A central question in cell biology is how gene expression is spatially and temporally regulated in response to stimuli. Neurons are particularly mystifying due to their complex morphology, wherein dendrites and axons that comprise most of the cell volume extend great distances (>10 mm in length) from the cell body. Paradoxically, neurons must respond in a fast and selective manner to accurately transmit synaptic signals across these distances to neighboring cells. In vivo genetic studies have demonstrated a clear requirement for newly synthesized proteins to drive long-term potentiation and depression, synaptic plasticity, and memory formation Indeed, all the components necessary for translation including mRNAs, ribosomes, and initiation factors, are localized within axons and dendrites. This raises the question: how are specific subsets of mRNAs translationally regulated in a selective, fast, and spatially localized manner to propagate distinct signals within neurons? Intriguingly, trans-acting factors known as ribosome-associated proteins (RAPs) have emerged as critical players in regulating translational specificity and subcellular localization that can rapidly fine-tune translation in response to extracellular signals. However, we lack the technologies to be able to precisely isolate and analyze the translational machinery at discrete locations within neurons. In this grant, we will apply new technologies to mark and characterize ribosomes in distinct subdomains of neurons for the first time. We will also directly delineate how RAP binding to the ribosome endows greater specificity in translational control to reflect unique cellular needs and diversity in subcellular space in neurons. In Aim1 we will develop a new technology known as ALIBi (AviTag-specific Location-restricted Inducible Biotinylation), which enables proximity-dependent biotin labeling for the isolation of ribosomes in a spatiotemporally targeted manner. With this technology we will be able to identify RAPs and study localized translation at an unprecedented subcellular resolution in a tunable and highly specific fashion. In Aim2 we will characterize a novel RAP that encodes an ATP-dependent helicase that is present on neuronal ribosomes. Neurons translate some of the longest transcripts in the body containing highly structured 5’UTRs that may require helicase activity for their translation. Here, we will address the outstanding question of whether RAP binding to neural ribosomes endows greater specificity to translational control. Together, this work will uncover the functional consequences of RAP-ribosome interactions with respect to localized translation and neural development utilizing new technologies that for the first time enable us to directly probe neural ribosomes and their functions in localized translational control.