NUCLEAR-LOCALIZATION IS REQUIRED FOR FUNCTION OF THE ESSENTIAL CLOCK PROTEIN FRQ

The frequency (frq) gene in Neurospora encodes central components of a circadian oscillator, a negative feedback loop involving frq mRNA and two forms of FRQ protein. Here we report that FRQ is a nuclear protei n and nuclear localization is essential for its function. Deletion of the nuclear localization signal (NLS) renders FRQ unable to enter into the nucleus and abolishes overt circadian rhythmicity, while reinsert ion of the NLS at a novel site Gear the N-terminus of FRQ restores its function, Each form of FRQ enters the nucleus soon after its synthesi s in the early subjective day; there is no evidence for regulated sequ estration In the cytoplasm prior to nuclear entry, The kinetics of the nuclear entry are consistent with previous elate showing rapid depres sion of frq transcript levels following the synthesis of FRQ, and sugg est that early in each circadian cycle, when FRQ is synthesized, it en ters the nucleus and depresses the level of its own transcript.