The nuclear transportation of CHRONO regulates the circadian rhythm.

The pace of the endogenous circadian clock is important for organisms to maintain homeostasis. CHRONO has been shown to be a core component of the mammalian clock and has recently been implicated to function in several important physiological aspects. To function properly, CHRONO needs to enter the nucleus to repress transcription. We have previously shown that the N-terminus of CHRONO is required for its nuclear entry. However, how CHRONO enters the nucleus and regulates the circadian clock remains unknown. Here, we report that a novel non-classical nuclear localization signal (NLS) in the N-terminus of CHRONO is responsible for its nuclear entry. Multiple nuclear transporters are identified that facilitate the nuclear import of CHRONO. We show that the Arg63 is the critical amino acid of the NLS. Using prime editing technology, we precisely edit the Arg63 to Ala at the genomic loci and demonstrate that this mutation prolongs the circadian period, which is similar to knockdown of CHRONO. By using the CHRONO knockout and R63A mutant cells, we also investigated the changes in the cytoplasmic/nuclear distribution of BMAL1. We show that BMAL1 localizes more in the cytoplasm in the deficiency of CHRONO nuclear entry. These results provide a model for CHRONO nuclear entry using a network of importins involved in the regulation of the circadian period.