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 all three branches of the project: (a) T-box riboswitches, (b) PKR-regulatory RNAs, and (c) R-loop recognition by proteins. (a) To gain insights into the T-box structural dynamics before and during tRNA binding, we carried out SAXS, fluorescence lifetime and mutational analyses, as well as single-molecule FRET analyses in collaboration with Hoi Sung Chungs lab at NIDDK. Our data suggest that free T-boxes assume several flexible, open conformations and tRNA binding drives domain closure and remodels the T-box structure. We further show that the pseudoknot structure acts as a geometric hub that organizes the overall T-box architecture to facilitate tRNA binding and domain closure. In addition, we have characterized the structure and tRNA-binding mechanisms of select non-canonical T-boxes of atypical architectures. (b) To understand the structure, dynamics, and interactions of the complete HIV TAR RNA, we determined the full-length structures of TAR and its complex with several peptide and small molecules. Using multi-probe fluorescence lifetime analysis, hairpin-mediated ligation probing, and 1D and 2D NMR analyses (through a collaboration), we further characterized the dynamic behavior of TAR RNA in solution. Using fluorescence polarization and kinase assays, we analyzed the effects of TAR on PKR and mapped the TAR regions important for PKR manipulation. Finally, we identified additional privileged binding sies on TAR for several small molecules. (c) To elucidate how S9.6 antibody recognizes R-loops, we first demonstrated that S9.6 exhibits robust selectivity in binding DNA-RNA hybrids over double-stranded (ds) RNA and in categorically rejecting dsDNA. We further determined the crystal structures of a S9.6 antigen-binding fragment (Fab) free and bound to a 13-bp hybrid duplex. The structures and attendant biophysical and mutational analyses detail how S9.6 achieves specific recognition of hybrids in R-loops, and provide a framework for S9.6 protein engineering. This work has been published in Nat. Commun.