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) tRNA-like structures in lncRNAs, and (c) Nucleic acid recognition by antibodies. (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 triggers nearly instantaneous domain closure and guides the folding of 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. This work has been provisionally accepted for publication in Nature Communications. b) To gain insights into the maturation of MALAT1 lncRNA and biogenesis of mascRNA, we have solved the first crystal structures of human mascRNA before and after RNase P processing. mascRNA employs a novel, unusually streamlined mode of partial tRNA mimicry to recruit tRNA-processing enzymes. Using in vitro RNase P, ELAC2, and aminoacylation assays, we have defined the structural determinants of mascRNA that drive its processing and identified its non-tRNA features that escape aminoacylation, preventing interference with translation. (c) To elucidate how S9.6 antibody recognizes R-loops, we recently reported the first 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 was published in Nat. Commun. To understand how the J2 antibody recognize dsRNA, we have determined its first co-crystal structure bound to a dsRNA duplex and are currently characterizing its binding interface and specificity.