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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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....

Protocol

1. Solution and cell preparation

  1. Solution preparation
    1. 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’s Modified Eagle’s Medium (DMEM).
      NOTE: Antibiotics can be added to the cell culture medium, but we do not use antibiotics.
    2. 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.......

Representative Results

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 .......

Discussion

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 .......

Acknowledgements

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).

....

Materials

NameCompanyCatalog NumberComments
0.5 M EDTA, pH 8.0Thermo Fisher ScientificAM9260G
1 M Tris, pH 7.0Thermo Fisher ScientificAM9855G
1 M Tris, pH 8.0Thermo Fisher ScientificAM9855G
1.7 mL microcentrifuge tubeAxygenMCT-175-C
10% Nonidet-p40 (NP-40)BiosolutionBN015
10% Urea-acrylamide gel solution7 M (w/v) Urea and 0.5X TBE, stored protected from light at 4 °C
10X DNA loading bufferTaKaRa9157
15 mL conical tubeSPL50015
3' adaptor5'-rApp NN NNT GGA ATT CTC GGG TGC CAA GG/3ddC/-3'
3 M Sodium Acetate pH 5.5Thermo Fisher ScientificAM9740
5' adaptor5'-GUU CAG AGU UCU ACA GUC CGA CGA UCN NNN-3'
5 M NaClThermo Fisher ScientificAM9760G
50 mL conical tubeSPL50050
Acid-phenol chloroform, pH 4.5Thermo Fisher ScientificAM9722
Agencourt AMPure XPBeckman CoulterA63881Magnetic beads DNA/RNA clean up
Antarctic alkaline phosphataseNew England BiolabsM0289S
Anti-DGCR8Made in house
Anti-PKR (D7F7)Cell signaling technology12297S
Anti-PKR (Milli)Millipore EMD07-151
ATP (100 mM)GE HealthcareGE27-2056-01
Bromophenol blue sodium saltSigma-aldrichB5525
Calf intestinal alkaline phosphataseTaKaRa2250A
Cell scraper 25 cm 2-positionSarstedt83.183
CMV promoter sequence5'-CGCAAATGGGCGGTAGGCGTG-3'
Dulbecco's modified eagle mediumWelgeneLM001-05
dNTP mixture (2.5 mM)TaKaRa4030
Ethanol, Absolute, ACS GradeAlfa-AesarA9951
Fetal bovine serumMerckM-TMS-013-BKR
FormamideMerck104008
GlycineBio-basicGB0235
GlycoBlue coprecipitant (15 mg/mL)Thermo Fisher ScientificAM9516
IsopropanolMerck8.18766.1000
NEBNext rRNA Depletion KitNew England BiolabsE6318rRNA Depletion Kit
NocodazoleSigma-AldrichM1404
Normal rabbit IgGCell signaling technology2729S
ParaformaldehydeSigma-Aldrich6148
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 mLAxygenPCR-02-C
Phosphate bufered saline (PBS) TabletTaKaRaT9181
Phusion high-fidelity DNA polymeraseNew England BiolabsM0530High-fidelity polymerase
PlateFuge microcentrifuge with swing-out rotorBenchmarkc2000
Polynucleotide kinase (PNK)TaKaRa2021A
Protease inhibitor cocktail set IIIMerck535140-1MLCN
Proteinase K, recombinant, PCR GradeSigma-Aldrich3115879001
qPCR primer sequence: CO1 HeavyForward/Reverse: 5′-GCCATAACCCAATACCAAACG-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′
qPCR primer sequence: CO1 LightForward/Reverse: 5′-TTGAGGTTGCGGTCTGTTAG-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′
qPCR primer sequence: CO2 HeavyForward/Reverse: 5′-CTAGTCCTGTATGCCCTTTTCC-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′
qPCR primer sequence: CO2 LightForward/Reverse: 5′-GTAAAGGATGCGTAGGGATGG-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′
qPCR primer sequence: CO3 HeavyForward/Reverse: 5′-CCTTTTACCACTCCAGCCTAG-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′
qPCR primer sequence: CO3 LightForward/Reverse: 5′-CTCCTGATGCGAGTAATACGG-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′
qPCR primer sequence: CYTB HeavyForward/Reverse: 5′-CAATTATACCCTAGCCAACCCC-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′
qPCR primer sequence: CYTB LightForward/Reverse: 5′-GGATAGTAATAGGGCAAGGACG -3′/5′-CGCAAATGGGCGGTAGGCGTG-3′
qPCR primer sequence: GAPDHForward/Reverse: 5′-CAACGACCACTTTGTCAAGC-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′
qPCR primer sequence: ND1 HeavyForward/Reverse: 5′-TCAAACTCAAACTACGCCCTG-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′
qPCR primer sequence: ND1 LightForward/Reverse: 5′-GTTGTGATAAGGGTGGAGAGG-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′
qPCR primer sequence: ND4 HeavyForward/Reverse: 5′-CTCACACTCATTCTCAACCCC-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′
qPCR primer sequence: ND4 LightForward/Reverse: 5′-TGTTTGTCGTAGGCAGATGG-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′
qPCR primer sequence: ND5 HeavyForward/Reverse: 5′-CTAGGCCTTCTTACGAGCC-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′
qPCR primer sequence: ND5 LightForward/Reverse: 5′-TAGGGAGAGCTGGGTTGTTT-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′
qPCR primer sequence: ND6 HeavyForward/Reverse: 5′-TCATACTCTTTCACCCACAGC-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′
qPCR primer sequence: ND6 LightForward/Reverse: 5′-TGCTGTGGGTGAAAGAGTATG-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′
Random hexamerThermo Fisher ScientificSO142
Recombinant Dnase I (Rnase-free) (5 U/μL)TaKaRa2270A
Recombinant Rnase inhibitor (40 U/μL)TaKaRa2313A
Ribo-Zero rRNA Removal KitIlluminaMRZH116rRNA Removal Kit
RotatorFINEPCR, ROTATOR AGD1.5-32
RT primer sequence: CO1 Heavy5′-CGCAAATGGGCGGTAGGCGTGTTGAGGTTGCGGTCTGTTAG-3′
RT primer sequence: CO1 Light5′-CGCAAATGGGCGGTAGGCGTGGCCATAACCCAATACCAAACG-3′
RT primer sequence: CO2 Heavy5′-CGCAAATGGGCGGTAGGCGTGGTAAAGGATGCGTAGGGATGG-3′
RT primer sequence: CO2 Light5′-CGCAAATGGGCGGTAGGCGTGCTAGTCCTGTATGCCCTTTTCC-3′
RT primer sequence: CO3 Heavy5′-CGCAAATGGGCGGTAGGCGTGCTCCTGATGCGAGTAATACGG-3′
RT primer sequence: CO3 Light5′-CGCAAATGGGCGGTAGGCGTGCCTTTTACCACTCCAGCCTAG-3′
RT primer sequence: CYTB Heavy5′-CGCAAATGGGCGGTAGGCGTGGGATAGTAATAGGGCAAGGACG-3′
RT primer sequence: CYTB Light5′-CGCAAATGGGCGGTAGGCGTGCAATTATACCCTAGCCAACCCC-3′
RT primer sequence: GAPDH5′-CGCAAATGGGCGGTAGGCGTGTGAGCGATGTGGCTCGGCT-3′
RT primer sequence: ND1 Heavy5′-CGCAAATGGGCGGTAGGCGTGGTTGTGATAAGGGTGGAGAGG-3′
RT primer sequence: ND1 Light5′-CGCAAATGGGCGGTAGGCGTGTCAAACTCAAACTACGCCCTG-3′
RT primer sequence: ND4 Heavy5′-CGCAAATGGGCGGTAGGCGTGTGTTTGTCGTAGGCAGATGG-3′
RT primer sequence: ND4 Light5′-CGCAAATGGGCGGTAGGCGTGCCTCACACTCATTCTCAACCC-3′
RT primer sequence: ND5 Heavy5′-CGCAAATGGGCGGTAGGCGTGTTTGGGTTGAGGTGATGATG-3′
RT primer sequence: ND5 Light5′-CGCAAATGGGCGGTAGGCGTGCATTGTCGCATCCACCTTTA-3′
RT primer sequence: ND6 Heavy5′-CGCAAATGGGCGGTAGGCGTGGGTTGAGGTCTTGGTGAGTG-3′
RT primer sequence: ND6 Light5′-CGCAAATGGGCGGTAGGCGTGCCCATAATCATACAAAGCCCC-3′
Siliconized polypropylene 1.5 mL G-tubeBio Plas4167SLS50
Sodium dedecyl sulfateBiosesangS1010
Sodium deoxycholateSigma-AldrichD6750
SUPERase In Rnase inhibitorThermo Fisher ScientificAM2694
SuperScript III reverse transcriptaseThermo Fisher Scientific18080093Reverse transcriptase for library preparation
SuperScript IV reverse transcriptaseThermo Fisher Scientific18090010Reverse transcriptase for qRT-PCR
SYBR gold nucleic acid gl stainThermo Fisher ScientificS11494
T4 polynucleotide kinaseNew England BiolabsM0201S
T4 RNA ligase 1 (ssRNA Ligase)New England BiolabsM0204
T4 RNA ligase 2, truncated KQNew England BiolabsM0373
ThermomixerEppendorf ThermoMixer C with ThermoTop
ThymidineSigma-AldrichT9250
Tris-borate-EDTA buffer (TBE)TaKaraT9122
Triton X-100PromegaH5142
Ultralink Protein A sepharose beadsThermo Fisher Scientific22810Protein A beads
UltrasonicatorBioruptor
UreaBio-basicUB0148
Vortex mixerDAIHAN ScientificVM-10
Xylene cyanolSigma-AldrichX4126
γ-32P-ATP (10 μCi/μL, 3.3 μM)PerkinElmerBLU502A100UC

References

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