STING at the nuclear envelope: novel partners contribute to innate immune responses
The innate immune response (IIR) is the first line of defence against pathogen infection and relies on the recognition of pathogen associated molecules by host cell sensors. STING (STimulator of INterferon Genes) is the essential adaptor protein in IIRs triggered by the recognition of cytoplasmic double-stranded DNA, a potent signal of pathogen infection or host cell DNA damage. Most STING resides in the endoplasmic reticulum and propagates IIR signalling cascades upon binding the second messenger, cGAMP, produced by the upstream cytosolic DNA sensor, cGAS. However, STING was initially identified as a nuclear envelope transmembrane protein and yet the function of STING within the nuclear envelope has been relatively understudied. Therefore, in this study I sought to investigate STING localisation and functions within the nuclear envelope. The nuclear envelope is a double membrane system comprising inner and outer nuclear membranes and, in this thesis, I present work showing for the first time that STING is present in both the inner and outer membranes by immunogold electron microscopy. Moreover, live-cell microscopy of GFP-tagged STING reveals that it increases mobility and redistributes to the outer nuclear membrane upon IIR stimulation by transfected dsDNA or the dsRNA mimic poly(I:C). Previously, immunoprecipitation of STING from isolated nuclear envelopes coupled with mass spectrometry identified a nuclear envelope-STING proteome consisting of known nuclear membrane proteins and enriched in DNA- and RNA-binding proteins. Seventeen of these nuclear envelope STING partners are known to bind direct interactors of the immune transcription factors, IRF3/7, and so it was hypothesised that these proteins could contribute to IIR through STING at the nuclear envelope. Therefore, I interrogated a subset of these for a role in IIR, finding that STING partners SYNCRIP, MEN1, DDX5, SNRNP70, RPS27A, and AATF are novel modulators of dsDNA triggered IIR. Moreover, through siRNA-mediated knockdown and CRISPR/Cas9 gene editing I found that SYNCRIP is a novel antagonist of the RNA virus, influenza A virus, potentially shedding light on reports of STING-mediated inhibition of RNA viruses. Thus, the work presented in this thesis expands our knowledge of STING’s role in IIR and adds to a growing literature which shows that STING’s functions are more extensive than its role in the cytoplasmic DNA sensing pathway.