Watakabe A, Tanaka K, Shimura Y
Department of Biophysics, Faculty of Science, Kyoto University, Japan.
Genes Dev. 1993 Mar;7(3):407-18. doi: 10.1101/gad.7.3.407.
Using mouse immunoglobulin mu (IgM) pre-mRNA as the model substrate for in vitro splicing, we have explored the role of exon sequences in splicing. We have found that deletion of the 5' portion of exon M2 of the IgM gene abolishes the splicing of its immediately upstream intron. Splicing was restored when a purine-rich sequence found within the deleted region was reinserted into the deletion construct. This M2 exon sequence was able to stimulate the splicing of a heterologous intron of the Drosophila doublesex pre-mRNA that contains a suboptimal 3' splice site sequence. These results show that the IgM M2 exon sequence functions as a splicing enhancer. We found that the assembly of the early splicing complex is stimulated by the M2 exon sequence. In vitro competition experiments show that this stimulatory effect is mediated by the interaction of some trans-acting factors. Our results suggest that the U1 snRNP is one such factor. We propose that recognition of an enhancer exon sequence by the components of splicing machinery plays a vital role in the selection of splice sites, not only for the IgM pre-mRNA but for other pre-mRNAs. We designate such a sequence as exon recognition sequence (ERS).
以小鼠免疫球蛋白μ（IgM）前体mRNA作为体外剪接的模型底物，我们探究了外显子序列在剪接中的作用。我们发现，IgM基因外显子M2的5'部分缺失会导致其紧邻上游内含子的剪接被阻断。当将缺失区域内发现的富含嘌呤的序列重新插入缺失构建体时，剪接得以恢复。该M2外显子序列能够刺激果蝇双性前体mRNA的异源内含子的剪接，该内含子含有一个次优的3'剪接位点序列。这些结果表明，IgM M2外显子序列作为剪接增强子发挥作用。我们发现，早期剪接复合体的组装受到M2外显子序列的刺激。体外竞争实验表明，这种刺激作用是由一些反式作用因子的相互作用介导的。我们的结果提示，U1 snRNP就是这样一种因子。我们提出，剪接机制的组分对增强子外显子序列的识别不仅对IgM前体mRNA，而且对其他前体mRNA的剪接位点选择都起着至关重要的作用。我们将这样的序列称为外显子识别序列（ERS）。
