Activation of RIG-I by hairpin RNA

N Shah1, SA Beckham1, D Luo2, MCJ Wilce1 and JA Wilce1

  1. Biomedicine Discovery Institute, Department of Biochemistry and Molecular Biology, Monash University, Wellington Road, Clayton VIC 3800, Australia
  2. Nanyang Technological University, Singapore

As part of our innate immune system, ATP dependent RIG-I like receptors (RLRs) within the host cytoplasm detect viral RNA. RIG-I (retinoic acid inducible gene-I receptor), in particular, senses dsRNA with a 5′-triphosphate overhang and, following a conformational rearrangement and release of its CARD domains, mediates the ultimate induction of type I interferons and pro-inflammatory cytokines. It has been shown in activation assays that a 5′ triphosphate-10mer dsRNA hairpin is able to activate the RIG-I, but a 5′ ppp-8mer does not (1). There is no biophysical evidence yet, however, of CARD release by the 10mer dsRNA and not the 8mer. Based on the question whether 10mer is the minimum length of dsRNA enough for RIG-I activation or 8mer dsRNA can also activate it, we are investigating this using size-exclusion chromatography-coupled small-angle X-ray scattering (SAXS) (2), and limited tryptic digest experiments. In addition, by using different ATP analogues, we are examining the importance of ATP on the conformational changes of RIG-I:RNA complexes. This study will help in better understanding of the molecular interactions necessary for RIG-I activation.
References: 1. Kohlway, A., Luo, D., Rawling, D. C., Ding, S. C., and Pyle, A. M. (2013) Defining the functional determinants for RNA surveillance by RIG-I. EMBO reports 14, 772-779. 2. Beckham, S. A., Brouwer, J., Roth, A., Wang, D., Sadler, A. J., John, M., Jahn-Hofmann, K., Williams, B. R., Wilce, J. A., and Wilce, M. C. (2013) Conformational rearrangements of RIG-I receptor on formation of a multiprotein:dsRNA assembly. Nucleic acids research 41, 3436-3445.