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

描述(由申请人提供)：来自体外系统的新证据表明，相当数量的mRNAs包含替代的翻译起始点，进一步放大了可以从单个RNA分子产生的蛋白质产品的数量和多样性。使用新开发的“核糖体足迹”方法，这种新的多样性可以在细胞培养中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应用到神经系统的研究中，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体中捕获核糖体。因此，目标1是确定替代翻译是否是复杂哺乳动物大脑的共同特征，以及它是如何在不同的细胞类型中受到调节的。此外，在酵母和其他体外系统中，替代翻译受到细胞压力和其他操作的调节。我们的第二个目标是确定神经元活动是否可以调节体内的替代翻译，使用一种新开发的双报告“CHOP-Trap”小鼠，它允许并行的光遗传操作和从目标细胞群中捕获核糖体，在这种情况下，中脑多巴胺能神经元。我们希望，开发工具来描述与奖赏和成瘾相关的CEL人群中的替代翻译将使我们能够在未来深入分析中枢神经系统障碍的这一过程。

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.