Sathyanarayanan Sriram, Zheng Xiangzhong, Kumar Shailesh, Chen Chun-Hong, Chen Dechun, Hay Bruce, Sehgal Amita
Howard Hughes Medical Institute, Department of Neuroscience, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.
Genes Dev. 2008 Jun 1;22(11):1522-33. doi: 10.1101/gad.1652308.
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 approximately 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.
生物钟调节许多不同的生理过程，并使其与环境的昼夜明暗周期同步。在果蝇中，光通过昼夜蓝光光感受器隐花色素（CRY）传递给生物钟。响应光信号时，CRY会促进生物钟蛋白无时间性（TIM）的降解，随后自身也会降解。为了鉴定参与昼夜节律调节的新基因，我们在果蝇细胞中进行了一次无偏向性的全基因组筛选，采用了一种灵敏的定量测定方法来检测光诱导的CRY降解。我们系统地敲低了约21,000个基因的表达，并鉴定出那些调节CRY稳定性的基因。这些基因包括泛素连接酶、信号转导分子和氧化还原分子。在筛选中鉴定出的许多基因对CRY降解具有特异性，并不影响TIM蛋白对光的响应性降解，这表明在很大程度上，这两条途径是不同的。我们通过检测这些基因各自的突变果蝇，进一步在体内验证了三个候选基因对CRY稳定性的影响。这项工作鉴定出了一个参与光依赖性CRY降解的新型调控网络，并证明了全基因组RNA干扰方法在理解昼夜节律生物学方面的作用。
