A multi-species approach to find regulators of deafness genes

项目总结 小鼠的基因筛查和人类自然发生的基因变异提供了宝贵的 识别与毛细胞功能和耳聋有关的基因的资源。两个例子是识别 与亚瑟综合征有关的基因，以及Myh9在这两种疾病中的作用 症状性和非症状性耳聋。我们已经利用果蝇遗传学的力量来识别新的 与听力和耳聋有关的基因。果蝇和脊椎动物的听觉器官有许多 尽管在进化时间上相距甚远，但分子和功能上的相似性。我们确认了 Ubr3，一种E3泛素连接酶的突变，导致感觉成分的物理分离 约翰斯顿的风琴从飞行天线。令人惊讶的是，这种表型与在基因突变中看到的相同。 果蝇肌球蛋白VIIa。由于人类肌球蛋白VIIa突变会导致亚瑟综合征IB型，所以有可能 Ubr3可能调节无脊椎动物和脊椎动物的肌球蛋白VIIa功能。我们的数据表明，Ubr3 基因上与肌球蛋白VIIa和Ubr3相互作用，肌球蛋白VIIa在物理上和基因上相互作用 果蝇与另外两个亚瑟综合征蛋白PCDH15和SANS的同源物。然而，我们发现， Ubr3不修饰肌球蛋白VIIa，而是单一泛素化非肌肉肌球蛋白II。 增加两个肌球蛋白之间的相互作用，微调这种相互作用的水平似乎对 肌球蛋白VIIa的功能。 在这项建议中，我们会扩大以果蝇作为了解失聪的模式系统。 研究Ubr3、肌球蛋白II和肌球蛋白VIIa在果蝇听力中的作用(目标1)。然后我们将测试 Ubr3在小鼠毛细胞发育和功能中的作用，并将测试是否存在相互作用 肌球蛋白II和肌球蛋白VIIa在小鼠体内是保守的(目标2)。最后，我们将进行3000人的基因筛查 利用新开发的蛋白质敲除技术获得可能与人类疾病有关的果蝇基因 识别果蝇听力起作用的基因的技术(目标3)。

PROJECT SUMMARY Genetic screens in mice and the naturally occurring genetic variation in humans have provided a valuable resource to identify genes implicated in hair cell function and deafness. Two examples is the identification of genes involved in Usher syndrome, the most common form of deaf-blindness, and the role of Myh9 in both syndromic and non-syndromic deafness. We have used the power of Drosophila genetics to identify new genes involved in hearing and deafness. The auditory organs of Drosophila and vertebrates have a number of molecular and functional similarities despite being widely separated in evolutionary time. We identified mutations in Ubr3, an E3 ubiquitin ligase, that cause a physical detachment of the sensory components of Johnston's organ from the fly antenna. Strikingly, this phenotype is identical to that seen in mutations in Drosophila Myosin VIIa. Since Myosin VIIa mutations in humans cause Usher Syndrome type IB, it is possible that Ubr3 may regulate Myosin VIIa function in invertebrates and vertebrates. Our data suggest that Ubr3 genetically interacts with Myosin VIIa and Ubr3 and Myosin VIIa physically and genetically interact with Drosophila homologues of two other Usher syndrome proteins, PCDH15 and Sans. However, we have found that Ubr3 does not modify Myosin VIIa, but instead mono-ubiquitinates non-muscle Myosin II. This modification increase an interaction between the two myosins, and fine-tuning the level of this interaction appears critical for Myosin VIIa function. In the present proposal, we will expand on the use of Drosophila as a model system to understand deafness by characterizing the roles of Ubr3, Myosin II and Myosin VIIa in hearing in Drosophila (Aim 1). We will then test the function of Ubr3 in the development and function of mouse hair cells, and will test whether the interaction of Myosin II and Myosin VIIa is conserved in mice (Aim 2). Finally, we will carry out a genetic screen of 3000 Drosophila genes that may be involved in human disease using newly developed protein knockdown technology to identify genes that play a role in hearing in Drosophila (Aim 3).