Noncoding RNA structures and interactions in cellular stress responses

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

We have made significant progresses in both arms of the project: (a) the T-box riboswitches and (b) PKR-regulatory RNAs. (a) We have solved the first co-crystal structure of a T-box riboswitch 3' discriminator domain in complex with an uncharged tRNA and the first full-length T-box-tRNA complex structure using cryo-EM (in collaboration with Wah Chius lab). These two structures, supported by small-angle X-ray scattering (SAXS), in vivo and in vitro analyses detail the exquisitely selective, tripartite interactions between the tRNA and the T-box mRNA. Specifically, the T-box clamps the tRNA, captures its universal 3'-CCA end using an elaborate discriminator structure, and interrogates its aminoacylation state using a steric device made of RNA. This work elucidates the core mechanisms of amino-acid sensing by all known T-box riboswitches. It also provides unprecedented structural details into how higher-order RNA-RNA interactions achieve multivalency and specificity, which may inform RNA engineering and targeting by small molecules, and inspire design of artificial RNA devices that act as sensors and conditional regulators of gene expression. (b) We have determined the first crystal structure of Adenovirus Virus-Associated RNA I (VA-I), the first described viral noncoding RNA and an extensively studied PKR inhibitor. We further performed extensive biochemical and mutational analyses to validate and extend the structural findings. The VA-I structure visualized all its elements necessary for PKR inhibition. We further discovered that the stacked apical stem and tetrastem form the minimal PKR-inhibitory core, and a central domain pseudoknot facilitates VA-I folding and prevents PKR activation. These structural features collectively define VA-I as an archetypal PKR inhibitor made of RNA. This work provides insights into how viruses circumnavigate cellular rules of self vs non-self RNAs to escape and compromise host innate immunity. This work has been published in Nature Communications.