Role of regulated mRNA degradation in the control of axonal mRNA localization and translation.

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 DESCRIPTION (provided by applicant): Intra-axonal protein synthesis is involved with the proper wiring of the nervous system and can have restorative or pathogenic effects in response to nerve injury and neurodegenerative stimuli. The set of axonally synthesized proteins, the axonal translatome, is regulated through the control of mRNA localization and translation. Targeting the axonal translatome could result in the development of novel therapies for the treatment of neurological disorders. Yet, the basic mechanisms regulating the specificity of axonal mRNA localization and translation need to be studied in more detail. We have found that axonal mRNAs are immediately degraded after being translated. Additionally, we have found depletion in axons of Pumilio-1/2 target mRNAs, with Pumilio-1 being expressed in axons and enriched in growth cones. This, together with the known role of Pumilio-1/2 as translational repressors and promoters of mRNA deadenylation suggests that Pumilio homologues regulate axonal mRNA localization through an exclusion mechanism. The goal of this project is to test our hypothesis that mRNA degradation is a major mechanism regulating both mRNA localization and translation of localized transcripts. For this purpose we have designed the following aims: i) to establish the role of mRNA degradation in the translational control of localized transcripts and ii) to determine the contributions of Pumilio-1/2 in the establishment of axonal mRNA localization through an exclusion mechanism. We will be using neuronal cultures in microfluidic devices for the isolation of pure axonal material, accompanied by high-throughput sequencing for an unbiased axonal mRNA quantification. We will interfere with Pumilio-1/2 function by RNAi and use microscopy for the analysis of neurodevelopmental defects associated with axon growth. The completion of this study will significantly advance our understanding of the regulation of intra-axonal protein synthesis and thus support the mission to increase understanding of biological processes that lay the foundation for advances in disease diagnosis, treatment, and prevention.