Noncoding RNA structures and interactions in cellular stress responses and immunity

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

We have made significant progresses in both branches of the project: (a) Gcn2 kinase, and (b) HIV-1 TAR RNAs. (a) To gain insights into Gcn2 structure, function, and mechanisms, we determined the first crystal structures of the HisRS domain of Gcn2 at 2.0-2.7 Å resolutions, which reveal that the HisRS-like domain of Gcn2 structurally mimics the authentic HisRS enzyme, but functionally repurposes its substrate-binding and catalytic domains to serve as a sensor and responder to stresses. We further mapped Gcn2 intra-domain and inter-domain interactions using crosslinking mass spec (XL-MS) analyses. Complementary genetics and biochemical analyses performed in Alan Hinnebusch’s lab (NICHD) provided functional contexts and support for the structural findings. Together these data exemplify how a metabolic enzyme is repurposed to host new local structures and sequences that confer a novel regulatory function. This work has been published in Proceedings of the National Academy of Sciences USA. b) To elucidate the structure, dynamics, and interactions of the complete HIV TAR RNA, we determined the first full-length, high-resolution crystal structures of HIV-1 TAR, and its complex with the HIV-1 Tat RNA-binding region in a previously unobserved pose. Comparison of these structures reveal that the full TAR RNA structure is polymorphic and dynamic. We find that the highly conserved lower stem of TAR exhibits conformational excursions that transiently open its terminal helical region. We affirmed these structural findings in solution using RNA Overhang Analysis by Ligation of Hairpin Oligonucleotides (ROALHO), a new enzymatic assay that we developed. Using time-correlated single photon counting (TCSPC) analyses of 2-aminopurines site-specifically incorporated into various locations of TAR, we observed differential local and global TAR structural remodeling by proteins including HIV-1 nucleocapsid, Tat, and PKR. Using fluorescence polarization and kinase assays, we clarified the effects of TAR on PKR kinase activity and mapped the TAR region important for PKR manipulation to its lower stem. Together, our data portray the structure, dynamics, and interactions of a complete HIV-1 TAR RNA, uncover a convergent RNA-based viral strategy to evade innate immunity by PKR, and provide potential avenues to develop antivirals that target a dynamic, multifunctional viral RNA. This work has been published in Nature Communications.