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Abstract

The circadian clock of the filamentous fungus Neurospora crassa (N. crassa) is based on a negative transcriptional-translational feedback loop (TTFL), in which the transcriptional activator White Collar Complex (WCC) activates expression of frequency (frq), the central negative regulator of the clock. FRQ, together with FRQ-interacting RNA helicase (FRH) and casein kinase 1a (CK1a), forms the FRQ-FRH-CK1a complex (FFC), which inhibits the transcriptional activator of FRQ, WCC, by phosphorylation, thus completing the transcription-translation feedback loop. CK1a, which is anchored to FRQ, hyperphosphorylates FRQ at its intrinsically disordered regions (IDRs) in a slow, temperature-independent manner, forming a module suitable for molecular time measurement. However, the molecular processes triggered by the hyperphosphorylation of FRQ and the way in which time is measured are not yet fully understood. The present work aims at understanding this mechanism in more detail and therefore focuses on the role of phosphorylation in the remodeling of the FRQ-FRH-CK1a complex. For this purpose, fluorescently tagged versions of the central clock proteins of N. crassa were heterologously overexpressed in cultured mammalian cells and their interaction and subcellular dynamics were investigated by live cell imaging. A major result of this work is the discovery of a new and unexpected function of FRH within the framework of the circadian clock. FRH like its Saccharomyces cerevisiae homolog, Mtr4p, has an essential role as a helicase in the exosome complex in RNA metabolism. In the circadian clock, FRH protects its interaction partner FRQ from premature degradation. In this work, I uncovered a new role of FRH. My data show that FRH decodes the phosphorylation state of FRQ, triggering a two-step remodeling of the FFC complex. The FRQ dimer binds initially two FRH molecules. This hetero-tetrameric complex is inactive because binding of FRH blocks the interaction of FRQ with WCC. Slow phosphorylation of FRQ leads with a delay to the dissociation of one bound FRH molecule, resulting in activation of FRQ by exposure of a WCC binding. The complex interacts with WCC and supports its phosphorylation and inactivation by FRQ-bound CK1a. Further phosphorylation of FRQ leads eventually to the release of the second FRH. The FRQ dimer is then rapidly exported from the nucleus and degraded in the cytosol. These mechanisms ensure precise activation and inactivation of FRQ and emphasize the central role of FRH as a decoder of time-dependent signals in the circadian rhythm of Neurospora.