Germline mutations in the NF2 gene predispose to tumors of multiple types as well as ocular features such as cataracts and retinal hamartomas. Although NF2 germline mutations are relatively rare, the NF2 gene is the single most commonly mutated gene in sporadic benign brain tumors. Other than its function as a recessive tumor suppressor based on genetic analyses and sequence homology to ERM proteins, little is known about the function of the NF2 gene product, called merlin or schwannomin. They have identified seven schwannomin/merlin binding proteins (SBkPs) using the yeast two-hybrid system. In addition to BII-spectrin or fodrin, six novel proteins have been identified.
Three specific aims are proposed to evaluate these proteins: 1) The interaction of schwannomin and SBPs will be verified using in vitro binding assays and in vivo co-immunoprecipitation. Interacting domains will be determined by testing partial fusions of schwannomin and SBPs using filter assays for B-galactosidase and in vitro binding assays. 2) Full length cDNAs for SBkPs will obtained and mapped to human chromosome regions using FISH and radiation hybrid mapping. tissue and subcellular distribution of SBPs by Northern and Western blot analyses as well as immunocytochemistry will be analyzed. Subcellular co-localization will be determined by confocal microscopy. 3) The functional importance of SBPs will be determined by analyzing decrease of binding to schwannomins containing naturally occurring missense mutations. After transfection of SBP cDNAs into cell lines and human schwannoma cells, changes of cell morphology, proliferation and schwannomin intracellular localization will be observed. Based on nucleolar localization of schwannomin in STS26T Schwann cells, its effect on RNA polymerase I transcription will be assayed in vitro using nuclear extracts, purified transcription factors and recombinant proteins.
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