Alternative splicing of intron 3 of the serine/arginine-rich protein 9G8 gene - Identification of flanking exonic splicing enhancers and involvement of 9G8 as a trans-acting factor

9G8 protein belongs to the conserved serine/arginine-rich (SR) protein fami ly, whose members exhibit multiple functions in constitutive and alternativ e splicing. We have previously shown that 9G8 primary transcripts are subje cted to alternative splicing by excision/retention of intron 3 and to a tis sue specific modulation. Because both 5'- and 3'-splice sites of intron 3 a ppear to be suboptimal in vertebrates, we tested the 9G8 intron 3 as a nove l model system of alternative splicing. By using an in vitro approach and a mutational analysis, we have identified two purine-rich exonic splicing en hancers (ESE) located in exon 4 and a (GAA)(3) enhancer located in exon 3. These elements act in concert to promote efficient splicing activation both in vitro and in vivo. Titration experiments with an excess of exonic enhan cers or SR-specific RNA targets strongly suggest that SR proteins are speci fically involved in the activation process. Although ASF/SF2 was expected t o interact the most efficiently with ESE according to the enhancer sequence s, UV cross-linking coupled or not to immunopurification demonstrates that 9G8 is highly recruited by the three ESE, followed by SC35. In contrast, AS F/SF2 only binds significantly to the (GAA)(3) motif. S100 complementation experiments with individual SR proteins demonstrate that only 9G8 is able t o fully restore splicing of intron 3. These results, and the fact that the exon 3 and 4 ESE sequences are conserved in vertebrates, strongly suggest t hat the alternative splicing of intron 3 represents an important step in th e regulation of the expression of 9G8.