PROJECT SUMMARY Local translation is a conserved molecular mechanism of concentrating proteins within distinct subcellular compartments. In morphologically distinct cells like neurons, mRNAs must travel great distances to reach their final destinations. RNA-binding proteins (RBPs) bind target mRNAs and coordinate mRNA fates including transport, anchoring, and translation. By binding target mRNAs, RBPs support a network of RNA-protein and protein-protein interactions called the messenger ribonucleoprotein complex (mRNP). While there has been significant work to investigate direct RNA-protein interactions, we do not know constituents of the mRNP that may interact with RBPs to regulate them. For this proposal, we will investigate the zipcode binding protein 1 (ZBP1) and its target mRNA, -actin, to evaluate how phosphorylation of RBPs alters the protein and RNA composition of the mRNP. We hypothesize that ZBP1 phosphorylation alters the β-actin mRNP composition, thereby causing dynamic changes in transport, anchoring, and translation. We will investigate this hypothesis using two distinct cell types: fibroblasts and neurons. First, we will test this hypothesis by combining proximity biotinylation with the MS2 RNA labeling system to capture global changes in the -actin mRNP in response ZBP1 phosphorylation in mouse embryonic fibroblasts. We will validate these findings using imaging techniques. To understand the molecular effects of -actin mRNP composition, we will follow how the transport, anchoring, and translation are affected by various ZBP1 phosphorylation mutants using a combination of single cell microscopy and molecular biology techniques. With cultured hippocampal neurons, we will evaluate whether the -actin mRNA motility and translation changes in response to introduced ZBP1 phosphorylation mutants. These studies will be important for determining how assembly of the -actin mRNP is altered by ZBP1 phosphorylation and identify the molecular consequences of this form of RBP regulation. This project will be completed under the mentorship of Dr. Robert H. Singer at Albert Einstein College of Medicine, a leading expert in RNA biology and single molecule microscopy. Under the advisement of Dr. Singer, Leti Nunez will receive a multi-disciplinary training in molecular and cellular biology combined with expertise in state-of-the-art microscopy. During the duration of this award, Leti will receive training to advance her career in academic medicine particularly relevant to the cellular basis of disease. For example, ZBP1’s role in mRNA localization has been implicated in memory and learning and tumor metastasis.