly progressing through S and G2/M. The synchronization protocol by thymidine and nocodazole (Thy-Noc) treatment blocks cells in early mitosis, and release from Thy-Noc mediated arrest provides a synchronized cellular population suitable for G1 phase and S phase-entry studies. Application of both procedures requires monitoring of the cell cycle distribution profiles, which is typically performed after propidium iodide (PI) staining of the cells and flow cytometry-mediated analysis of DNA content. We show that the combined use of two synchronization protocols is a robust approach to clearly determine the transcriptional profiles of genes that are differentially regulated in the cell cycle (i.e. E2F1 and E2F7), and consequently to have a better understanding of their role in cell cycle processes. Furthermore, we show that this approach is useful for the study of mechanisms underlying drug-based therapies (i.e. mitomycin C, an anticancer agent), because it allows to discriminate genes that are responsive to the genotoxic agent from those solely affected by cell cycle perturbations imposed by the agent.

We report two cell synchronization protocols that provide a context for studying events related to specific phases of the cell cycle. We show that this approach is useful for analyzing the regulation of specific genes in an unperturbed cell cycle or upon exposure to agents affecting the cell cycle.

The gene expression program of the cell cycle represents a critical step for understanding cell cycle-dependent processes and their role in diseases such ...

Genetics

mRNA Interactome Capture from Plant Protoplasts

RNA-binding proteins (RBPs) determine the fates of RNAs. They participate in all RNA biogenesis pathways and especially contribute to post-transcriptional gene regulation (PTGR) of messenger RNAs (mRNAs). In the past few years, a number of mRNA-bound proteomes from yeast and mammalian cell lines have been successfully isolated through the use of a novel method called &#34;mRNA interactome capture,&#34; which allows for the identification of mRNA-binding proteins (mRBPs) directly from a physiological environment. The method is composed of in vivo ultraviolet (UV) crosslinking, pull-down and purification of messenger ribonucleoprotein complexes (mRNPs) by oligo(dT) beads, and the subsequent identification of the crosslinked proteins by mass spectrometry (MS). Very recently, by applying the same method, several plant mRNA-bound proteomes have been reported simultaneously from different Arabidopsis tissue sources: etiolated seedlings, leaf tissue, leaf mesophyll protoplasts, and cultured root cells. Here, we present the optimized mRNA interactome capture method for Arabidopsis thaliana leaf mesophyll protoplasts, a cell type that serves as a versatile tool for experiments that include various cellular assays. The conditions for optimal protein yield include the amount of starting tissue and the duration of UV irradiation. In the mRNA-bound proteome obtained from a medium-scale experiment (107 cells), RBPs noted to have RNA-binding capacity were found to be overrepresented, and many novel RBPs were identified. The experiment can be scaled up (109 cells), and the optimized method can be applied to other plant cell types and species to broadly isolate, catalog, and compare mRNA-bound proteomes in plants.

Here, we present an interactome capture protocol applied to Arabidopsis thaliana leaf mesophyll protoplasts. This method critically relies on in vivo UV crosslinking and allows for the isolation and identification of plant mRNA-binding proteins from a physiological environment.

RNA-binding proteins (RBPs) determine the fates of RNAs. They participate in all RNA biogenesis pathways and especially contribute to post-transcriptional ...

Monitoring Activation of the Antiviral Pattern Recognition Receptors RIG-I And PKR By Limited Protease Digestion and Native PAGE

Host defenses to virus infection are dependent on a rapid detection by pattern recognition receptors (PRRs) of the innate immune system. In the cytoplasm, the PRRs RIG-I and PKR bind to specific viral RNA ligands. This first mediates conformational switching and oligomerization, and then enables activation of an antiviral interferon response. While methods to measure antiviral host gene expression are well established, methods to directly monitor the activation states of RIG-I and PKR are only partially and less well established.
Here, we describe two methods to monitor RIG-I and PKR stimulation upon infection with an established interferon inducer, the Rift Valley fever virus mutant clone 13 (Cl 13). Limited trypsin digestion allows to analyze alterations in protease sensitivity, indicating conformational changes of the PRRs. Trypsin digestion of lysates from mock infected cells results in a rapid degradation of RIG-I and PKR, whereas Cl 13 infection leads to the emergence of a protease-resistant RIG-I fragment. Also PKR shows a virus-induced partial resistance to trypsin digestion, which coincides with its hallmark phosphorylation at Thr 446. The formation of RIG-I and PKR oligomers was validated by native polyacrylamide gel electrophoresis (PAGE). Upon infection, there is a strong accumulation of RIG-I and PKR oligomeric complexes, whereas these proteins remained as monomers in mock infected samples.
Limited protease digestion and native PAGE, both coupled to western blot analysis, allow a sensitive and direct measurement of two diverse steps of RIG-I and PKR activation. These techniques are relatively easy and quick to perform and do not require expensive equipment.

Innate defenses to virus infections are triggered by pattern recognition receptors (PRRs). The two cytoplasmic PRRs RIG-I and PKR bind to viral signature RNAs, change conformation, oligomerize, and activate antiviral signaling. Methods are described which allow to conveniently monitor the conformational switching and the oligomerization of these cytoplasmic PRRs.

Host defenses to virus infection are dependent on a rapid detection by pattern recognition receptors (PRRs) of the innate immune system. In the cytoplasm, ...

Immunology and Infection

Monitoring Protein-RNA Interaction Dynamics In Vivo at High Temporal Resolution Using &#967;CRAC

The interaction between RNA-binding proteins (RBPs) and their RNA substrates exhibits fluidity and complexity. Within its lifespan, a single RNA can be bound by many different RBPs that will regulate its production, stability, activity, and degradation. As such, much has been done to understand the dynamics that exist between these two types of molecules. A particularly important breakthrough came with the emergence of &#8216;cross-linking and immunoprecipitation&#8217; (CLIP). This technique allowed stringent investigation into which RNAs are bound by a particular RBP. In short, the protein of interest is UV cross-linked to its RNA substrates in vivo, purified under highly stringent conditions, and then the RNAs covalently cross-linked to the protein are converted into cDNA libraries and sequenced. Since its conception, many derivative techniques have been developed in order to make CLIP amenable to particular fields of study. However, cross-linking using ultraviolet light is notoriously inefficient. This results in extended exposure times that make the temporal study of RBP-RNA interactions impossible. To overcome this issue, we recently designed and built much-improved UV irradiation and cell harvesting devices. Using these new tools, we developed a protocol for time-resolved analyses of RBP-RNA interactions in living cells at high temporal resolution: Kinetic CRoss-linking and Analysis of cDNAs (&#967;CRAC). We recently used this technique to study the role of yeast RBPs in nutrient stress adaptation. This manuscript provides a detailed overview of the &#967;CRAC method and presents recent results obtained with the Nrd1 RBP.

Kinetic cross-linking and analysis of cDNA is a method that allows investigation of the dynamics of protein-RNA interactions in living cells at high temporal resolution. Here the protocol is described in detail, including the growth of yeast cells, UV cross-linking, harvesting, protein purification, and next generation sequencing library preparation steps.

The interaction between RNA-binding proteins (RBPs) and their RNA substrates exhibits fluidity and complexity. Within its lifespan, a single RNA can be ...

Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates

Polynucleotide kinases (PNKs) are enzymes that catalyze the phosphorylation of the 5&#39; hydroxyl end of DNA and RNA oligonucleotides. The activity of PNKs can be quantified using direct or indirect approaches. Presented here is a direct, in vitro approach to measure PNK activity that relies on a fluorescently-labeled oligonucleotide substrate and polyacrylamide gel electrophoresis. This approach provides resolution of the phosphorylated products while avoiding the use of radiolabeled substrates. The protocol details how to set up the phosphorylation reaction, prepare and run large polyacrylamide gels, and quantify the reaction products. The most technically challenging part of this assay is pouring and running the large polyacrylamide gels; thus, important details to overcome common difficulties are provided. This protocol was optimized for Grc3, a PNK that assembles into an obligate pre-ribosomal RNA processing complex with its binding partner, the Las1 nuclease. However, this protocol can be adapted to measure the activity of other PNK enzymes. Moreover, this assay can also be modified to determine the effects of different components of the reaction, such as the nucleoside triphosphate, metal ions, and oligonucleotides.

This protocol describes a nonradioactive assay to measure kinase activity of polynucleotide kinases (PNKs) on small DNA and RNA substrates.

Polynucleotide kinases (PNKs) are enzymes that catalyze the phosphorylation of the 5&#39; hydroxyl end of DNA and RNA oligonucleotides. The activity of ...

Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such probes typically consist of variants of CFP and YFP, intervened by a phosphorylatable sequence and a phospho-binding domain. Upon phosphorylation, the probe changes conformation, which results in a change of the distance or orientation between CFP and YFP, leading to a change in FRET efficiency (Fig 1). Several probes have been published during the last decade, monitoring the activity balance of multiple kinases and phosphatases, including reporters of PKA2, PKB3, PKC4, PKD5, ERK6, JNK7, Cdk18, Aurora B9 and Plk19. Given the modular design, additional probes are likely to emerge in the near future10. 
Progression through the cell cycle is affected by stress signaling pathways 11. Notably, the cell cycle is regulated differently during unperturbed growth compared to when cells are recovering from stress12.Time-lapse imaging of cells through the cell cycle therefore requires particular caution. This becomes a problem particularly when employing ratiometric imaging, since two images with a high signal to noise ratio are required to correctly interpret the results. Ratiometric FRET imaging of cell cycle dependent changes in kinase and phosphatase activities has predominately been restricted to sub-sections of the cell cycle8,9,13,14.
Here, we discuss a method to monitor FRET-based probes using ratiometric imaging throughout the human cell cycle. The method relies on equipment that is available to many researchers in life sciences and does not require expert knowledge of microscopy or image processing.

FRET-based reporters are increasingly used to monitor kinase and phosphatase activities in live cells. Here we describe a method on how to use FRET-based reporters to assess cell cycle-dependent changes in target phosphorylation.

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such ...

Biology

Positive Regulator Molecules

Mitotic cell division results in daughter cells that exactly resemble the parent cell. However, errors in the DNA replication or distribution of genetic material may lead to genetic mutations that may be passed down to every new cell formed from the resulting abnormal cell. Propagation of such mutant cells is restricted through checkpoint mechanisms present at different stages of the cell cycle. These checkpoints involve regulator molecules that either promote or demote cell cycle events.
Proteins, such as cyclin and cyclin-dependent kinases, are positive regulator molecules responsible for the cell cycle&#8217;s progression through various checkpoints. The cyclins were initially named such because their synthesis and degradation assume a cyclical pattern. There are at least four functional cyclins whose concentration fluctuates predictably across the cell cycle. When a cell gets promoted to the next stage, the cyclins of the previous phase get degraded. It is the changes in the cyclin concentration that trigger various cell cycle events.
Cyclins form an active complex with protein kinases that can phosphorylate specific target proteins during the cell cycle. Because these kinases need cyclin for activation, they are called cyclin-dependent kinases or Cdks. In the absence of cyclin, the Cdks are inactive, and in the absence of a fully activated cyclin/Cdk complex, the cell fails to pass through the checkpoints.
The positive regulator molecules are expressed by genes that belong to a group called proto-oncogenes. When mutated, these become oncogenes that cause the cell to become cancerous. For instance, a mutation that causes the Cdks to become active even in the absence of cyclin can cause the mutant cell to pass uninterrupted through the checkpoints leading to uncontrolled growth and proliferation.

Positive Regulators of Cell Cycle: Cyclins and Cdks

Mitotic cell division results in daughter cells that exactly resemble the parent cell. However, errors in the DNA replication or distribution of genetic ...

Education

JoVE Core

Molecular Biology

Unit 2

Cell Proliferation

KEY TERMS AND CONCEPTS

Inhibition of Cdk Activity

The orderly progression of the cell cycle depends on the activation of Cdk protein by binding to its cyclin partner. However, the cell cycle must be restricted when undergoing abnormal changes. Most cancers correlate to the deregulated cell cycle, and since Cdks are a central component of the cell cycle, Cdk inhibitors are extensively studied to develop anticancer agents. For instance, cyclin D associates with several Cdks, such as Cdk 4/6, to form an active complex. The cyclin D-Cdk4/6 complex then phosphorylates and inactivates the tumor suppressor retinoblastoma protein (Rb) to promote the G1-to-S phase transition of the cell cycle. In normal cells, Rb protein is reactivated through the regulation of Cdk activity, thus, preventing abnormal cell cycle transitions. 
There are at least three known mechanisms by which Cdk activity is regulated- cyclin degradation, inhibitory phosphorylation, or binding of inhibitory proteins. Mutations that prevent these mechanisms lead to Cdk-mediated tumorigenesis.
Because Cdk 4/6 plays a substantial role in tumor formation, several Cdk inhibitors have been developed for clinical use. The most recent ones are selective for Cdk4 and Cdk6. There are at least three clinically approved Cdk 4/6 inhibitors: abemaciclib, ribociclib, and palbociclib. These inhibitors bind to the ATP pocket of Cdk 4 and 6, inactivating the Cyclin D-Cdk4/6 complexes, leading to Rb protein activation and subsequent cell cycle arrest. In some cases, the inhibitor-mediated cell cycle arrest causes an increase in apoptosis in tumor cells.
Inhibition of the cell cycle and subsequent programmed cell death are the most common mechanisms of Cdk4/6 inhibitors. However, a recent study in mouse models of breast cancer showed that Cdk4/6 inhibition could also lead to severe immunogenic effects. During the study, the Cdk inhibitor seemed to enhance tumor cells&#39; antigen-presenting ability, thereby allowing cytotoxic T cells to recognize and destroy the tumor cells.

Inhibition of CDK Activity

The orderly progression of the cell cycle depends on the activation of Cdk protein by binding to its cyclin partner. However, the cell cycle must be ...

PI3K/mTOR/AKT Signaling Pathway

The mammalian target of rapamycin&#160; (mTOR) is a serine/threonine kinase that regulates growth, proliferation, and cell survival in response to hormones, growth factors, or nutrient availability. This kinase exists in two structurally and functionally distinct forms: mTOR complex 1&#160; (mTORC1) and mTOR complex 2&#160; (mTORC2). The first form (mTORC1) is composed of a rapamycin-sensitive Raptor and proline-rich Akt substrate, PRAS40. In contrast,&#160; mTORC2 consists of a rapamycin-insensitive companion called Rictor, mammalian stress-activated protein map kinase-interacting protein 1 (mSin1 or MAPKAP1).&#160; Mammalian lethal with SEC13 protein 8 (mLST8) is a common protein of both complexes.
The binding of insulin to the insulin receptor, an RTK, initiates the mTOR pathway. The activated receptors auto-phosphorylate tyrosine 960 on its cytoplasmic domain. The phosphorylated tyrosine forms a docking site for the insulin receptor substrate (IRS) that binds the receptor via their phospho-tyrosine binding (PTB) domain. In addition, IRS contains a PH domain that helps them bind phosphoinositides on the plasma membrane.&#160; At least four types of IRS (IRS-1 to 4) assist in insulin receptor signaling. Once bound to the receptor, multiple tyrosines on the IRS are phosphorylated, which recruits the SH2 domain-containing phosphatidylinositol-3-kinase or PI-3K to the cytosolic surface of the plasma membrane. The PI-3K binds to the phosphatidylinositol (4,5)-bisphosphate&#160; (PIP2) and produces phosphatidylinositol (3,4,5)-trisphosphate or PIP3. Adjacent PIP3 functions as docking sites for protein kinase B (PKB) or AKT and 3-phosphoinositide-dependent kinase 1 or PDK1. As mTORC2 phosphorylates AKT at serine 473, it undergoes a conformational change and exposes a threonine 308 residue, which subsequently leads to phosphorylation of the AKT by PDK1.
Activated AKT dissociates from the membrane to phosphorylate a series of downstream targets to promote cell growth, proliferation, and other responses. For example, the forkhead transcription factor, FOXO3a, is a transcription factor that activates the transcription of pro-apoptotic proteins.&#160; Phosphorylation of FOXO3a by AKT induces its binding to 14-3-3 protein and its subsequent sequestration in the cytosol, thereby promoting cell survival. Activated Akt also inhibits tuberous sclerosis protein 2 or Tsc 2 and promotes Rheb activation. Activated Rheb (Rheb-GTP) further stimulates mTORC1 and induces cell growth.&#160;

Studying RNA Interactors of Protein Kinase RNA-Activated during the Mammalian Cell Cycle

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