DEVELOPMENT OF A NOVEL APPROACH TO STUDY ALTERNATIVE TRANSLATION IN THE CNS

描述（由申请人提供）：体外系统中出现的证据表明，大量mRNA含有替代翻译起始位点，进一步增加了可由单个RNA分子产生的蛋白质产物的数量和多样性。这种新的多样性可以在细胞培养中使用新开发的“核糖体足迹”方法以高通量和系统的方式进行研究。“在这里，我们建议将这些新方法引入神经系统的研究中，使用我们的工具和专业知识从小鼠大脑中遗传定义的细胞群中捕获核糖体。因此，目标1是确定替代翻译是否是复杂的哺乳动物大脑的共同特征，以及它如何在不同的细胞类型中受到调节。此外，在酵母和其他体外系统中，替代翻译受细胞应激和其他操作的调节。我们的第二个目标是确定神经元活性是否可以调节体内的替代翻译，使用新开发的双报告基因“CHOP-TRAP”小鼠，其允许从靶向细胞群体（在这种情况下是中脑多巴胺能神经元）进行平行光遗传学操作和核糖体捕获。我们希望，开发工具来表征与奖励和成瘾相关的细胞群体中的替代翻译，将允许未来深入分析CNS疾病中的这一过程。

DESCRIPTION (provided by applicant): There is emerging evidence from in vitro systems that a remarkable number of mRNAs contain alternative translation initiation sites, further amplifying the number and diversity of protein products that can be generated from a single RNA molecule. This new diversity can be studied in a high throughput and systematic manner in cell culture using newly developed methods for "ribosome footprinting." Here, we propose to bring these new methods into the study of the nervous system using our tools and expertise in capturing ribosomes from genetically defined cell populations in the mouse brain. Thus, Aim 1 is to determine if alternative translation is a common feature of the complex mammalian brain, and how it is regulated across distinct cell types. Furthermore, in yeast and other in vitro systems, alternative translation is regulated by cellular stress and other manipulations. Our second aim is to determine whether neuronal activity can regulate alternative translation in vivo, using a newly developed dual-reporter "CHOP-TRAP" mouse that permits parallel optogenetic manipulation and ribosome capture from targeted cell populations, in this case midbrain Dopaminergic neurons. It is our hope that the development of tools to characterize alternative translation in cel populations relevant to reward and addiction will permit future in-depth analysis of this process in CNS disorder.