DESCRIPTION (provided by applicant): This Maximizing Investigators' Research Award will be focused on the understanding of mechanisms of how fundamental biological phenomena: circadian clock and RNA interference. Circadian clocks control a wide variety of fundamental cellular, physiological, and behavioral processes in eukaryotic organisms. The molecular machinery that permits the measurement of time is referred to as the "circadian clock" and its output as circadian rhythms. Our long-term goal is to understand the molecular and biochemical mechanisms of eukaryotic circadian clocks. RNA interference (RNAi) is a post-transcriptional/transcriptional gene silencing mechanism conserved from fungi to humans. In RNAi pathways, small non-coding RNAs (sRNAs) with sizes ranging from 20-30 nucleotides (nt), including microRNAs (miRNAs) and various small interfering RNAs (siRNAs), associate with and guide Argonaute family proteins to messenger RNA targets, resulting in the silencing of gene expression in diverse biological processes. The filamentous fungus Neurospora crassa offers a powerful experimentally-accessible system for both circadian clock and RNAi mechanisms. Our previous studies have made fundamental contributions to both circadian and RNAi fields. For our future circadian clock research, we propose to focus on three different key aspects of the circadian oscillator mechanism. In Specific Aim 1, we will determine how phosphorylation of FRQ regulates its activity and its structural conformation. This study will help establish a biochemical mechanism critical for the circadian negative feedback process in Neurospora. In Specific Aim 2, we will determine the mechanism for how CATP regulates frq transcription by regulating the chromatin structure. In Specific Aim 3, we will determine the mechanism for how antisense transcription of qrf transcription regulates frq expression by transcriptional interference. Together, these objectives take advantage of a well-established model system to address three fundamental questions that are critical for our understanding of eukaryotic circadian clocks and will elucidate the genetic, biochemical, and molecular mechanism of the Neurospora clock. Because of the conservation between the Neurospora and animal clocks, our results will provide important insights into eukaryotic clock mechanisms. For our future RNAi and small RNA research, we will focus on the mechanisms of quelling, milRNA and dicer-independent small RNA pathways. In Specific Aim 4, we determine the mechanism of how DNA damage induces of qiRNA production and how qiRNAs promote homologous recombination. In Specific Aim 5, we will determine the milRNA production pathways and decipher the design principles of milRNAs. In Specific Aim 6, we will determine the biogenesis pathway and function of disiRNAs. Together, these studies address several fundamental questions in small RNA biogenesis and will significantly expand our current knowledge of sRNA biogenesis pathways and sRNA function.

PUBLIC HEALTH RELEVANCE Circadian rhythms and RNAi are both known to regulate numerous biochemical, cellular, developmental, and behavioral activities observed in most organisms. The importance of biological clocks in human physiology and mental health is evident from their ubiquitous influence on a wide range of cellular and organismal processes. The malfunction of the clock is known to be associated with several forms of human psychiatric illness and with sleep disorders. On the other hand, in addition to its wide use as a tool for study of gene function and gene identification, RNAi technologies have been extensively used in pharmaceutical target validation and in therapeutic development. A better understanding of these two fundamental mechanisms will potentially lead to new therapeutic approaches for treating human diseases.