Mechanistic investigations of the role of RNA in gene regulation

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RNA molecules are involved in all steps of gene expression. This proposal describes the application of organic and biophysical chemistry to the study of a recently characterized mechanism of gene regulation involving short RNAs. The introduction of DNA, RNA, or nucleic acid mimics into cells has been intensively investigated by chemists as a means to modulate gene expression in biological systems. In 1998, Fire and colleagues reported the fascinating observation that injection of double-stranded RNA (dsRNA) into the worm. C. elegans resulted in a specific silencing of the homologous gene's expression. This phenomenon, termed RNA interference or RNAi, was subsequently shown to involve RNA degradation by short dsRNA molecules. RNAi is believed to represent a cellular response to viral dsRNA; however, it had previously also been known that short cellular RNAs were involved in regulation of development in higher organisms. Subsequent to the discovery of RNAi, several hundred "micro RNAs" (miRNAs) were cloned from worms, flies, and humans. It is now clear that a large number of miRNAs are involved in regulation of gene expression in diverse higher organisms. Recently, it has been shown that bacteria express short RNAs that confer resistance to infection by bacterial viruses or phage. The bacterial system appears to target foreign DNA for degradation as opposed to the targeting of RNA as described above. We are studying the structure and function of protein and protein-RNA complexes involved in bacterial RNA interference. Using synthetic RNA substrates, we are probing the protein-RNA interactions that occur in the interference pathway. High-resolution structural analysis will allow us to determine the bases of specific RNA recognition and the interference mechanism. These studies will enhance our understanding of catalytic mechanisms as well as protein and RNA structure and help us further elucidate the role of small RNAs in gene regulation.