Poster abstracts

Poster number 5 submitted by Alok Sharma

The role of alternative splicing of NF1 exon 23a in cardiomyocyte differentiation and function.

Alok S Sharma (Department of Genetics and Genome Sciences), Melissa Hinman (Department of Genetics and Genome Sciences), Guangbin Luo (Department of Genetics and Genome Sciences, Case Comprehensive Cancer Center), Hua Lou (Department of Genetics and Genome Sciences, Case Comprehensive Cancer Center, Center for RNA Molecular Biology)

Abstract:
Germline mutations in neurofibromatosis type 1 (NF1) result in NF1 disease, which presents with multiple symptoms including an increased risk of cardiovascular diseases. Studies using several different mouse models further established the important role of Nf1 in the cardiovascular system. Neurofibromin, the protein encoded by Nf1, functions as a Ras-GAP (GTPase-activating protein). It promotes the conversion of active Ras-GTP to inactive Ras-GDP via its GAP-related domain (GRD). One of its alternatively expressed exons, exon 23a, lies in the middle of the GRD of neurofibromin. The impact of expression of this exon on Ras-GAP activity of endogenous Nf1 is poorly understood. Our lab has generated two ES cell lines through gene targeting that express exon 23a at either 0% or 100% and demonstrated that expression of exon 23a controls the active Ras output in ES cells with low exon 23a expression correlating with high active Ras level. To investigate the role of Nf1 exon 23a expression in the cardiovascular system, the two ES cell lines were differentiated into beating cardiomyocytes. The primary beating cardiomyocytes and the wild-type ES cell derived cardiomyocytes show a similar level of Nf1 exon 23a expression at 70%. The mutated cells maintain the expected pattern of exon 23a expression throughout the process of differentiation. They also show the expected differences in active Ras-levels and phosphorylation levels of downstream targets of the Ras signaling pathway. Surprisingly, the cardiomyocytes differentiated from these cells exhibit statistically significant differences in both the number of beating embryoid bodies (EBs) and the beating frequency. The cardiomyocytes with no expression of exon 23a beat faster and have higher percentage of beating EBs than their counterparts with 100% expression of exon 23a. In addition, real time RT-PCR analysis examining the transcript levels of progenitor and differentiated cardiomyocyte markers during a time course of differentiation suggests that the cells with no expression of exon 23a differentiate faster and these cardiomyocytes show the highest expression of cardiomyocyte specific genes. These studies indicate that proper splicing of NF1 exon 23a is important for cardiomyocyte differentiation by maintaining the appropriate Ras-GTP levels.

Keywords: alternative splicing, NF1