Control of hnRNP A1 alternative splicing: an intron element represses use of the common 3 ' splice site

Alternative splicing of exon 7B in the hnRNP A1 pre-mRNA produces mRNAs enc oding two proteins: hnRNP A1 and the less abundant A1B. We have reported th e identification of several intron elements that contribute to exon 7B skip ping. In this study, we report the activity of a novel element, conserved e lement 9 (CE9), located in the intron downstream of exon 7B. We show that m ultiple copies of CE9 inhibit exon 7B-exon 8 splicing in vitro. When CE9 is inserted between two competing 3' splice sites, a single copy of CE9 decre ases splicing to the distal 3' splice site. Our in vivo results also suppor t the conclusion that CE9 is a splicing modulator. First, inserting multipl e copies of CE9 into an A1 minigene compromises the production of fully spl iced products. Second, one copy of CE9 stimulates the inclusion of a short internal exon in a derivative of the human beta-globin gene. In this case, in vitro splicing assays suggest that CE9 decreases splicing of intron 1, a n event that improves splicing of intron 2 and decreases skipping of the sh ort internal exon. The ability of CE9 to act on heterologous substrates, co mbined with the results of a competition assay, suggest that the activity o f CE9 is mediated by a trans-acting factor. Our results indicate that CE9 r epresses the use of the common 3' splice site in the hnRNP A1 alternative s plicing unit.