Paper Title
Alternative Splicing Patterns of Fox-2 Alternative Isoforms During Neuronal Differentiation

Abstract
P19 cells are widely used to understand the mechanism of differentiation, and the development of cells into neurons. In mammals, it has been reported that Fox proteins has three family members, which are Fox-1, Fox-2 and Fox-3. Both the Fox-1 and Fox-2 are closely related since they have an identical RNA recognition motif (RRM) domain. We analyzed Fox proteins in neuronal differentiation of P19 cells. It is known that 90% of human genes undergo alternative splicing. Fox-1 was specifically observed in the neural cell stage (Day 7) of P19 cells. We found that Fox-1 was able to regulate alternative splicing of a transcription factor, Mef2c. Mef2c gene consists of thirteen exons, including three different alternative splicings. The mutually exclusive alternative splicings occur in exons 1 and 2. The skipping type alternative splicing occurs in exon , located in the middle of transcriptional activation domain. The 3' splice site selection type of alternative splicing, the  region, is located in the last exon of Mef2c. It was suggested that neural-specific inclusion of Mef2c exon was promoted by Fox-1. Fox-2 showed similar function in promoting Mef2c exon inclusion. However, Fox-2 expression was observed in undifferentiated cells and increased slightly in the differentiation. Our result of Exon Array analysis suggested Fox-2 has one of alternative exons, which changed in neuronal differentiation. Essentially, long isoform of Fox-2, expressing in undifferentiated P19 cells, was replaced into short isoforms of Fox-2 in the differentiation. Our preliminary result suggested that short isoforms strongly affected alternative splicing in one of known targets of Fox-1, compared with long isoform of Fox-2. Taken together, Fox-1 and alternative isoforms of Fox-2 were expressed during neuronal differentiation of P19 cells, and contributed to regulate neural splicing event. Keywords� alternative splicing, Fox proteins, neuronal differentiation