Gallego M E, Balvay L, Brody E
Centre de Génétique Moléculaire, Centre National de la Recherche Scientifique, Gif-sur-Yvette, France.
Mol Cell Biol. 1992 Dec;12(12):5415-25. doi: 10.1128/mcb.12.12.5415-5425.1992.
The chicken beta-tropomyosin gene contains an internal pair of mutually exclusive exons (6A and 6B) that are selected in a tissue-specific manner. Exon 6A is incorporated in fibroblasts and smooth muscle cells, whereas exon 6B is skeletal muscle specific. In this study we show that two different regions in the intron between the two mutually exclusive exons are important for this specific selection in nonmuscle cells. Sequences in the 3' end of the intron have a negative effect in the recognition of the 3' splice site, while sequences in the 5' end of the intron have a positive effect in the recognition of the 5' splice site. First, sequences in exon 6B as well as in the intron upstream of exon 6B are both able to inhibit splicing when placed in a heterologous gene. The sequences in the polypyrimidine stretch region contribute to splicing inhibition of exons 5 or 6A to 6B through a mechanism independent of their implication in the previously described secondary structure around exon 6B. Second, we have identified a sequence of 30 nucleotides in the intron just downstream of exon 6A that is essential for the recognition of the 5' splice site of exon 6A. This is so even after introduction of a consensus sequence into the 5' splice site of this exon. Deletion of this sequence blocks splicing of exon 6A to 6B after formation of the presplicing complex. Taken together, these results suggest that both the mutually exclusive behavior and the choice between exons 6A and 6B of the chicken beta-tropomyosin gene are trans regulated.
鸡β-原肌球蛋白基因包含一对内部相互排斥的外显子（6A和6B），它们以组织特异性方式被选择。外显子6A被整合到成纤维细胞和平滑肌细胞中，而外显子6B是骨骼肌特异性的。在本研究中，我们表明在这两个相互排斥的外显子之间的内含子中的两个不同区域对于非肌肉细胞中的这种特异性选择很重要。内含子3'端的序列对3'剪接位点的识别有负面影响，而内含子5'端的序列对5'剪接位点的识别有正面影响。首先，当置于异源基因中时，外显子6B以及外显子6B上游内含子中的序列都能够抑制剪接。多嘧啶延伸区域中的序列通过一种独立于它们在先前描述的外显子6B周围二级结构中的作用的机制，对从外显子5或6A到6B的剪接抑制有贡献。其次，我们在紧挨着外显子6A下游的内含子中鉴定出一段30个核苷酸的序列，它对于识别外显子6A的5'剪接位点至关重要。即使在将一个共有序列引入该外显子的5'剪接位点之后也是如此。删除该序列会在形成剪接前复合物后阻断外显子6A到6B的剪接。综上所述，这些结果表明鸡β-原肌球蛋白基因的相互排斥行为以及外显子6A和6B之间的选择都是反式调节的。
