br Introduction The alternative splicing of
Introduction The alternative splicing of pre-mRNA is essential for generating multiple matured mRNAs from a single gene by selectively removing introns. Neural thip show many examples of regulated alternative splicing events, which produce structural changes in proteins important for the development of synaptic connections and for communicating between cells electrically and chemically (Cáceres and Kornblihtt, 2002, Grabowski and Black, 2001). The serine/arginine rich (SR) proteins, major components of the spliceosomes, are critical for alternative splicing and are regulated in various physiological and pathological conditions including neural development (Fu, 1995, Lopez, 1998). Transformer 2 (Tra2), an SR protein, has been cloned in Drosophila and mammals. In Drosophila melanogaster, Tra2 cooperates with Transformer (Tra) to regulate sexual development (Amrein et al., 1994, Baker, 1989, Tacke and Manley, 1999, Tian and Maniatis, 1992, Tian and Maniatis, 1993, Tian and Maniatis, 1994). In mammals, two forms of Tra2 (Tra2α and Tra2β) have been identified as sequence-specific activators of pre-mRNA splicing (Beil et al., 1997, Dauwalder et al., 1996, Matsuo et al., 1995, Nayler et al., 1998, Segade et al., 1996, Tacke et al., 1998). Five isoforms of Tra2β transcripts have been identified (Nayler et al., 1998). Tra2βl, the major isoform, encodes the full-length Tra2β protein. Tra2β3, 4 and 5 theoretically encode truncated Tra2 proteins. The intracellular distributions of recombinant truncated proteins have been studied, although the native truncated proteins have not been detected in vivo (Nayler et al., 1998). It appears that Tra2βl is functionally the dominant isoform. Tra2βl is a sequence-specific pre-mRNA splicing activator in mammalians (Tacke et al., 1998) and has been indicated to play a role in neural diseases associated with the aberrant pre-mRNA splicing. For example, it is known that a short form of survival motor neuron 2 (SMN2) proteins due to the abnormal splicing of exon 7 of SMN2 is associated with spinal muscular atrophy (SMA), a common motor neuron disease (Hofmann et al., 2000). Tra2βl reportedly increases the expression of full-length SMN2 proteins by enhancing the use of exon 7 via binding to an AG-rich exonic splicing enhancer in SMN exon 7, suggesting that Tra2βl may be associated with SMA (Hofmann et al., 2000). FTDP-17 (frontotemporal dementia with Parkinsonism linked to chromosome 17), an autosomal dominant hereditary neurodegenerative disorder, is due to the abnormal exon 10 splicing of Tau pre-mRNA caused by the mutations of AG-rich splicing enhancer in the exon 10. Recent study shows that the exonic splicing enhancer is the target of Tra2βl proteins, indicating the important role of Tra2βl in FTDP-17 (Jiang et al., 2003). Besides its role in neural diseases, recent studies show that Tra2βl transcripts are developmentally regulated in mammalian neural tissues (Chen et al., 2003, Daoud et al., 1999, Nayler et al., 1998), suggesting that Tra2βl might be involved in the regulation of neural development. To study the potential function of Tra2βl in neural development, in the present study, we focused on the questions whether the neural expression of Tra2βl proteins is developmentally regulated and what is the effect of Tra2βl on neural differentiation. To investigate whether Tra2βl plays a role in regulating the splicing of the genes is important for development and neural function, we also examined the effect of Tra2βl proteins on alterative splicing of FGF-2R (fibroblast growth factor receptor 2) and GluR-B (glutamate receptor subunit B) minigenes. The FGF-2R gene is important in both neural and non-neural development (Ford et al., 2001). The exclusive splicing of two exons (K-SAM and BEK) in the third immunoglobulin-like domain of FGF-2R pre-mRNA is regulated developmentally (Ford et al., 2001, Gilbert et al., 1993, Johnson et al., 1991). GluR-B is one of four AMPA receptor subunits (GluR-A–GluR-D) and is developmentally regulated (Seeburg, 1993). The exclusive splicing of the Flop and Flip exons of GluR-B pre-mRNA regulates AMPA receptor function (Koike et al., 2000, Seeburg, 1993). Both minigene models have been well characterized and were used in the present study.