GABAergic Neuron Differentiation in C. elegans

描述(由申请人提供)：本次更新申请的长期目标是确定保守的神经元DLK-1激酶途径中基于RNA的调节机制。通过对线虫进行遗传学研究，我的实验室发现了几条潜在的突触形成的保守路径。在当前阶段，我们主要关注MAPKKK DLK-1(双亮氨酸拉链承载激酶)。我们发现DLK-1激酶级联的一个关键下游靶点是CEBP-1，它是CCAAT/增强子结合蛋白家族中的一个bZip蛋白。DLK-1的激活可以稳定CEBP-1mRNA，促进CEBP-1的轴突合成，两者都需要CEBP-1mRNA的3‘非翻译区(3’UTR)。在平行的工作中，我们证明了新的核蛋白SydN-1在突触形成中显示出特定的作用，并对前mRNA核聚腺苷酸化(NPolyA)进行负性调节。我们最近发现了DLK-1蛋白的两个异构体之间的异构体结合控制其活性的机制。抑制性DLK-1S异构体是利用内部多聚腺苷酸化位点(PAS)产生的。我们的初步数据表明，DLK-1s mRNA的PAS选择受SydN-1途径的调控。在目标1中，我们将识别DLK-1S mRNA中的顺式调节元件，并确定它们与反式中的Sydn-1/NPolyA成分的相互作用。我们还将鉴定其他神经元转录本，这些转录本正在经历由SydN-1/NPolyA调控的选择性多聚腺苷基化。在目标2中，我们将重点研究CEBP-1mRNA调控的机制。我们将研究局部和身体合成的CEBP-1的功能输出。我们提出的实验结合了多种方法来解决广泛研究领域感兴趣的基本问题。越来越多的研究支持DLK激酶在神经元发育和轴突损伤反应中的重要性。许多人类疾病，包括智力低下和肌肉营养不良，都是由各种RNA结合蛋白的基因突变引起的。我们的发现将为理解维持神经系统完整性的基本机制以及理解疾病管理提供重要的见解。

DESCRIPTION (provided by applicant): The long-term goal of this renewal application is to define the mechanisms of RNA based regulation in the conserved neuronal DLK-1 kinase pathway. Taking genetic approaches in C. elegans, my laboratory has uncovered several conserved pathways underlying synapse formation. In the current period we have focused on the MAPKKK DLK-1 (Dual-Leucine-zipper bearing kinase). We discovered that a key downstream target of the DLK-1 kinase cascade is CEBP-1, a bZip protein of the CCAAT/Enhancer binding protein family. Activation of DLK-1 stabilizes the cebp-1 mRNA and can promote axonal synthesis of CEBP-1, both of which require the 3' untranslated region (3' UTR) of cebp-1 mRNA. In a parallel line of work, we showed that the novel nuclear protein SYDN-1 exhibits specific effects in synapse formation and acts to negatively regulate pre-mRNA nuclear polyadenylation (NpolyA). We have recently identified a mechanism in which heteromeric binding between two isoforms of DLK-1 protein controls its activity. The inhibitory DLK-1S isoform is produced using an internal polyadenylation site (PAS). Our preliminary data show that the PAS choice of dlk-1S mRNA is regulated by the SYDN-1 pathway. In Aim 1 we will identify cis-regulatory elements in the dlk- 1S mRNA and determine their interactions with the SYDN-1/NpolyA components in trans. We will also identify other neuronal transcripts undergoing alternative polyadenylation regulated by SYDN-1/NpolyA. In Aim 2 we will focus on the mechanism underlying cebp-1 mRNA regulation. We will investigate the functional outputs of locally and somatically synthesized CEBP-1. Our proposed experiments combine multiple approaches to tackle fundamental questions of central interest to broad research fields. Increasing studies have supported the importance of the DLK kinases in neuronal development and axon injury responses. Numerous human diseases, including mental retardation and muscular dystrophy, are caused by genetic mutations in diverse RNA binding proteins. Our findings will provide significant insights both to the understanding of the basic mechanisms maintaining the integrity of the nervous system and also to the understanding of disease management.