Poster abstracts

Poster number 27 submitted by Melissa Hinman

A novel mouse and embryonic stem cell model for studying the biological role of neurofibromatosis type 1 exon 23a alternative splicing

Melissa N. Hinman (Department of Genetics, Case Western Reserve University), Victoria A. Barron (Department of Genetics, Case Western Reserve University), Guangbin Luo (Department of Genetics, Case Comprehensive Cancer Center, Case Western Reserve University), Hua Lou (Department of Genetics, Case Comprehensive Cancer Center, Center for RNA Molecular Biology, Case Western Reserve University)

Abstract:
Neurofibromatosis type 1 (NF1) functions in the regulation of cell proliferation and differentiation, particularly in the nervous system. Mutations in NF1 lead to a dominant disorder with variable phenotypes including benign tumors of the peripheral and optic nerves, café au lait spots, and learning disabilities. NF1 is a tumor suppressor that inactivates the oncogene Ras through its GTPase activating protein (GAP)-related domain (GRD). NF1 alternative exon 23a falls within the GRD, and its inclusion decreases Ras-GAP activity. Exon 23a is mainly included in most tissues but skipped in neurons. Throughout development, exon 23a inclusion is tightly regulated, with a decrease in inclusion that correlates with neuronal differentiation. We hypothesize that regulation of exon 23a inclusion is important for the biological functions of NF1.
Mice lacking exon 23a exhibit learning defects but normal development and tumor incidence. We will further investigate the biological importance of exon 23a inclusion by ablating its regulation. Using a splicing reporter system, we mutated the 5’ splice site and polypyrimidine tract of exon 23a to more closely match consensus sequences. In a variety of cell types, including primary neurons, exon 23a inclusion from the mutant splicing reporter approaches 100%, and is not affected by overexpression of negative splicing regulators from the Hu and CELF families. We targeted these mutations into the endogenous Nf1 locus of mouse embryonic stem (ES) cells, and are currently generating mutant mice. Mutant ES cells show nearly 100% exon 23a inclusion, even after neuronal differentiation. We will examine mutant ES cells for alterations in Ras GAP activity, proliferation, and neuronal differentiation, and mutant mice for phenotypes such as increased tumor incidence and defects in neuronal development. These studies will illustrate the biological significance of this evolutionarily conserved, regulated alternative splicing event.

Keywords: Alternative Splicing