Roscigno R F, Weiner M, Garcia-Blanco M A
Department of Microbiology and Immunology, School of Medicine, Duke University Medical Center, Durham, North Carolina 27710.
J Biol Chem. 1993 May 25;268(15):11222-9.
The polypyrimidine (py) tract of introns is required for efficient spliceosome assembly and splicing of pre-mRNAs. A detailed mutational analysis of the py tract of an adenovirus 2 intron was carried out. Utilizing a "precursor in pieces" vector system, it was possible to synthesize py tract mutant pre-mRNAs that were otherwise identical. The mutant pre-mRNAs that were otherwise identical. The mutant pre-mRNAs were analyzed for in vitro splicing, for formation of splicing complexes, and for binding to proteins in the HeLa nuclear extract. Chimeric pre-mRNAs that contained the yeast branch point consensus sequence (UAC-UAAC) and altered py tracts were also analyzed. Mutational analysis showed the following. First, any mutation in the py tract that affected splicing did so by interferring with complex A formation in spliceosome assembly. Second, introduction of purines into the py tract is detrimental only if the length of the tract is shortened and if there is a reduction in the number of consecutive uracil residues. Third, uracil and cytosine do not have equivalent functions in the py tract. Our results with chimeric pre-mRNAs also show that a strong py tract can partially replace a weak branch point sequence and a strong branch point sequence can partially replace a weak py tract. Finally, the one surprising finding obtained when examining protein binding was that a mutant pre-mRNA did not bind to heterogeneous nuclear ribonucleoprotein C proteins and yet spliced close to wild type level.
内含子的多嘧啶（py）序列对于前体mRNA高效组装剪接体和剪接是必需的。我们对腺病毒2内含子的py序列进行了详细的突变分析。利用“片段前体”载体系统，可以合成其他方面均相同的py序列突变前体mRNA。对这些突变前体mRNA进行了体外剪接、剪接复合体形成以及与HeLa细胞核提取物中蛋白质结合的分析。还分析了含有酵母分支点共有序列（UAC-UAAC）和改变的py序列的嵌合前体mRNA。突变分析结果如下。首先，py序列中任何影响剪接的突变都是通过干扰剪接体组装过程中A复合体的形成来实现的。其次，只有当py序列长度缩短且连续尿嘧啶残基数量减少时，向py序列中引入嘌呤才是有害的。第三，尿嘧啶和胞嘧啶在py序列中不具有等效功能。我们对嵌合前体mRNA的研究结果还表明，强py序列可以部分替代弱分支点序列，强分支点序列也可以部分替代弱py序列。最后，在检测蛋白质结合时得到的一个惊人发现是，一种突变前体mRNA不与不均一核核糖核蛋白C蛋白结合，但剪接水平却接近野生型。
