Smulders R H, Kokke B P, Gijsen M L, de Jong W W
Department of Biochemistry, University of Nijmegen, The Netherlands.
Mol Biol Rep. 1998 Nov;25(4):225-30. doi: 10.1023/a:1006897910253.
alphaA-Crystallin is a member of the small heat shock protein family that is abundantly expressed as a structural component in the vertebrate eye lens. In lenses of rodents and some other mammals, there occurs a minor variant of alphaA-crystallin, which has an insertion of 23 amino acid residues. This variant, alphaA(ins)-crystallin, results from differential integration of an optional exon into a small fraction of the mRNA. We have studied whether this alternative splicing is caused by a non-consensus cytosine in the 5' splice site adjacent to the optional exon. After replacement of the aberrant cytosine in the hamster alphaA-crystallin gene by a consensus thymine, 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 alternative splicing of the alphaA-crystallin primary transcript is mainly due to a non-consensus 5' splice site nucleotide. However, we conclude that the small size of the optional exon is probably an additional contributing factor and therefore it seems that the splicing mechanism is based on recognition of exons rather than introns.
αA-晶体蛋白是小热休克蛋白家族的成员，在脊椎动物眼晶状体中作为结构成分大量表达。在啮齿动物和其他一些哺乳动物的晶状体中，存在αA-晶体蛋白的一种次要变体，它插入了23个氨基酸残基。这种变体αA(ins)-晶体蛋白是由一个可选外显子差异整合到一小部分mRNA中产生的。我们研究了这种可变剪接是否由与可选外显子相邻的5'剪接位点中的非共有胞嘧啶引起。在用共有胸腺嘧啶取代仓鼠αA-晶体蛋白基因中的异常胞嘧啶，并将该基因瞬时转染到中国仓鼠卵巢细胞中后，可选外显子确实几乎完全剪接到成熟mRNA中。相反，用腺嘌呤或鸟嘌呤取代胞嘧啶会完全消除可选外显子的剪接。我们的结果证实，αA-晶体蛋白初级转录本的可变剪接主要是由于非共有5'剪接位点核苷酸。然而，我们得出结论，可选外显子的小尺寸可能是另一个促成因素，因此剪接机制似乎基于外显子而非内含子的识别。
