5'-Triphosphate guanosine RNAs recruit GTP-binding proteins to suppress RIG-I/IFN type I signaling

The interferon (IFN) response is crucial for antiviral activity, but excessive activation can contribute to tissue damage and autoimmune disorders. The cytoplasmic receptor retinoic acid-inducible gene I (RIG-I) detects viral double-stranded RNAs (dsRNAs) and endogenous RNA polymerase III (RNA Pol III) transcripts carrying a 5'-triphosphate (5'-ppp) moiety, triggering IRF3 phosphorylation and IFN response. Many viral RNAs initiate with 5'-triphosphate adenosine (5'-pppA), whereas most endogenous RNA Pol III transcripts in higher eukaryotes start with 5'-triphosphate guanosine (5'-pppG), yet no reason for this bias is known. Here, we show that 5'-pppA dsRNAs trigger stronger RIG-I/IFN responses than 5'-pppG dsRNAs, despite comparable RIG-I stimulation in vitro. Using RNA pull-down and mass spectrometry, we identify GTP-binding proteins that preferentially interact with 5'-pppG dsRNAs. Guanosine supplementation, which increases intracellular GTP levels, decreases the immunogenic difference between 5'-pppG and 5'-pppA dsRNAs. Our findings provide insights into sequence-dependent activation of the RIG-I/IFN pathway, with implications for evolutionary pressures on RNA sequences, RNA-driven autoimmunity, viral immunogenicity, and the rational design of RNA therapeutics.