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

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 0.1% (v/v) paraformaldehyde by adding 1x PBS.
      CAUTION: Perform all steps in a fume hood and be careful not to boil the paraformaldehyde solutio....

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 .......

Materials

List of materials used in this article

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
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
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