Molecular functions of the double-sided and inverted ubiquitin-interacting motif found in Xenopus tropicalis cryptochrome 6.

Cryptochromes (CRYs) are multifunctional molecules that act as a circadian clock oscillating factor, a blue-light sensor, and a light-driven magnetoreceptor. Cry genes are classified into several groups based on the evolutionary relationships. Cryptochrome 6 gene (Cry6) is present in invertebrates and lower vertebrates such as amphibians and fishes. Here we identified a Cry6 ortholog in Xenopus tropicalis (XtCry6). XtCRY6 retains a conserved long N-terminal extension (termed CRY N-terminal extension; CNE) that is not found in any CRY in the other groups. A structural prediction suggested that CNE contained unique structures; a tetrahelical fold structure topologically related to KaiA/RbsU domain, overlapping nuclear- and nucleolar-localizing signals (NLS/NoLS), and a novel motif (termed DI-UIM) overlapping a double-sided ubiquitin-interacting motif (DUIM) and an inverted ubiquitin-interacting motif (IUIM). Potential activities of the NLS/NoLS and DI-UIM were examined to infer the molecular function of XtCRY6. GFP-NLS/NoLS fusion protein exogenously expressed in HEK293 cells was mostly observed in the nucleolus, while GFP-XtCRY6 was observed in the cytoplasm. A glutathione S-transferase (GST) pull-down assay suggested that the DI-UIM physically interacts with polyubiquitin. Consistently, protein docking simulations implied that XtCRY6 DI-UIM binds two ubiquitin molecules in a relationship of a twofold rotational symmetry with the symmetry axis parallel or perpendicular to the DI-UIM helix. These results strongly suggested that XtCRY6 does not function as a circadian transcriptional repressor and that it might have another function such as photoreceptive molecule regulating light-dependent protein degradation or gene expression through a CNE-mediated interaction with ubiquitinated proteins in the cytoplasm and/or nucleolus.

Midnight/midday-synchronized expression of cryptochrome genes in the eyes of three teleost species, zebrafish, goldfish, and medaka.

Photoperiodic responses are observed in many organisms living in the temperate zones. The circadian clock is involved in photoperiodic time measurement; however, the underlying molecular mechanism for detection of the day length remains unknown. We previously compared the expression profiles of the Cryptochrome(Cry) genes in the zebrafish eye and reported that Cry1ab has a double peak with variable expression duration depending on the photoperiod. In this study, to understand commonalities and differences in the photoperiodic responses of ocular Cry genes, we identified Cryptochrome genes in two other teleost species, goldfish and medaka, living in temperate zones, and measured ocular Cry mRNA levels in all of the three species, under different photoperiods (long-day [14 h light: 10 h dark] and short-day [10 h light: 14 h dark] and in constant darkness. Cry1ab mRNA levels did not show dual peaks in goldfish or medaka under the examined conditions; however, the mRNA expression profiles of many Crys were altered in all three species, depending on the day length and light condition. Based on their expression profiles, Cry mRNA peaks were classified into three groups that better synchronize to sunrise (light-on), midnight/midday (middle points of the dark/light periods), or sunset (light-off). These results suggest the presence of multiple oscillators that oscillate independently or a complex oscillator in which Cry expression cycles change in a photoperiod-dependent manner in the eye.