The rodent alpha A-crystallin gene: mutagenesis of a non-consensus 5 '-splice site to study alternative splicing in vivo

alpha A-Crystallin is a member of the small heat shock protein family that is abundantly expressed as a structural component in the vertebrate eye len s. In lenses of rodents and some other mammals, there occurs a minor varian t of alpha A-crystallin, which has an insertion of 23 amino acid residues. This variant, alpha A(ins)-crystallin, results from differential integratio n of an optional exon into a small fraction of the mRNA. We have studied wh ether this alternative splicing is caused by a non-consensus cytosine in th e 5' splice site adjacent to the optional exon. After replacement of the ab errant cytosine in the hamster alpha A-crystallin gene by a consensus thymi ne, and transient transfection of this gene in Chinese Hamster Ovary cells, the optional exon is indeed almost completely spliced into the mature mRNA . In contrast, replacement of the cytosine by adenine or guanine completely abolishes the splicing of the optional exon. Our results confirm that alte rnative splicing of the alpha A-crystallin primary transcript is mainly due to a non-consensus 5' splice site nucleotide. However, we conclude that th e small size of the optional exon is probably an additional contributing fa ctor and therefore it seems that the splicing mechanism is based on recogni tion of exons rather than introns.