ROLE OF THE NUCLEAR MATRIX PROTEIN NRB/B IN BRAIN

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DESCRIPTION(Verbatim from Applicant's Abstract): The nuclear matrix is defined as the insoluble framework of the nucleus and has been implicated in modulating gene expression, the cell cycle and nuclear structural integrity via linkage to intermediate filaments of the cytoskeleton. We have discovered and characterized a novel neuronal nuclear matrix protein, NRP/B (nuclear restricted protein/brain), which contains two major structural elements: a BTB domain-like structure in the predicted N-terminus, and a "kelch motif" in the predicted C-terminal domain. NRP/B mRNA (5.5 kb) is predominantly expressed in human fetal and adult brain. During mouse embryogenesis, NRP/B mRNA expression is upregulated in the nervous system. The NRP/B protein is also expressed in rat primary hippocampal neurons, but not in primary astrocytes. During the differentiation of murine Neuro 2A and human SH-SY5Y neuroblastoma cells, NRP/B protein expression is upregulated. Overexpression of NRP/B in these cells significantly induces growth arrest and augments neuronal process formation, while treatment with antisense NRP/B oligodeoxynucleotides inhibits neurite development of rat primary hippocampal neurons as well as neuronal process formation during differentiation of SH-SY5Y and Neuro 2A neuroblastoma cells. In addition, we have observed that microinjection of anti-NRP/B antibodies inhibited neurite outgrowth of PC12 cells in response to NGF stimulation, while microinjection of NRP/B cDNA promoted the neurite outgrowth of these cells. Both in vivo and in vitro experiments demonstrate that NRP/B can be phosphorylated and bind to the functionally active hypophosphorylated form of p110RB during neuronal differentiation of SH-SY5Y neuroblastoma cells induced by retinoic acid. The interaction of NRP/B with p110RB is mediated via the BTB/POZ domain of NRP/B. Furthermore, direct association of NRP/B with nuclear actin was observed, and this association is mediated via the "kelch" repeats. These data demonstrate that NRP/B is a novel nuclear matrix protein, specifically expressed in primary neurons, that interacts with p110RB and participates in the regulation of neuronal process formation. We aim to further characterize this novel neuronal nuclear matrix protein with regard to its role in regulatory pathways that control neuronal differentiation. We propose to specifically address: (1) the biological function(s) of the NRP/B protein; (2) the DNA binding properties of NRP/B and its structure/function relationships; and (3) the characterization of NRP/B protein during neuronal growth and differentiation. These studies should provide novel insights into the role of this nuclear matrix protein in neuronal differentiation.