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Identification of novel genes involved in light-dependent CRY degradation through a genome-wide RNAi screen

¹ Howard Hughes Medical Institute, Department of Neuroscience, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA; ² Division of Biology, California Institute of Technology, Pasadena, California 91125, USA

Circadian clocks regulate many different physiological processes and synchronize these to environmental light:dark cycles. In Drosophila, light is transmitted to the clock by a circadian blue light photoreceptor CRYPTOCHROME (CRY). In response to light, CRY promotes the degradation of the circadian clock protein TIMELESS (TIM) and then is itself degraded. To identify novel genes involved in circadian entrainment, we performed an unbiased genome-wide screen in Drosophila cells using a sensitive and quantitative assay that measures light-induced degradation of CRY. We systematically knocked down the expression of 21,000 genes and identified those that regulate CRY stability. These genes include ubiquitin ligases, signal transduction molecules, and redox molecules. Many of the genes identified in the screen are specific for CRY degradation and do not affect degradation of the TIM protein in response to light, suggesting that, for the most part, these two pathways are distinct. We further validated the effect of three candidate genes on CRY stability in vivo by assaying flies mutant for each of these genes. This work identifies a novel regulatory network involved in light-dependent CRY degradation and demonstrates the power of a genome-wide RNAi approach for understanding circadian biology.

[Keywords: RNAi; circadian rhythms; clock proteins; degradation; photoreceptors]

Received January 16, 2008; revised version accepted April 3, 2008.

⁴ Present address: Molecular Oncology, Merck Research Laboratories, Boston, MA 02115, USA.

⁵ Corresponding author.

E-MAIL amita{at}mail.med.upenn.edu; FAX (215) 746-0232.

Supplemental material is available at http://www.genesdev.org.

Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.1652308.

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