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Fiegen, Ann

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Fiegen

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Ann

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Fiegen, Ann

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2014 - 20163

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Author's Publications: search.filters.isAuthorOfPublication.9c69a93d-d6b3-4da0-b083-1e44b9937ff1

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    Diverse intracellular pathogens activate Type III Interferon expression from peroxisomes
    (2014) Odendall, Charlotte; Dixit, Evelyn; Stavru, Fabrizia; Bierne, Helene; Franz, Kate M.; Fiegen, Ann; Boulant, Steeve; Gehrke, Lee; Cossart, Pascale; Kagan, Jonathan
    Type I Interferon (IFN) responses are considered the primary means by which viral infections are controlled in mammals. Despite this view, several pathogens activate antiviral responses in the absence of Type I IFNs. The mechanisms controlling Type I IFN-independent responses are undefined. We have found that RIG-I like Receptors (RLRs) induce Type III IFN expression in a variety of human cell types, and identified factors that differentially regulate Type I and III IFN expression. We identified peroxisomes as a primary site that initiates Type III IFN expression, and revealed that the process of intestinal epithelial cell differentiation upregulates peroxisome biogenesis and promotes robust Type III IFN responses in human cells. These findings highlight the interconnections between innate immunity and cell biology.
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    RNAs Containing Modified Nucleotides Fail To Trigger RIG-I Conformational Changes for Innate Immune Signaling
    (American Society for Microbiology, 2016) Fiegen, Ann; Wang, Chen; Marcotrigiano, Joseph; Gehrke, Lee
    ABSTRACT 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.
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    Nucleotide Modifications of RNA Suppress RIG-I Antiviral Signaling by Unique Mechanisms
    (2016-05-02) Fiegen, Ann; Kagan, Jonathan; Cunningham, James; Bartel, David; RajBhandary, Uttam
    In order to counter pathogen infection while preventing autoimmune responses, the human innate immune system must be precisely regulated to distinguish “self” from “non-self”. Pattern recognition receptors detect “non-self” pathogen RNAs and initiate antiviral signaling. Accumulated evidence suggests that host “self” RNAs contain modified nucleotides that evade or suppress immune signaling; however, the precise mechanisms are not understood. Defining these mechanisms is relevant toward understanding the biology of immunity as well as the applied use of RNAs as therapeutic molecules, where reducing ligand immunogenicity is essential. Evidence from our lab and others’ suggests that the cytosolic RNA helicase RIG-I (retinoic acid inducible gene-I) detects not only the 5’ terminus and double-stranded nature of RNA, but also the presence/absence of modified nucleotides. In the present study, we use a model RNA ligand (polyU/UC), derived from the 3’ untranslated region of the hepatitis C virus RNA, to dissect the mechanisms by which RNAs containing nucleotide modifications suppress or evade RIG-I signaling. Five assays were developed to test our hypothesis that eight different nucleotide modifications, both natural and synthetic, share a common mechanism of innate immune evasion. In vitro transcribed 5’-triphosphate polyU/UC RNA containing canonical nucleotides potently activates the RIG-I signaling pathway in transfected cells, culminating in an antiviral state. When transcribed with any of eight modified nucleotides, the polyU/UC RNA suppressed the RIG-I antiviral response. Unexpectedly, the modified nucleotides had different effects on RIG-I:RNA binding affinity, as well as RIG-I conformational change. The data suggest that multiple RIG-I evasion/suppression mechanisms associated with different modified nucleotides may have evolved to effect a common result of interrupting innate immune signaling responses to “self” RNA. Our findings hold implications for understanding the co-evolution of the innate immune response and RNA modification pathways across domains of life, as well as for defining approaches for testing the multitude of naturally occurring and synthetic nucleotides that may have utility in the design of therapeutic RNAs.