Project Summary Regulating mRNA in space and time is an ancient, conserved operation cells perform to precisely coordinate the flow of genetic information available for translation. RNA transcription, localization and local translation is implicated in neuronal outgrowth, plasticity, and repair, and dysregulation of these RNA processes leads to diverse neurological disorders. Mutations in several RNA binding proteins are associated with neurodevelopmental diseases and axon defects, yet the role of mRNA regulation in synaptogenesis remains unclear. Additionally, most neuronal RNA localization studies focus on postsynaptic compartments, and very few studies have been conducted in vivo in the context of intact physiology. This project takes advantage of the genetic accessibility and transparency of the nematode C. elegans to investigate mRNA regulation as synaptogenesis occurs in the living animal, focusing on the conserved essential presynaptic active zone gene syd-2/liprin-alpha. This will be accomplished using a combination of high-resolution imaging and mass spectrometry, which will characterize the dynamics of syd-2 mRNA and identify novel candidates of mRNA regulation at the transcriptional and trafficking level. Proof-of-principle preliminary data suggests that live imaging of endogenous RNA transcription and mRNA granule movement can be performed in neurons of the intact worm using the MS2 binding site/MS2 coat protein system to label endogenous mRNA with fluorescent proteins. Ongoing work will pair this system with an active zone marker, an orthologous high-resolution protein imaging system, and a proximity labeling enzyme to probe the relationship between mRNA regulation and synaptogenesis and uncover novel regulatory mechanisms. Studying neuronal mRNA regulation using complementary live imaging and unbiased biochemical approaches like mass spectrometry will reveal how these complex neurodevelopmental processes are coordinated and identify regulatory candidates for future therapeutic targeting. The studies proposed here (via a co-mentorship) will synergize Singer lab RNA imaging technology with Kurshan lab expertise in presynaptic assembly to further our understanding of neuronal mRNA regulation in living organisms. This proposal will facilitate the achievement of Jackson’s career goal to become an excellent physician-scientist and leader of a laboratory, harnessing the exceptional learning environment of Albert Einstein College of Medicine to foster his scientific and professional growth.