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.
This project seeks to define the roles of RNA-based regulation in controlling signal transduction involving the conserved DLK-1 kinase in synapse formation and function. The proposed experiments combine genetic analysis with molecular and cellular manipulations to tackle two poorly understood topics, alternative polyadenylation and localized mRNA in neurons. The outcome will provide fundamental insights into the multifaceted control of neuronal function and identify key molecular pathways underlying neuronal dysfunction in mental retardation, brain trauma and injury.
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