Information encoded in the DNA of an animal's genome is used to generate all the proteins found in their cells. This instruction set must be "read out" by each cell in a selective way, so that cells only generate the proteins they need according to what type of cell they are going to become, how early or late along the developmental timeline it is, and where in the embryo they are. These DNA instructions direct the formation of messenger RNA templates that are used to build the proteins. This project investigates how cells in the nervous system (neurons) interpret this genetic information in a more subtle way, to make unique, longer RNAs that are not found in other non-nervous system cells. A new gene-editing technology called CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) will be used to eliminate the genetic information that causes these longer RNAs to be constructed in fruit fly neurons, to see how this affects the wiring of the developing nervous system. Experiments will discover the mechanisms by which molecules expressed in neurons control the generation of these longer RNAs. Such knowledge is vital for understanding an entire class of mechanisms underlying brain cell development that is currently very poorly understood. This project has a broader impact of training undergraduate and graduate students in neuroscience and molecular biology in Nevada, a state with a historically low percentage of adults with bachelors or advanced degrees. An outreach program called, "Editing the future", will be implemented in AP biology classes at a local high school. Students will learn about the huge potential impact of CRISPR gene-editing technology, and receive hands-on experience performing experiments on fruit flies, including using a technique called polymerase-chain reaction (PCR) to amplify and visualize DNA from CRISPR-mutated flies.
The 3´ Untranslated Region (3´ UTR) can regulate the stability, subcellular localization and translation of an mRNA. This post-transcriptional control is particularly important for neurons given their highly polarized morphology and capacity for localized translation in axons and dendrites. Most genes in higher organisms express at least two alternative 3´ UTR isoforms generated by alternative cleavage and polyadenylation (APA). In Drosophila, hundreds of genes express extended 3´ UTRs that are specific to neural tissues; however, the functional importance of this is unknown. Many genes involved in axon guidance generate extended 3´ UTR isoforms in a manner dependent on the neural-expressed RNA binding protein ELAV. This project tests the hypothesis that extended 3' UTR isoforms generated by ELAV are required for correct axon guidance. A novel approach using CRISPR genome editing to functionally remove 3´ UTR isoforms of axon guidance genes will be carried out. Characterization of neurodevelopment in these mutants will be performed. The impact of extended 3´ UTRs on RNA localization and translation will be determined. In parallel, the precise mechanisms of 3´ UTR extension of axon guidance genes by the RNA-binding protein ELAV will be elucidated. Extended 3´ UTRs are found for nine genes involved in axon guidance, and this project will focus on three - comm, Dscam1 and fasI. The project has the potential to transform our understanding of APA by establishing it as an essential component of nervous system development.
