Poster abstracts

Poster number 8 submitted by Victoria Barron

Complex control of NF1 exon 23a splicing

Victoria A. Barron (Department of Genetics, Case Western Reserve University), Hui Zhu (Department of Genomic Medicine, The Cleveland Clinic), Melissa N. Hinman (Department of Genetics, Case Western Reserve University), Andrea N. Ladd (The Cleveland Clinic), Hua Lou (Department of Genetics, Case Western Reserve University)

Abstract:
Two families of RNA binding proteins that are involved in splicing regulation are the CUG-BP and ETR-3 like factors (CELF) and the Muscleblind-like proteins (MBNL). The CELF protein family consists of six highly conserved family members and are known to bind to UG-rich RNA elements. The MBNL family has three family members and bind to the YGCU(U/G)Y motif (Y, pyrimidine) with a preference for stem-loop RNA secondary structures. Although these proteins have distinct splicing targets, MBNL and CELF proteins act as antagonists in six pre-mRNA targets. We have identified the neurofibromatosis type I (NF1) pre-mRNA as a novel target of CELF protein-mediated splicing regulation. The NF1 mRNA encodes a 2,800 amino acid protein called neurofibromin. Exon 23a, which is skipped in neuronal tissues and included in other tissue types, is especially interesting, because it falls within the GAP-related domain of the NF1 protein. The type II isoform, which contains exon 23a, is associated with low GAP activity while the type I isoform, in which exon 23a is skipped, is associated with high GAP activity. We show that over-expression of CELF proteins in HeLa cells promoted skipping of exon 23a in both a NF1 splicing reporter and the endogenous NF1. SiRNA knockdown of endogenous ETR-3 knockdown promoted inclusion of NF1 exon 23a. Over-expression of a dominant-negative CELF protein promoted inclusion of exon 23a, in CA77 cells, providing further evidence that CELF proteins function as negative regulators of NF1 exon 23a. UV-crosslinking immunoprecipitation assays have confirmed that CUG-BP1 and ETR-3 bind, strongly, to the NF1 pre-mRNA. In vitro splicing and binding analyses show that CELF proteins block splicing of NF1 exon 23a. We have identified two MBNL binding motifs in the intronic sequence upstream to NF1 exon 23a. The RNA sequence within this region is predicted to form a stem-loop structure. Over-expression of MBNL1 promoted NF1 exon 23a inclusion in CA77 cells, suggesting that they are positive regulators. Future work will focus on the regulation of this event by MBNL and how MBNL and CELF proteins function as antagonists.

Keywords: Alternative Splicing, Neurofibromatosis Type I, CELF and MBNL