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RNAs Containing Modified Nucleotides Fail To Trigger RIG-I Conformational Changes for Innate Immune Signaling
Open accessAbstract
UNLABELLED: Invading pathogen nucleic acids are recognized and bound by cytoplasmic (retinoic acid-inducible gene I [RIG-I]-like) and membrane-bound (Toll-like) pattern recognition receptors to activate innate immune signaling. Modified nucleotides, when present in RNA molecules, diminish the magnitude of these signaling responses. However, mechanisms explaining the blunted signaling have not been elucidated. In this study, we used several independent biological assays, including inhibition of virus replication, RIG-I:RNA binding assays, and limited trypsin digestion of RIG-I:RNA complexes, to begin to understand how RNAs containing modified nucleotides avoid or suppress innate immune signaling. The experiments were based on a model innate immune activating RNA molecule, the polyU/UC RNA domain of hepatitis C virus, which was transcribed in vitro with canonical nucleotides or with one of eight modified nucleotides. The approach revealed signature assay responses associated with individual modified nucleotides or classes of modified nucleotides. For example, while both N-6-methyladenosine (m6A) and pseudouridine nucleotides correlate with diminished signaling, RNA containing m6A modifications bound RIG-I poorly, while RNA containing pseudouridine bound RIG-I with high affinity but failed to trigger the canonical RIG-I conformational changes associated with robust signaling. These data advance understanding of RNA-mediated innate immune signaling, with additional relevance for applying nucleotide modifications to RNA therapeutics. IMPORTANCE: The innate immune system provides the first response to virus infections and must distinguish between host and pathogen nucleic acids to mount a protective immune response without activating autoimmune responses. While the presence of nucleotide modifications in RNA is known to correlate with diminished innate immune signaling, the underlying mechanisms have not been explored. The data reported here are important for defining mechanistic details to explain signaling suppression by RNAs containing modified nucleotides. The results suggest that RNAs containing modified nucleotides interrupt signaling at early steps of the RIG-I-like innate immune activation pathway and also that nucleotide modifications with similar chemical structures can be organized into classes that suppress or evade innate immune signaling steps. These data contribute to defining the molecular basis for innate immune signaling suppression by RNAs containing modified nucleotides. The results have important implications for designing therapeutic RNAs that evade innate immune detection.
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“by incorporating naturally occurring modified nucleotides into the IVT mRNA, its innate immune activity could be drastically reduced [6,7]”
From The dawn of mRNA vaccines: the COVID-19 case (2021) · cites this paper as [7]
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Metadata
- Authors
- Durbin, A. F., Wang, C., Marcotrigiano, J., & Gehrke, L.
- Affiliations
- Harvard University (US)
- Rutgers, The State University of New Jersey (US)
- Corresponding author
- Gehrke, Lee
- Venue
- mBio
- Publisher
- American Society for Microbiology
- Published
- 2016-11-02
- Volume
- 7
- Issue
- 5
- ISSN
- 2150-7511 (ISSN-L)
- 2161-2129
- PMC
- PMC5030355
- OpenAlex
- W2522303658
- Cited by
- 238
- References
- 58
- Citations by year
- 2026: 13 · 2025: 22 · 2024: 25 · 2023: 27 · 2022: 28 · 2021: 49 · 2020: 30 · 2019: 23 · 2018: 13 · 2017: 8
- FWCI
- 8.88
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- mBio (publishedVersion, cc-by)
- Europe PMC (PubMed Central) (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Digital Access to Scholarship at Harvard (DASH) (Harvard University) (submittedVersion, cc-by)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
- DSpace@MIT (Massachusetts Institute of Technology) (submittedVersion, cc-by-nc)
- Funders
- HHS | U.S. Public Health Service (CA159132, AI100190)
- Topics
- Life Sciences › Immunology and Microbiology › Immunology › interferon and immune responses
- Life Sciences › Immunology and Microbiology › Immunology › Immune Response and Inflammation
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
- Keywords
- RIG-I, modified nucleotides, innate immune signaling, pseudouridine, RNA conformational change, RNA therapeutics, immune evasion, viral RNA recognition
- MeSH terms
- DEAD Box Protein 58, Cell Line, Humans, Immunity, Innate, Models, Molecular, Ribonucleosides, RNA, Viral, Signal Transduction, Hepacivirus, Interferon-beta
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