Activation of PKR by a short-hairpin RNA.

eIF-2 Kinase / metabolism Humans RNA, Double-Stranded / metabolism RNA, Small Interfering / genetics RNA-Binding Proteins / metabolism Adenosine Deaminase / metabolism Enzyme Activation DEAD-box RNA Helicases / metabolism HEK293 Cells Gene Knockdown Techniques
Double-stranded RNA PKR RNA interference RNAi dsRNA

Journal

Scientific reports
ISSN: 2045-2322
Titre abrégé: Sci Rep
Pays: England
ID NLM: 101563288

Informations de publication

Date de publication:
09 10 2024
Historique:
received: 14 05 2024
accepted: 26 09 2024
medline: 10 10 2024
pubmed: 10 10 2024
entrez: 9 10 2024
Statut: epublish

Résumé

Recognition of viral infection often relies on the detection of double-stranded RNA (dsRNA), a process that is conserved in many different organisms. In mammals, proteins such as MDA5, RIG-I, OAS, and PKR detect viral dsRNA, but struggle to differentiate between viral and endogenous dsRNA. This study investigates an shRNA targeting DDX54's potential to activate PKR, a key player in the immune response to dsRNA. Knockdown of DDX54 by a specific shRNA induced robust PKR activation in human cells, even when DDX54 is overexpressed, suggesting an off-target mechanism. Activation of PKR by the shRNA was enhanced by knockdown of ADAR1, a dsRNA binding protein that suppresses PKR activation, indicating a dsRNA-mediated mechanism. In vitro assays confirmed direct PKR activation by the shRNA. These findings emphasize the need for rigorous controls and alternative methods to validate gene function and minimize unintended immune pathway activation.

Identifiants

DOI: 10.1038/s41598-024-74477-3 PMID: 39384561
pubmed: 39384561
doi: 10.1038/s41598-024-74477-3
pii: 10.1038/s41598-024-74477-3
doi:

Substances chimiques

eIF-2 Kinase EC 2.7.11.1
RNA, Double-Stranded 0
EIF2AK2 protein, human EC 2.7.11.1
RNA, Small Interfering 0
RNA-Binding Proteins 0
Adenosine Deaminase EC 3.5.4.4
ADAR protein, human EC 3.5.4.37
DEAD-box RNA Helicases EC 3.6.4.13

Types de publication

Journal Article

Langues

eng

Sous-ensembles de citation

IM

Pagination

23533

Subventions

Organisme : NIH HHS
ID : K99MD016946
Pays : United States
Organisme : NIH HHS
ID : R35GM141262
Pays : United States
Organisme : National Institutes of Health, Pakistan
ID : R01CA262804

Informations de copyright

© 2024. The Author(s).

Références

Rehwinkel, J. & Gack, M. U. RIG-I-like receptors: their regulation and roles in RNA sensing. Nat. Rev. Immunol. 20, 537–551. https://doi.org/10.1038/s41577-020-0288-3 (2020).
doi: 10.1038/s41577-020-0288-3 pubmed: 32203325 pmcid: 7094958
Hovanessian, A. G. & Justesen, J. The human 2’-5’oligoadenylate synthetase family: unique interferon-inducible enzymes catalyzing 2’-5’ instead of 3’-5’ phosphodiester bond formation. Biochimie 89, 779–788. https://doi.org/10.1016/j.biochi.2007.02.003 (2007).
doi: 10.1016/j.biochi.2007.02.003 pubmed: 17408844
García, M. A., Meurs, E. F. & Esteban, M. The dsRNA protein kinase PKR: virus and cell control. Biochimie 89, 799–811. https://doi.org/10.1016/j.biochi.2007.03.001 (2007).
doi: 10.1016/j.biochi.2007.03.001 pubmed: 17451862
Cottrell, K. A., Andrews, R. J. & Bass, B. L. The competitive landscape of the dsRNA world. Mol. Cell 84, 107–119. https://doi.org/10.1016/j.molcel.2023.11.033 (2024).
doi: 10.1016/j.molcel.2023.11.033 pubmed: 38118451
Reich, D. P. & Bass, B. L. Mapping the dsRNA World. Cold Spring Harb. Perspect. Biol. https://doi.org/10.1101/cshperspect.a035352 (2019).
doi: 10.1101/cshperspect.a035352 pubmed: 30824577 pmcid: 6396333
Shang, R., Lee, S., Senavirathne, G. & Lai, E. C. microRNAs in action: Biogenesis, function and regulation. Nat. Rev. Genet. 24, 816–833. https://doi.org/10.1038/s41576-023-00611-y (2023).
doi: 10.1038/s41576-023-00611-y pubmed: 37380761 pmcid: 11087887
Moore, C. B., Guthrie, E. H., Huang, M. T. & Taxman, D. J. Short hairpin RNA (shRNA): Design, delivery, and assessment of gene knockdown. Methods Mol. Biol. 629, 141–158. https://doi.org/10.1007/978-1-60761-657-3_10 (2010).
doi: 10.1007/978-1-60761-657-3_10 pubmed: 20387148 pmcid: 3679364
Birmingham, A. et al. 3’ UTR seed matches, but not overall identity, are associated with RNAi off-targets. Nat. Methods 3, 199–204. https://doi.org/10.1038/nmeth854 (2006).
doi: 10.1038/nmeth854 pubmed: 16489337
Jackson, A. L. et al. Widespread siRNA “off-target” transcript silencing mediated by seed region sequence complementarity. RNA 12, 1179–1187. https://doi.org/10.1261/rna.25706 (2006).
doi: 10.1261/rna.25706 pubmed: 16682560 pmcid: 1484447
Lewis, B. P., Burge, C. B. & Bartel, D. P. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 120, 15–20. https://doi.org/10.1016/j.cell.2004.12.035 (2005).
doi: 10.1016/j.cell.2004.12.035 pubmed: 15652477
Meurs, E. F., Galabru, J., Barber, G. N., Katze, M. G. & Hovanessian, A. G. Tumor suppressor function of the interferon-induced double-stranded RNA-activated protein kinase. Proc. Natl. Acad. Sci. U S A 90, 232–236. https://doi.org/10.1073/pnas.90.1.232 (1993).
doi: 10.1073/pnas.90.1.232 pubmed: 7678339 pmcid: 45634
Pindel, A. & Sadler, A. The role of protein kinase R in the interferon response. J. Interferon Cytokine Res. 31, 59–70. https://doi.org/10.1089/jir.2010.0099 (2011).
doi: 10.1089/jir.2010.0099 pubmed: 21166592
Qiao, H. et al. Cell fate determined by the activation balance between PKR and SPHK1. Cell Death Differ. 28, 401–418. https://doi.org/10.1038/s41418-020-00608-8 (2021).
doi: 10.1038/s41418-020-00608-8 pubmed: 32801355
Ucci, J. W., Kobayashi, Y., Choi, G., Alexandrescu, A. T. & Cole, J. L. Mechanism of interaction of the double-stranded RNA (dsRNA) binding domain of protein kinase R with short dsRNA sequences. Biochemistry 46, 55–65. https://doi.org/10.1021/bi061531o (2007).
doi: 10.1021/bi061531o pubmed: 17198375
Su, Q. et al. Tyrosine phosphorylation acts as a molecular switch to full-scale activation of the eIF2alpha RNA-dependent protein kinase. Proc. Natl. Acad. Sci. USA 103, 63–68. https://doi.org/10.1073/pnas.0508207103 (2006).
doi: 10.1073/pnas.0508207103 pubmed: 16373505
Galabru, J. & Hovanessian, A. Autophosphorylation of the protein kinase dependent on double-stranded RNA. J. Biol. Chem. 262, 15538–15544 (1987).
doi: 10.1016/S0021-9258(18)47759-9 pubmed: 3479429
Dey, M., Mann, B. R., Anshu, A. & Mannan, M. A. Activation of protein kinase PKR requires dimerization-induced cis-phosphorylation within the activation loop. J. Biol. Chem. 289, 5747–5757. https://doi.org/10.1074/jbc.M113.527796 (2014).
doi: 10.1074/jbc.M113.527796 pubmed: 24338483
Nanduri, S., Carpick, B. W., Yang, Y., Williams, B. R. & Qin, J. Structure of the double-stranded RNA-binding domain of the protein kinase PKR reveals the molecular basis of its dsRNA-mediated activation. EMBO J. 17, 5458–5465. https://doi.org/10.1093/emboj/17.18.5458 (1998).
doi: 10.1093/emboj/17.18.5458 pubmed: 9736623 pmcid: 1170871
Schmedt, C. et al. Functional characterization of the RNA-binding domain and motif of the double-stranded RNA-dependent protein kinase DAI (PKR). J. Mol. Biol. 249, 29–44. https://doi.org/10.1006/jmbi.1995.0278 (1995).
doi: 10.1006/jmbi.1995.0278 pubmed: 7776374
Husain, B., Mukerji, I. & Cole, J. L. Analysis of high-affinity binding of protein kinase R to double-stranded RNA. Biochemistry 51, 8764–8770. https://doi.org/10.1021/bi301226h (2012).
doi: 10.1021/bi301226h pubmed: 23062027
Kim, Y. et al. PKR senses nuclear and mitochondrial signals by interacting with endogenous double-stranded RNAs. Mol. Cell 71, 1051-1063.e1056. https://doi.org/10.1016/j.molcel.2018.07.029 (2018).
doi: 10.1016/j.molcel.2018.07.029 pubmed: 30174290
Cottrell, K. A., Ryu, S., Torres, L. S., Schab, A. M. & Weber, J. D. Induction of viral mimicry upon loss of DHX9 and ADAR1 in breast cancer cells. bioRxiv https://doi.org/10.1101/2023.02.27.530307 (2023).
doi: 10.1101/2023.02.27.530307 pubmed: 37662237 pmcid: 10002699
Kung, C. P. et al. Evaluating the therapeutic potential of ADAR1 inhibition for triple-negative breast cancer. Oncogene 40, 189–202. https://doi.org/10.1038/s41388-020-01515-5 (2021).
doi: 10.1038/s41388-020-01515-5 pubmed: 33110236
Nicholson, D. W. et al. Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature 376, 37–43. https://doi.org/10.1038/376037a0 (1995).
doi: 10.1038/376037a0 pubmed: 7596430
Lazebnik, Y. A., Kaufmann, S. H., Desnoyers, S., Poirier, G. G. & Earnshaw, W. C. Cleavage of poly(ADP-ribose) polymerase by a proteinase with properties like ICE. Nature 371, 346–347. https://doi.org/10.1038/371346a0 (1994).
doi: 10.1038/371346a0 pubmed: 8090205
Cottrell, K. A. et al. Induction of viral mimicry upon loss of DHX9 and ADAR1 in breast cancer cells. Cancer Res. Commun. 4, 986–1003. https://doi.org/10.1158/2767-9764.CRC-23-0488 (2024).
doi: 10.1158/2767-9764.CRC-23-0488 pubmed: 38530197 pmcid: 10993856
Safran, S. A., Eckert, D. M., Leslie, E. A. & Bass, B. L. PKR activation by noncanonical ligands: a 5’-triphosphate requirement versus antisense contamination. RNA 25, 1192–1201. https://doi.org/10.1261/rna.071910.119 (2019).
doi: 10.1261/rna.071910.119 pubmed: 31239298 pmcid: 6800522
Kostura, M. & Mathews, M. B. Purification and activation of the double-stranded RNA-dependent eIF-2 kinase DAI. Mol. Cell Biol. 9, 1576–1586. https://doi.org/10.1128/mcb.9.4.1576-1586.1989 (1989).
doi: 10.1128/mcb.9.4.1576-1586.1989 pubmed: 2725516 pmcid: 362574
Liu, H. et al. Tumor-derived IFN triggers chronic pathway agonism and sensitivity to ADAR loss. Nat. Med. 25, 95–102. https://doi.org/10.1038/s41591-018-0302-5 (2019).
doi: 10.1038/s41591-018-0302-5 pubmed: 30559422
Ishizuka, J. J. et al. Loss of ADAR1 in tumours overcomes resistance to immune checkpoint blockade. Nature 565, 43–48. https://doi.org/10.1038/s41586-018-0768-9 (2019).
doi: 10.1038/s41586-018-0768-9 pubmed: 30559380
Gannon, H. S. et al. Identification of ADAR1 adenosine deaminase dependency in a subset of cancer cells. Nat. Commun. 9, 5450. https://doi.org/10.1038/s41467-018-07824-4 (2018).
doi: 10.1038/s41467-018-07824-4 pubmed: 30575730 pmcid: 6303303
Murayama, T. et al. Targeting DHX9 triggers tumor-intrinsic interferon response and replication stress in small cell lung cancer. Cancer Discov. 14, 468–491. https://doi.org/10.1158/2159-8290.CD-23-0486 (2024).
doi: 10.1158/2159-8290.CD-23-0486 pubmed: 38189443 pmcid: 10905673
Zhang, Z., Weinschenk, T., Guo, K. & Schluesener, H. J. siRNA binding proteins of microglial cells: PKR is an unanticipated ligand. J. Cell Biochem. 97, 1217–1229. https://doi.org/10.1002/jcb.20716 (2006).
doi: 10.1002/jcb.20716 pubmed: 16315288
Sledz, C. A., Holko, M., de Veer, M. J., Silverman, R. H. & Williams, B. R. Activation of the interferon system by short-interfering RNAs. Nat. Cell Biol. 5, 834–839. https://doi.org/10.1038/ncb1038 (2003).
doi: 10.1038/ncb1038 pubmed: 12942087
Armstrong, M. E. et al. Small interfering RNAs induce macrophage migration inhibitory factor production and proliferation in breast cancer cells via a double-stranded RNA-dependent protein kinase-dependent mechanism. J. Immunol. 180, 7125–7133. https://doi.org/10.4049/jimmunol.180.11.7125 (2008).
doi: 10.4049/jimmunol.180.11.7125 pubmed: 18490711
Puthenveetil, S. et al. Controlling activation of the RNA-dependent protein kinase by siRNAs using site-specific chemical modification. Nucleic Acids Res. 34, 4900–4911. https://doi.org/10.1093/nar/gkl464 (2006).
doi: 10.1093/nar/gkl464 pubmed: 16982647 pmcid: 1635244
Marques, J. T. et al. A structural basis for discriminating between self and nonself double-stranded RNAs in mammalian cells. Nat. Biotechnol. 24, 559–565. https://doi.org/10.1038/nbt1205 (2006).
doi: 10.1038/nbt1205 pubmed: 16648842
Mayo, C. B., Wong, C. J., Lopez, P. E., Lary, J. W. & Cole, J. L. Activation of PKR by short stem-loop RNAs containing single-stranded arms. RNA 22, 1065–1075. https://doi.org/10.1261/rna.053348.115 (2016).
doi: 10.1261/rna.053348.115 pubmed: 27208315 pmcid: 4911914
Kaempfer, R. Interferon-gamma mRNA attenuates its own translation by activating PKR: a molecular basis for the therapeutic effect of interferon-beta in multiple sclerosis. Cell Res. 16, 148–153. https://doi.org/10.1038/sj.cr.7310020 (2006).
doi: 10.1038/sj.cr.7310020 pubmed: 16474427
Heinicke, L. A. et al. RNA dimerization promotes PKR dimerization and activation. J. Mol. Biol. 390, 319–338. https://doi.org/10.1016/j.jmb.2009.05.005 (2009).
doi: 10.1016/j.jmb.2009.05.005 pubmed: 19445956 pmcid: 2763119
Zuker, M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31, 3406–3415 (2003).
doi: 10.1093/nar/gkg595 pubmed: 12824337 pmcid: 169194

Auteurs

Kyle A Cottrell (KA)

Department of Medicine, Division of Molecular Oncology, Washington University School of Medicine, 660 South Euclid Avenue, Campus Box 8069, St. Louis, MO, 63110, USA. cottrellka@pudue.edu.
ICCE Institute, Washington University School of Medicine, Saint Louis, MO, USA. cottrellka@pudue.edu.
Department of Biochemistry, Purdue University, S University St, West Lafayette, IN, 201, USA. cottrellka@pudue.edu.

Sua Ryu (S)

Department of Medicine, Division of Molecular Oncology, Washington University School of Medicine, 660 South Euclid Avenue, Campus Box 8069, St. Louis, MO, 63110, USA.
ICCE Institute, Washington University School of Medicine, Saint Louis, MO, USA.

Helen Donelick (H)

Department of Biochemistry, University of Utah, Salt Lake City, UT, USA.

Hung Mai (H)

Department of Medicine, Division of Molecular Oncology, Washington University School of Medicine, 660 South Euclid Avenue, Campus Box 8069, St. Louis, MO, 63110, USA.
ICCE Institute, Washington University School of Medicine, Saint Louis, MO, USA.

Addison A Young (AA)

Department of Biochemistry, Purdue University, S University St, West Lafayette, IN, 201, USA.

Jackson R Pierce (JR)

Department of Biochemistry, Purdue University, S University St, West Lafayette, IN, 201, USA.

Brenda L Bass (BL)

Department of Biochemistry, University of Utah, Salt Lake City, UT, USA.

Jason D Weber (JD)

Department of Medicine, Division of Molecular Oncology, Washington University School of Medicine, 660 South Euclid Avenue, Campus Box 8069, St. Louis, MO, 63110, USA. jweber@wustl.edu.
Department of Cell Biology and Physiology, Washington University School of Medicine, Saint Louis, MO, USA. jweber@wustl.edu.
Department of Biology, Siteman Cancer Center, Washington University School of Medicine, Saint Louis, MO, USA. jweber@wustl.edu.
ICCE Institute, Washington University School of Medicine, Saint Louis, MO, USA. jweber@wustl.edu.

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Classifications MeSH

questionsmedicales.fr › Acides aminés, peptides et protéines › Protéines › Protéines et peptides de signalisation intracellulaire › Protein-Serine-Threonine Kinases › eIF-2 Kinase › Activation of PKR by a short-hairpin RNA.
questionsmedicales.fr › Eucaryotes › Animaux › Chordés › Vertébrés › Mammifères › Eutheria › Primates › Haplorhini › Catarrhini › Hominidae › Humains › Activation of PKR by a short-hairpin RNA.
questionsmedicales.fr › Phénomènes génétiques › Structures génétiques › Conformation d'acide nucléique › ARN double brin › Activation of PKR by a short-hairpin RNA.
questionsmedicales.fr › Acides nucléiques, nucléotides et nucléosides › Acides nucléiques › ARN › ARN non traduit › Petit ARN non traduit › Petit ARN interférent › Activation of PKR by a short-hairpin RNA.
questionsmedicales.fr › Acides aminés, peptides et protéines › Protéines › Nucléoprotéines › Protéines de liaison à l'ARN › Activation of PKR by a short-hairpin RNA.
questionsmedicales.fr › Enzymes et coenzymes › Enzymes › Hydrolases › Aminohydrolases › Nucleoside deaminases › Adenosine deaminase › Activation of PKR by a short-hairpin RNA.
questionsmedicales.fr › Métabolisme › Activation enzymatique › Activation of PKR by a short-hairpin RNA.
questionsmedicales.fr › Enzymes et coenzymes › Enzymes › Transferases › Phosphotransferases › Nucleotidyltransferases › RNA nucleotidyltransferases › RNA helicases › DEAD-box RNA helicases › Activation of PKR by a short-hairpin RNA.
questionsmedicales.fr › Cellules › Cellules épithéliales › Cellules HEK293 › Activation of PKR by a short-hairpin RNA.
questionsmedicales.fr › Techniques d'investigation › Techniques génétiques › Ciblage de gène › Techniques de knock-down de gènes › Activation of PKR by a short-hairpin RNA.