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Incorporation of pseudouridine into mRNA enhances translation by diminishing PKR activation
Open accessAbstract
Previous studies have shown that the translation level of in vitro transcribed messenger RNA (mRNA) is enhanced when its uridines are replaced with pseudouridines; however, the reason for this enhancement has not been identified. Here, we demonstrate that in vitro transcripts containing uridine activate RNA-dependent protein kinase (PKR), which then phosphorylates translation initiation factor 2-alpha (eIF-2α), and inhibits translation. In contrast, in vitro transcribed mRNAs containing pseudouridine activate PKR to a lesser degree, and translation of pseudouridine-containing mRNAs is not repressed. RNA pull-down assays demonstrate that mRNA containing uridine is bound by PKR more efficiently than mRNA with pseudouridine. Finally, the role of PKR is validated by showing that pseudouridine- and uridine-containing RNAs were translated equally in PKR knockout cells. These results indicate that the enhanced translation of mRNAs containing pseudouridine, compared to those containing uridine, is mediated by decreased activation of PKR.
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“Substitution of chemically modified nucleotides in messenger ribonucleic acid (mRNA) enhances stability and transfection capability, and decreases immunogenicity1–3”
From Complete substitution with modified nucleotides in self-amplifying RNA suppresses the interferon response and increases potency (2025) · cites this paper as [3]
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“Native RNA activates pattern recognition receptors such as toll-like receptors and triggers type I interferon (IFN) production, resulting in translational shutoff and systemic inflammation3.”
From Complete substitution with modified nucleotides in self-amplifying RNA suppresses the interferon response and increases potency (2025) · cites this paper as [3]
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“This can be circumvented by the incorporation of naturally occurring nucleosides and by applying stringent purification to remove double-stranded RNA contaminants (19–21).”
From mRNA vaccine with unmodified uridine induces robust type I interferon-dependent anti-tumor immunity in a melanoma model (2022) · cites this paper as [21]
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“Transferring this concept to in vitro transcribed mRNA, for instance, pseudouridine-containing mRNAs reduced activation of known RNA sensors substantially.”
From Sequence-engineered mRNA without chemical nucleoside modifications enables an effective protein therapy in large animals (2015) · cites this paper as [22]
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“The two uridine analogs pseudouridine (Ψ) or 1-methyl-pseudouridine (m1Ψ) are widely used for mRNA therapeutics because they can increase mRNA stability and help modulate the host immune response31,32.”
From Human 5′ UTR design and variant effect prediction from a massively parallel translation assay (2019) · cites this paper as [32]
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“Although pseudouridine is primarily found in tRNA, rRNA, and small nuclear RNAs, pseudouridine-containing mRNAs were still translated and produced even more protein compared to unmodified mRNA.”
From Sequence-engineered mRNA without chemical nucleoside modifications enables an effective protein therapy in large animals (2015) · cites this paper as [22]
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“In addition, nucleoside modifications are well known to limit or even inhibit RNA sensor engagement.20,21,22”
From Sequence-engineered mRNA without chemical nucleoside modifications enables an effective protein therapy in large animals (2015) · cites this paper as [22]
In the citing paper
“Nucleoside-modified mRNA can reduce the activation of TLRs, retinoic acid-inducible gene I (RIG-I), protein kinase R (PKR), and 2′-5′-oligoadenylate synthetase (OAS). Additionally, nucleoside modification increases translation activity and resistance against RNase L-mediated degradation.54,81,173–176”
From Advances in COVID-19 mRNA vaccine development (2022) · cites this paper as [174]
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Metadata
- Authors
- Anderson, B. R., Muramatsu, H., Nallagatla, S. R., Bevilacqua, P. C., Sansing, L. H., Weissman, D., & Karikó, K.
- Affiliations
- Pennsylvania State University (US)
- Corresponding author
- Karikó, Katalin
- ORCID
- Venue
- Nucleic Acids Research
- Publisher
- Oxford University Press (OUP)
- Published
- 2010-05-10
- Volume
- 38
- Issue
- 17
- Pages
- 5884-5892
- ISSN
- 0305-1048 (ISSN-L)
- 1362-4962
- PMC
- PMC2943593
- OpenAlex
- W2156788486
- Cited by
- 682
- References
- 60
- Citations by year
- 2026: 67 · 2025: 99 · 2024: 143 · 2023: 90 · 2022: 77 · 2021: 52 · 2020: 22 · 2019: 22 · 2018: 26 · 2017: 17 · 2016: 21 · 2015: 13 · 2014: 6 · 2013: 12 · 2012: 8
- FWCI
- 3.49
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- Nucleic Acids Research (publishedVersion, cc-by-nc)
- PubMed Central (publishedVersion, cc-by-nc)
- PubMed (publishedVersion)
- HighWire Press Open Archive (submittedVersion)
- Funders
- Cleveland Clinic
- Cleveland Clinic Foundation
- National Institutes of Health
- National Heart, Lung, and Blood Institute (R42HL087688)
- National Institute of General Medical Sciences (T32GM007229, R01GM058709)
- National Institute of Allergy and Infectious Diseases (R01AI050484)
- National Institute of Diabetes and Digestive and Kidney Diseases (T32DK007748)
- National Institute of Dental and Craniofacial Research (R21DE019059)
- National Center for Research Resources (T32RR007063)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA modifications and cancer
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
- Keywords
- pseudouridine, mRNA translation, PKR, RNA-dependent protein kinase, eIF2α phosphorylation, translation initiation, uridine, in vitro transcribed mRNA, RNA-protein interactions
- MeSH terms
- Animals, Cell Line, Cells, Cultured, Humans, Pseudouridine, RNA, Messenger, Protein Biosynthesis, eIF-2 Kinase, Mice
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