Alternative splicing appears as a widespread means for producing polypeptide diversity from a single gene (Black 2003). In human fibronectin (FN), for example, up to 20 different polypeptide variants arise from alternative splicing in three regions of a single gene (Gutman and Kornblihtt 1987). However, this figure remains modest when compared with that of the Drosophila dscam gene, where an extremely complex array of alternative exons could potentially give rise to 38,016 DSCAM proteins (Schmucker et al. 2000). In spite of the estimation that 60% of human genes are expressed through alternative splicing and the sophisticated functional, cell-type, and developmental specificities documented in many cases, the mechanisms of alternative splicing regulation are poorly understood. A key role in splice site choice regulation is played by members of the SR (Ser/Argrich) family of proteins. These proteins participate both in constitutive and alternative splicing. By binding to splicing enhancers they can stimulate or repress spliceosome assembly at adjacent splice sites. It is conceivable that alternative splicing in different cell types or different points in time is regulated by variation in the relative abundance of SR proteins. However, although relative proportions of SR proteins and their antagonistic splicing factors (namely, heterogeneous nuclear ribonucleoprotein) vary naturally in several rat tissues and cell lines in culture (Hanamura et al. 1998), SR proteins do not seem to have a highly specific tissue distribution, which suggests the existence of more complex regulatory mechanisms.
The demonstration that differences in promoter structure lead to differences in alternative splicing of the transcript (Cramer et al. 1997) supports the concept that splicing and transcription are coupled and that this coupling may offer an additional level of regulation of alternative splicing. The system analyzed in our laboratory involved transient transfection of mammalian cells with minigenes carrying the EDI exon, which encodes a facultative repeat of FN. EDI contains an exonic splicing enhancer (ESE), which is targeted by the SR proteins SF2/ASF and 9G8. Overexpression of SF2/ASF and 9G8 markedly stimulates EDI inclusion, but the effect of these proteins is modulated by the promoter (Cramer et al. 1999). These effects are not the trivial consequence of different mRNA levels produced by each promoter (promoter strength) but depend on some qualitative properties conferred by promoters to the transcription/RNA processing machinery. The promoter effect is also observed in cell lines stably transfected with the same minigenes used as episomal templates, indicating that a physiological chromatin assembly of the integrated minigenes is compatible with the promoter mechanism (Kadener et al. 2001).
The promoter effect is not restricted to the FN EDI exon. Similar effects have been found independently in other genes. Reporter minigenes whose products are subject to alternative splicing decisions in the CD44 and the calcitonin gene related product (CGRP) genes were put under the control of steroid-sensitive promoters (mouse mammary tumor virus and synthetic promoters containing either the progesterone or the estrogen response elements) or promoters that do not respond to steroid hormones (CMV and thymidine kinase). Steroid hormones affected splice site selection only of pre-mRNAs produced by the first type of promoters. As in the case of FN EDI, promoter-dependent hormonal effects on splicing were not a consequence of an increase in transcription rate or of a saturation of the splicing machinery (Auboeuf et al. 2002). Promoter-dependent alternative splicing patterns have been also found when reporter minigenes for the cystic fibrosis transmembrane regulator (CFTR) exon 9 (Pagani et al. 2003) or for the fibroblast growth factor receptor 2 (Robson-Dixon and García-Blanco 2004) were expressed in mammalian cells.