Structure, function, and dynamics of viral RNAs and RNA-containing complexes

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PROJECT SUMMARY Eukaryotic cells use diverse mechanisms to regulate translation, many of which depend on structured RNA elements that interact directly with the protein synthesis machinery. This ubiquitous and powerful strategy for regulating gene expression is also adopted by viral RNAs, which must use the same biological machinery as cellular RNAs. Despite their importance, the detailed mechanisms that underlie both cellular and viral RNA structure-dependent translational control are poorly understood. To address this gap, we propose to build on our previous discoveries of internal ribosome entry site (IRES) RNA function by studying two different but related classes of viral RNAs that interface directly with the cellular translation machinery. The first comprises a diverse class of viral RNAs called tRNA-like structures (TLSs) that mimic tRNAs in mysterious ways and use this to bind a variety of cellular proteins; in so doing they enhance translation by unknown means. As new putative tRNA mimics are being discovered, this work promises insight of broad applicability. The second are termination upstream ribosome binding sites (TURBS) RNAs, which exist at the interface between two open reading frames where they drive ribosome reinitiation using unknown RNA structure. Evidence suggests that RNA sequence and structure can play an important role in ribosome reinitiation, thus studies of these viral TURBS promise to reveal foundational principles of this process. For these studies, we propose a ‘structure-forward’ strategy that exploits our substantial expertise in three-dimensional RNA structure determination, coupled with biochemistry to link structure with function and develop detailed new mechanistic models. In addition, we will use our structural discoveries to guide novel bioinformatics searches aimed at finding new versions of these structured elements in both cellular and viral RNAs. We anticipate that our work will inspire new questions about the nature, diversity, distribution, and power of RNA structure-dependent control of translation, suggest new systems to study, and help further understand broad underlying principles.