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Cited by 238interferon and immune responsesImmune Response and InflammationRNA regulation and disease

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RNAs Containing Modified Nucleotides Fail To Trigger RIG-I Conformational Changes for Innate Immune Signaling

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Durbin, Ann Fiegen, Wang, Chen, Marcotrigiano, Joseph, Gehrke, Lee·2016·mBio
DOI 10.1128/mbio.00833-16PMC PMC5030355
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Abstract

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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Figures

FIG 1 RNAmod and RIG-I-mediated IFN-β induction. (A) Huh7 cells were first transfected with luciferase reporter plasmids and then later mock transfected or transfected with 400 ng of polyU/UC RNA containing canonical nucleotides (can) or polyU/UC RNA containing the indicated modified nucleotides. (B) Following reporter plasmid transfection, Huh7 cells were transfected with EGFP mRNA containing a Cap-1 structure (Cap1) or 5′ppp terminus (5ppp) and canonical (can) nucleotides or pseudouridine substitution (Ψ). (A and B) After an additional 16 to 24 h of incubation, cell lysates were analyzed for IFN-β-promoter-driven firefly luciferase activity, and the data were normalized to constitutively produced Renilla luciferase values to control for transfection efficiency and then normalized to the mock-treated control condition. Error bars represent the standard deviations of results determined among technical triplicates, with results of a representative experiment shown. The statistical table reports the P values of results of paired two-tailed t tests comparing fold IFN responses between (A) canonical polyU/UC RNA and indicated modified RNAs or between (B) the indicated mRNAs, from multiple independent experiments. Additional data are provided in Fig. S1 and S2 in the supplemental material. (C) Huh7 cells were transfected with 400 ng polyU/UC RNA containing the indicated modified nucleotides. After 16 h of incubation, cells were infected with VSV-GFP at an MOI of 3. At 6 h postinfection, GFP expression was visualized by live-cell microscopy. Additional data are provided in Fig. S3 and S4 in the supplemental material. BF, bright field.
FIG 1 RNAmod and RIG-I-mediated IFN-β induction. (A) Huh7 cells were first transfected with luciferase reporter plasmids and then later mock transfected or transfected with 400 ng of polyU/UC RNA containing canonical nucleotides (can) or polyU/UC RNA containing the indicated modified nucleotides. (B) Following reporter plasmid transfection, Huh7 cells were transfected with EGFP mRNA containing a Cap-1 structure (Cap1) or 5′ppp terminus (5ppp) and canonical (can) nucleotides or pseudouridine substitution (Ψ). (A and B) After an additional 16 to 24 h of incubation, cell lysates were analyzed for IFN-β-promoter-driven firefly luciferase activity, and the data were normalized to constitutively produced Renilla luciferase values to control for transfection efficiency and then normalized to the mock-treated control condition. Error bars represent the standard deviations of results determined among technical triplicates, with results of a representative experiment shown. The statistical table reports the P values of results of paired two-tailed t tests comparing fold IFN responses between (A) canonical polyU/UC RNA and indicated modified RNAs or between (B) the indicated mRNAs, from multiple independent experiments. Additional data are provided in Fig. S1 and S2 in the supplemental material. (C) Huh7 cells were transfected with 400 ng polyU/UC RNA containing the indicated modified nucleotides. After 16 h of incubation, cells were infected with VSV-GFP at an MOI of 3. At 6 h postinfection, GFP expression was visualized by live-cell microscopy. Additional data are provided in Fig. S3 and S4 in the supplemental material. BF, bright field.CC BY · PubMed Central
FIG 2 RNAmod and RIG-I binding affinity. (A) Radiolabeled polyU/UC RNA was incubated with purified recombinant RIG-I to allow complex formation and then applied to a nitrocellulose membrane filter, which retains RNA-protein complexes, while unbound RNA passes through the membrane. The fraction of bound RNA was normalized to the maximum observed signal. Combined data from at least three independent experiments per ligand are presented with solid lines indicating the best-fit nonlinear regression and dashed lines indicating 95% confidence intervals. (B) Calculated equilibrium binding dissociation constants (Kd) derived from data shown in panel A, including 95% confidence interval, goodness of fit (R2), and statistical test (P value), demonstrating a significant difference between the two curve parameters Kd and slope for each comparison of RNAmod versus RNAcan. For the m6A data (indicated by an asterisk [*]), the experimental maximum observed signal was normalized to 100% RNA bound, although a binding plateau was not observed. Therefore, the accurate RIG-I:RNAm6A binding constant is likely higher (lower affinity) than that presented. Additional data are provided in Fig. S5 in the supplemental material. (C) Biotinylated polyU/UC RNA with the indicated modified nucleotides was added to Huh7 cell lysate. Negative controls included biotinylated X-RNA (X) and nonbiotinylated polyU/UC (-btn). RIG-I:RNA complexes that were captured with streptavidin-conjugated paramagnetic beads (BND) were detected by Western blotting with anti-RIG-I, relative to the RIG-I in 10% input (INP). The signal was quantified in the BND fraction relative to the INP fraction and normalized to canonical RNA (100%).
FIG 2 RNAmod and RIG-I binding affinity. (A) Radiolabeled polyU/UC RNA was incubated with purified recombinant RIG-I to allow complex formation and then applied to a nitrocellulose membrane filter, which retains RNA-protein complexes, while unbound RNA passes through the membrane. The fraction of bound RNA was normalized to the maximum observed signal. Combined data from at least three independent experiments per ligand are presented with solid lines indicating the best-fit nonlinear regression and dashed lines indicating 95% confidence intervals. (B) Calculated equilibrium binding dissociation constants (Kd) derived from data shown in panel A, including 95% confidence interval, goodness of fit (R2), and statistical test (P value), demonstrating a significant difference between the two curve parameters Kd and slope for each comparison of RNAmod versus RNAcan. For the m6A data (indicated by an asterisk [*]), the experimental maximum observed signal was normalized to 100% RNA bound, although a binding plateau was not observed. Therefore, the accurate RIG-I:RNAm6A binding constant is likely higher (lower affinity) than that presented. Additional data are provided in Fig. S5 in the supplemental material. (C) Biotinylated polyU/UC RNA with the indicated modified nucleotides was added to Huh7 cell lysate. Negative controls included biotinylated X-RNA (X) and nonbiotinylated polyU/UC (-btn). RIG-I:RNA complexes that were captured with streptavidin-conjugated paramagnetic beads (BND) were detected by Western blotting with anti-RIG-I, relative to the RIG-I in 10% input (INP). The signal was quantified in the BND fraction relative to the INP fraction and normalized to canonical RNA (100%).CC BY · PubMed Central
FIG 3 RIG-I:RNA conformation as probed by limited trypsin digestion time course. (A to D) RIG-I fragments were detected by Western blotting with a monoclonal antibody to the helicase domain. Cell extracts were incubated with trypsin in the absence (A [–RNA]) or presence of RNA ligands, including nonbinding control yeast tRNA (B [tRNA]), polyI:C (C), or polyU/UC (D), and with 1 mM ATP or AMP-PNP. Aliquots were removed from the reaction at various time points (minutes) post-addition of trypsin. Additional data are provided in Fig. S6 in the supplemental material. (E) Biotinylated RNAs were used in pulldown experiments to test for RNA binding by the 80-kDa and 55-kDa RIG-I fragments. Trypsin digests (+ tryp) and control lysates (− tryp) were incubated without RNA (-RNA), with nonbiotinylated polyU/UC RNA (-btn), with biotinylated polyU/UC RNA (can), or with biotinylated X RNA (X) in the presence of AMP-PNP. After 1.5 h, trypsin digestions were quenched by adding protease inhibitor and incubated with streptavidin paramagnetic beads. RIG-I present in the bead-bound fraction (BND) versus the input fraction (INP) was detected by Western blotting.
FIG 3 RIG-I:RNA conformation as probed by limited trypsin digestion time course. (A to D) RIG-I fragments were detected by Western blotting with a monoclonal antibody to the helicase domain. Cell extracts were incubated with trypsin in the absence (A [–RNA]) or presence of RNA ligands, including nonbinding control yeast tRNA (B [tRNA]), polyI:C (C), or polyU/UC (D), and with 1 mM ATP or AMP-PNP. Aliquots were removed from the reaction at various time points (minutes) post-addition of trypsin. Additional data are provided in Fig. S6 in the supplemental material. (E) Biotinylated RNAs were used in pulldown experiments to test for RNA binding by the 80-kDa and 55-kDa RIG-I fragments. Trypsin digests (+ tryp) and control lysates (− tryp) were incubated without RNA (-RNA), with nonbiotinylated polyU/UC RNA (-btn), with biotinylated polyU/UC RNA (can), or with biotinylated X RNA (X) in the presence of AMP-PNP. After 1.5 h, trypsin digestions were quenched by adding protease inhibitor and incubated with streptavidin paramagnetic beads. RIG-I present in the bead-bound fraction (BND) versus the input fraction (INP) was detected by Western blotting.CC BY · PubMed Central
FIG 4 RNAmod and RIG-I trypsin sensitivity. (A to D) Digestion of 293T cell lysate for 2 h in the presence of polyU/UC RNA with the indicated modifications or canonical nucleotides (can), at increasing polyU/UC RNA concentrations (0, 12.5, 25, 50, 100, 200, 400, 800, and 1,600 nM), in the presence of 2 mM ATP or AMP-PNP. Data represent results of Western blotting for 55-kDa and 80-kDa fragments of RIG-I with anti-helicase antibody. (E) Digestions of 293T cell lysate performed for 1.5 h in the presence of AMP-PNP and polyU/UC RNA with the indicated modifications, at increasing concentrations (0, 33, 100, 300, and 900 nM), or mass equivalent of polyI:C.
FIG 4 RNAmod and RIG-I trypsin sensitivity. (A to D) Digestion of 293T cell lysate for 2 h in the presence of polyU/UC RNA with the indicated modifications or canonical nucleotides (can), at increasing polyU/UC RNA concentrations (0, 12.5, 25, 50, 100, 200, 400, 800, and 1,600 nM), in the presence of 2 mM ATP or AMP-PNP. Data represent results of Western blotting for 55-kDa and 80-kDa fragments of RIG-I with anti-helicase antibody. (E) Digestions of 293T cell lysate performed for 1.5 h in the presence of AMP-PNP and polyU/UC RNA with the indicated modifications, at increasing concentrations (0, 33, 100, 300, and 900 nM), or mass equivalent of polyI:C.CC BY · PubMed Central
FIG 5 Model of RIG-I interaction with RNAcan and RNAmod. (Step 1) RIG-I is in an autorepressed conformation in the absence of ligand. (Step 2) 5′ppp RNA binds the RIG-I CTD. RNAm6A binds RIG-I with lower affinity than RNAcan. (Step 3) The RIG-I helicase domain binds the RNA, triggering a protein conformational change. RNAs containing Ψ, mΨ, or 5mC fail to induce RIG-I conformational change. (Step 4) The released CARDs of the activated RIG-I:RNA complex are ubiquitinated and signal downstream. This process is hypothesized to be inefficient for RIG-I:RNA2FdN, possibly due to ATPase negative regulation of RNA binding. Ubiquitinated RIG-I induces MAVS aggregation on the surface of the mitochondria. (Step 5) Mitochondrial complexes mediate downstream antiviral signaling. Among the RNAs tested, we observed that only RNAcan triggered RIG-I antiviral signaling.
FIG 5 Model of RIG-I interaction with RNAcan and RNAmod. (Step 1) RIG-I is in an autorepressed conformation in the absence of ligand. (Step 2) 5′ppp RNA binds the RIG-I CTD. RNAm6A binds RIG-I with lower affinity than RNAcan. (Step 3) The RIG-I helicase domain binds the RNA, triggering a protein conformational change. RNAs containing Ψ, mΨ, or 5mC fail to induce RIG-I conformational change. (Step 4) The released CARDs of the activated RIG-I:RNA complex are ubiquitinated and signal downstream. This process is hypothesized to be inefficient for RIG-I:RNA2FdN, possibly due to ATPase negative regulation of RNA binding. Ubiquitinated RIG-I induces MAVS aggregation on the surface of the mitochondria. (Step 5) Mitochondrial complexes mediate downstream antiviral signaling. Among the RNAs tested, we observed that only RNAcan triggered RIG-I antiviral signaling.CC BY · PubMed Central

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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
ORCID
Venue
mBio
Publisher
American Society for Microbiology
Published
2016-11-02
Volume
7
Issue
5
ISSN
  • 2150-7511 (ISSN-L)
  • 2161-2129
DOI
10.1128/mbio.00833-16
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
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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