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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 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 (eIF2α) 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.
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 (eIF2α) kinase family and phosphorylates eIF2α 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 eIF2α, PKR can also phosphorylate p53, insulin receptor substrate 1, inhibi....
1. Solution and cell preparation
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 .......
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 .......
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).
....Name | Company | Catalog Number | Comments |
0.5 M EDTA, pH 8.0 | Thermo Fisher Scientific | AM9260G | |
1 M Tris, pH 7.0 | Thermo Fisher Scientific | AM9855G | |
1 M Tris, pH 8.0 | Thermo Fisher Scientific | AM9855G | |
1.7 mL microcentrifuge tube | Axygen | MCT-175-C | |
10% Nonidet-p40 (NP-40) | Biosolution | BN015 | |
10% Urea-acrylamide gel solution | 7 M (w/v) Urea and 0.5X TBE, stored protected from light at 4 °C | ||
10X DNA loading buffer | TaKaRa | 9157 | |
15 mL conical tube | SPL | 50015 | |
3' adaptor | 5'-rApp NN NNT GGA ATT CTC GGG TGC CAA GG/3ddC/-3' | ||
3 M Sodium Acetate pH 5.5 | Thermo Fisher Scientific | AM9740 | |
5' adaptor | 5'-GUU CAG AGU UCU ACA GUC CGA CGA UCN NNN-3' | ||
5 M NaCl | Thermo Fisher Scientific | AM9760G | |
50 mL conical tube | SPL | 50050 | |
Acid-phenol chloroform, pH 4.5 | Thermo Fisher Scientific | AM9722 | |
Agencourt AMPure XP | Beckman Coulter | A63881 | Magnetic beads DNA/RNA clean up |
Antarctic alkaline phosphatase | New England Biolabs | M0289S | |
Anti-DGCR8 | Made in house | ||
Anti-PKR (D7F7) | Cell signaling technology | 12297S | |
Anti-PKR (Milli) | Millipore EMD | 07-151 | |
ATP (100 mM) | GE Healthcare | GE27-2056-01 | |
Bromophenol blue sodium salt | Sigma-aldrich | B5525 | |
Calf intestinal alkaline phosphatase | TaKaRa | 2250A | |
Cell scraper 25 cm 2-position | Sarstedt | 83.183 | |
CMV promoter sequence | 5'-CGCAAATGGGCGGTAGGCGTG-3' | ||
Dulbecco's modified eagle medium | Welgene | LM001-05 | |
dNTP mixture (2.5 mM) | TaKaRa | 4030 | |
Ethanol, Absolute, ACS Grade | Alfa-Aesar | A9951 | |
Fetal bovine serum | Merck | M-TMS-013-BKR | |
Formamide | Merck | 104008 | |
Glycine | Bio-basic | GB0235 | |
GlycoBlue coprecipitant (15 mg/mL) | Thermo Fisher Scientific | AM9516 | |
Isopropanol | Merck | 8.18766.1000 | |
NEBNext rRNA Depletion Kit | New England Biolabs | E6318 | rRNA Depletion Kit |
Nocodazole | Sigma-Aldrich | M1404 | |
Normal rabbit IgG | Cell signaling technology | 2729S | |
Paraformaldehyde | Sigma-Aldrich | 6148 | |
PCR forward primer (RP1) | 5'-AAT GAT ACG GCG ACC ACC GCG ATC TAC ACG TTC AGA GTT CTA CAG TCC GA-3' | ||
PCR index reverse primer (RPI) | 5'-CAA GCA GAA GAC GGC ATA CGA GAT NNN NNN GTG ACT GGA GTT CCT TGG CAC CCG AGA ATT CCA-3' | ||
PCR tubes with flat cap, 0.2 mL | Axygen | PCR-02-C | |
Phosphate bufered saline (PBS) Tablet | TaKaRa | T9181 | |
Phusion high-fidelity DNA polymerase | New England Biolabs | M0530 | High-fidelity polymerase |
PlateFuge microcentrifuge with swing-out rotor | Benchmark | c2000 | |
Polynucleotide kinase (PNK) | TaKaRa | 2021A | |
Protease inhibitor cocktail set III | Merck | 535140-1MLCN | |
Proteinase K, recombinant, PCR Grade | Sigma-Aldrich | 3115879001 | |
qPCR primer sequence: CO1 Heavy | Forward/Reverse: 5′-GCCATAACCCAATACCAAACG-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′ | ||
qPCR primer sequence: CO1 Light | Forward/Reverse: 5′-TTGAGGTTGCGGTCTGTTAG-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′ | ||
qPCR primer sequence: CO2 Heavy | Forward/Reverse: 5′-CTAGTCCTGTATGCCCTTTTCC-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′ | ||
qPCR primer sequence: CO2 Light | Forward/Reverse: 5′-GTAAAGGATGCGTAGGGATGG-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′ | ||
qPCR primer sequence: CO3 Heavy | Forward/Reverse: 5′-CCTTTTACCACTCCAGCCTAG-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′ | ||
qPCR primer sequence: CO3 Light | Forward/Reverse: 5′-CTCCTGATGCGAGTAATACGG-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′ | ||
qPCR primer sequence: CYTB Heavy | Forward/Reverse: 5′-CAATTATACCCTAGCCAACCCC-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′ | ||
qPCR primer sequence: CYTB Light | Forward/Reverse: 5′-GGATAGTAATAGGGCAAGGACG -3′/5′-CGCAAATGGGCGGTAGGCGTG-3′ | ||
qPCR primer sequence: GAPDH | Forward/Reverse: 5′-CAACGACCACTTTGTCAAGC-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′ | ||
qPCR primer sequence: ND1 Heavy | Forward/Reverse: 5′-TCAAACTCAAACTACGCCCTG-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′ | ||
qPCR primer sequence: ND1 Light | Forward/Reverse: 5′-GTTGTGATAAGGGTGGAGAGG-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′ | ||
qPCR primer sequence: ND4 Heavy | Forward/Reverse: 5′-CTCACACTCATTCTCAACCCC-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′ | ||
qPCR primer sequence: ND4 Light | Forward/Reverse: 5′-TGTTTGTCGTAGGCAGATGG-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′ | ||
qPCR primer sequence: ND5 Heavy | Forward/Reverse: 5′-CTAGGCCTTCTTACGAGCC-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′ | ||
qPCR primer sequence: ND5 Light | Forward/Reverse: 5′-TAGGGAGAGCTGGGTTGTTT-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′ | ||
qPCR primer sequence: ND6 Heavy | Forward/Reverse: 5′-TCATACTCTTTCACCCACAGC-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′ | ||
qPCR primer sequence: ND6 Light | Forward/Reverse: 5′-TGCTGTGGGTGAAAGAGTATG-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′ | ||
Random hexamer | Thermo Fisher Scientific | SO142 | |
Recombinant Dnase I (Rnase-free) (5 U/μL) | TaKaRa | 2270A | |
Recombinant Rnase inhibitor (40 U/μL) | TaKaRa | 2313A | |
Ribo-Zero rRNA Removal Kit | Illumina | MRZH116 | rRNA Removal Kit |
Rotator | FINEPCR, ROTATOR AG | D1.5-32 | |
RT primer sequence: CO1 Heavy | 5′-CGCAAATGGGCGGTAGGCGTGTTGAGGTTGCGGTCTGTTAG-3′ | ||
RT primer sequence: CO1 Light | 5′-CGCAAATGGGCGGTAGGCGTGGCCATAACCCAATACCAAACG-3′ | ||
RT primer sequence: CO2 Heavy | 5′-CGCAAATGGGCGGTAGGCGTGGTAAAGGATGCGTAGGGATGG-3′ | ||
RT primer sequence: CO2 Light | 5′-CGCAAATGGGCGGTAGGCGTGCTAGTCCTGTATGCCCTTTTCC-3′ | ||
RT primer sequence: CO3 Heavy | 5′-CGCAAATGGGCGGTAGGCGTGCTCCTGATGCGAGTAATACGG-3′ | ||
RT primer sequence: CO3 Light | 5′-CGCAAATGGGCGGTAGGCGTGCCTTTTACCACTCCAGCCTAG-3′ | ||
RT primer sequence: CYTB Heavy | 5′-CGCAAATGGGCGGTAGGCGTGGGATAGTAATAGGGCAAGGACG-3′ | ||
RT primer sequence: CYTB Light | 5′-CGCAAATGGGCGGTAGGCGTGCAATTATACCCTAGCCAACCCC-3′ | ||
RT primer sequence: GAPDH | 5′-CGCAAATGGGCGGTAGGCGTGTGAGCGATGTGGCTCGGCT-3′ | ||
RT primer sequence: ND1 Heavy | 5′-CGCAAATGGGCGGTAGGCGTGGTTGTGATAAGGGTGGAGAGG-3′ | ||
RT primer sequence: ND1 Light | 5′-CGCAAATGGGCGGTAGGCGTGTCAAACTCAAACTACGCCCTG-3′ | ||
RT primer sequence: ND4 Heavy | 5′-CGCAAATGGGCGGTAGGCGTGTGTTTGTCGTAGGCAGATGG-3′ | ||
RT primer sequence: ND4 Light | 5′-CGCAAATGGGCGGTAGGCGTGCCTCACACTCATTCTCAACCC-3′ | ||
RT primer sequence: ND5 Heavy | 5′-CGCAAATGGGCGGTAGGCGTGTTTGGGTTGAGGTGATGATG-3′ | ||
RT primer sequence: ND5 Light | 5′-CGCAAATGGGCGGTAGGCGTGCATTGTCGCATCCACCTTTA-3′ | ||
RT primer sequence: ND6 Heavy | 5′-CGCAAATGGGCGGTAGGCGTGGGTTGAGGTCTTGGTGAGTG-3′ | ||
RT primer sequence: ND6 Light | 5′-CGCAAATGGGCGGTAGGCGTGCCCATAATCATACAAAGCCCC-3′ | ||
Siliconized polypropylene 1.5 mL G-tube | Bio Plas | 4167SLS50 | |
Sodium dedecyl sulfate | Biosesang | S1010 | |
Sodium deoxycholate | Sigma-Aldrich | D6750 | |
SUPERase In Rnase inhibitor | Thermo Fisher Scientific | AM2694 | |
SuperScript III reverse transcriptase | Thermo Fisher Scientific | 18080093 | Reverse transcriptase for library preparation |
SuperScript IV reverse transcriptase | Thermo Fisher Scientific | 18090010 | Reverse transcriptase for qRT-PCR |
SYBR gold nucleic acid gl stain | Thermo Fisher Scientific | S11494 | |
T4 polynucleotide kinase | New England Biolabs | M0201S | |
T4 RNA ligase 1 (ssRNA Ligase) | New England Biolabs | M0204 | |
T4 RNA ligase 2, truncated KQ | New England Biolabs | M0373 | |
Thermomixer | Eppendorf ThermoMixer C with ThermoTop | ||
Thymidine | Sigma-Aldrich | T9250 | |
Tris-borate-EDTA buffer (TBE) | TaKara | T9122 | |
Triton X-100 | Promega | H5142 | |
Ultralink Protein A sepharose beads | Thermo Fisher Scientific | 22810 | Protein A beads |
Ultrasonicator | Bioruptor | ||
Urea | Bio-basic | UB0148 | |
Vortex mixer | DAIHAN Scientific | VM-10 | |
Xylene cyanol | Sigma-Aldrich | X4126 | |
γ-32P-ATP (10 μCi/μL, 3.3 μM) | PerkinElmer | BLU502A100UC |
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