Abstract

Alternative splicing of the primary RNA transcript is a common mechanism for generating protein diversity. A model system was developed to study this process in vitro that is useful for evaluation of splicing of transcripts expressed in cells that do not grow well in culture. The system was used to analyze skipping of exon 4 of the amelogenin message, normally expressed in ameloblast cells for a short interval during tooth enamel development. Amelogenins are highly conserved proteins resulting from extensive alternative splicing, with domains involved in a range of functions, including mineral formation and intercellular signaling. In the bovine gene, the very short intron 4 was predicted to inhibit inclusion of exon 4, because in murine ameloblasts, exon 4 is detectably included in mRNA, and intron 4 is longer than the bovine counterpart. Bovine intron 4 was lengthened, and this size increase enhanced exon 4 inclusion sixfold to eightfold, although splice site selection was inaccurate. Intron length, therefore, is not the sole determinant controlling amelogenin exon 4 inclusion, and cis-acting inhibitory elements may also be involved in exon skipping. This vector system allows evaluation of splicing of a tissue-specific RNA by focusing on exons of interest through transfection of heterologous cultured cells without complications attributable to background transcription of the gene being evaluated.