A novel bipartite intronic splicing enhancer promotes the inclusion of a mini-exon in the AMP deaminase 1 gene

Alternative splicing of the 12-base exon 2 of the adenosine monophosphate d eaminase (AMPD) gene is subject to regulation by both cis- and trans-regula tory signals. The extent of exon 2 inclusion is stage- and cell type-specif ic and is subject to the physiological state of the cell. In adult skeletal muscle, a cell type that regulates the activity of this allosteric enzyme at several levels, the exon S-plus form of AMPD, predominates. We have perf ormed a systematic analysis of the cis-acting regulatory sequences that res ide in the intron immediately downstream of this mini-exon. A complex eleme nt comprising sequences that enhance exon 2 inclusion and sequences that co unteract this effect resides in the middle of this intron. We demonstrate t hat the enhancing component is bipartite, with more than a kilobase of sequ ence separating the two functional sites. The presence of even minimal leve ls the mini-exon in the fully processed AMPD mRNA requires both of these si tes, neither of which appears in any other published splicing enhancer. An RNA binding activity derived from a muscle cell line requires both of the e nhancing sites. Mutations in either of the sites that eliminate exon 2 incl usion abrogate this binding activity.