Small noncoding RNAs are central components of the gene regulatory circuit in organisms. For proper health and development, organisms employ protein complexes containing small endogenous RNAs to control thousands of other genes through silencing mechanism. Genetics in model organisms implicate a link among small RNAs, Argonaute proteins, and processes that regulate chromatin structure, suggesting that RNA interference (RNAi) pathways can impact large domains of the genome. ? ? The candidate's search for an analogous link in mammals directed him to purify from rat testes the piRNA complex (piRC), which consists of mammalian Piwi, the Piwi-interacting RNAs, and a RecQ1 DNA helicase. Piwi-interacting RNAs (piRNAs) are a diverse class of small RNAs that are longer than microRNAs and derive from intergenic clusters of between 1-100 kb long. Piwi, a subclade of the Argonaute family, is essential for male fertility, while RecQ1 is related to a Neurospora gene with gene silencing roles. Despite these features, a rigorous understanding of piRC's molecular function is still lacking. ? ? This application seeks to dissect the molecular function of the piRNAs and piRC through these specific aims: 1) in vitro functional analysis of the piRNA complex; 2) delineating the function and regulation of piRNA cluster chromatin; and 3) understanding the genesis of piRNAs. The candidate will apply his current biochemical techniques to probe piRC's functional activity, and develop genetic and cell biology methodologies to understand the role of piRNA clusters. This application seeks support that will facilitate investigations of this new mechanism for RNAi in animals, and describes an integrative plan that will develop the career and transition of the candidate towards an independent research position at an academic institution. A deeper understanding of piRC's function will yield insight to fundamental gene and chromatin regulatory processes that impact human development and fertility. ? ? ?
