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 two branches of the project: (a) T-box riboswitches, and (b) RNA interactions in HIV/AIDS. (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 elucidate the molecular basis and specificities of HIV-1 Gag interactions with host tRNAs, we have applied rational RNA design and engineering to solve the first HIV-1 MA-tRNA complex structure. Further, we have characterized MA-tRNA interactions using mutational, ITC, AUC and fluorescence analyses. Complementary analyses of MA-tRNA interactions using ChIP-seq, live cell imaging and virology tools have been carried out in Paul Bieniaszs lab at Rockefeller University. We find that HIV-1 MAs global head has evolved a highly specific binding determinant, consisting of only four basic and aromatic residues, to achieve high-affinity, selective recognition of the unique elbow structure of host tRNAs. Mutant Gag that cannot bind tRNAs are prematurely localized to the plasma membrane and reduced HIV-1 replication. Together, these findings define the elusive RNA-binding specificities of HIV matrix, and reveal that this novel matrix-tRNA interface allows HIV-1 to exploit host tRNAs to regulate the membrane location of its major structural protein, Gag, and thus the assembly of new virions. These findings may potentially provide a novel avenue to perturb HIV particle assembly using RNA biologics. This work has recently been published in Cell Host & Microbe.