NUCLEAR SPECKLE LOCALIZATION OF RNA BINDING MOTIF PROTEIN 5: AN IMMEDIATE EARLY GENE UP-REGULATED BY VASCULAR ENDOTHELIAL GROWTH FACTOR.

Abstract

Angiogenesis is a fundamental process playing an important role in various pathological and physiological conditions including organ development and tumor metastasis. Blood vessels are primarily composed of endothelial cells. These cells are mostly quiescent in adults, but can be stimulated by an excess of pro-angiogenic factors. One specific pro-angiogenic factor, VEGF-A (specifically VEGF165) plays a key role in a variety of pathological and physiological conditions. However, the molecular and cellular mechanisms by which VEGF-A regulates the complex fundamental process of blood vessel formation is unknown. This study was undertaken to identify the immediate early genes (IEGs) induced by VEGF-A, a hitherto uncharted research avenue. Differential screening of a suppression subtractive hybridization cDNA library potentially enriched in the IEGs induced by VEGF-A, led to the identification of 17 IEG transcripts. Surprisingly, this new putative VEGF-A response gene set consisted of transcripts with diverse functions, including those of various transcription factors such as ATF3, EGR2 and IER2, growth factors, enzymes and novel genes. RNA Binding Motif protein 5 (RBM5) was the only putative tumor suppressor gene and differential apoptotic regulator identified in this new putative VEGF-A response gene set. This dissertation further focuses on the then novel multi-modular protein RBM5, whose up-regulation by VEGF-A seemed paradoxical. Correct localization of any protein is not only essential for its function but can also provide vital clues about its potential cellular role. Although, RBM5 was previously shown to be localized in the nucleus, its subnuclear localization was not defined. Localization of RBM5 in nuclear speckles was determined using enhanced green fluorescent protein (EGFP) fusion constructs and colocalization studies. Based on these results it can be hypothesized that RBM5 may function in pre-mRNA metabolism. Additionally, site-directed mutagenesis and colocalization studies confirmed that nuclear localization of RBM5 is mediated by only the C-terminal bipartite NLS, absent in all RBM5 splice variants.