Poster abstracts
Poster number 46 submitted by Prabar Ghosh
3′UTR-mediated localization of mRNAs to the rear of migrating aortic endothelial cells
Prabar K. Ghosh (Department of Cardiovascular & Metabolic Sciences, Lerner Research Institute, Cleveland Clinic), Debjit Khan (Equal contribution, Department of Cardiovascular & Metabolic Sciences, Lerner Research Institute, Cleveland Clinic), Jennifer Taylor, Vasu Kommireddy (Department of Cardiovascular & Metabolic Sciences, Lerner Research Institute, Cleveland Clinic), Dalia Halawani (Department of Neuroscience, Icahn School of Medicine, Mount Sinai), Linda Graham (Department of Biomedical Engineering, Lerner Research Institute, Cleveland Clinic), Paul L. Fox (Department of Cardiovascular & Metabolic Sciences, Lerner Research Institute, Cleveland Clinic)
Abstract:
Endothelial cell (EC) migration is the rate determining step for wound healing in injured blood vessels as well as angiogenesis. Defects in repair contribute to vascular pathogenesis including atherosclerosis. Although the importance of asymmetric protein localization in cell migration is well known, e.g., caveolin-1 (Cav-1) accumulation in the cell rear, essentially nothing is known about polarization of mRNA to cell rear, or mechanisms directing localization. To investigate the global significance of rear-localized transcripts, we isolated forward and rear segments of planar-migrating ECs by laser-capture microdissection, and analyzed localized mRNA abundance by microarray. Remarkably, transcripts with highest rear-to-front ratios encoded proteins that, like Cav-1, are associated with cholesterol metabolism, e.g., HMG-CoA reductase (HMGCR, rate-determining enzyme of cholesterol biosynthesis). Deletion of the 3'UTR from CAV1 mRNA prevents localization of Cav-1 protein to EC-rear, indicating localization of the protein is RNA-dependent. Fusion of either HMGCR or CAV1 3′UTR is sufficient to drive reporter expression to the rear of a migrating EC. We identified the first 112 nt of HMGCR 3′UTR as a minimal element for its rear localization, while the cis-element of CAV1 is present within the first 483 nt of its 3′UTR. In a candidate approach to discover protein interactors of rear-localized 3′UTRs, we observed reporter-3′UTR mRNA association with NSAP1. NSAP1 was localized in the EC-rear and endogenous rear-localized mRNAs were shown to bind NSAP1. Its depletion markedly reduced wound closure of an EC monolayer, confirming a critical role of NSAP1 in cell migration. Colchicine, an inhibitor of microtubule (MT) polymerization, disrupted rear-localization of HMGCR and Cav-1 proteins, implicating the MT system in cell-rear transcript localization. Association of HMGCR and CAV1 mRNAs as well as NSAP1 with MT-directed kinesin-1 motor complexes was observed suggesting a NSAP1-kinesin 1 transport complex for MT-dependent rear-localization of mRNAs. These results elucidate a novel regulatory system in motile cells that establishes mRNA polarization.
Keywords: mRNA polarization, cell migration, 3UTR