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神经元DLK-1激酶通路中基于RNA的调控机制。采用遗传方法对C.我的实验室已经发现了几条突触形成的保守途径。在当前阶段，我们重点关注MAPKKK DLK-1（双亮氨酸拉链激酶）。我们发现DLK-1激酶级联的关键下游靶标是CEBP-1，其是CCAAT/增强子结合蛋白家族的bZip蛋白。DLK-1的激活可稳定cebp-1 mRNA并可促进CEBP-1的轴突合成，这两者都需要cebp-1 mRNA的3'非翻译区（3' UTR）。在平行的工作中，我们发现新的核蛋白SYDN-1在突触形成中表现出特异性作用，并对前mRNA核多聚腺苷酸化（NpolyA）进行负调节。我们最近已经确定了一种机制，其中DLK-1蛋白的两种亚型之间的异聚体结合控制其活性。抑制性DLK-1 S同种型使用内部聚腺苷酸化位点（PAS）产生。我们的初步数据表明，PAS选择dlk-1 S mRNA的调节SYDN-1途径。在目的1中，我们将确定dlk-1 S mRNA中的顺式调控元件，并确定它们与SYDN-1/NpolyA组件的相互作用。我们还将确定其他神经元转录物，这些转录物经历由SYDN-1/NpolyA调控的多聚腺苷酸化。在目标2中，我们将重点关注cebp-1 mRNA调控的机制。我们将研究局部和体细胞合成的CEBP-1的功能输出。我们提出的实验结合了联合收割机多种方法来解决广泛研究领域的核心利益的基本问题。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，包括精神发育迟滞和肌肉萎缩症，是由不同的RNA结合蛋白的基因突变引起的。我们的研究结果将为理解维持神经系统完整性的基本机制和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.