Sensitivity of splice sites to antisense oligonucleotides in vivo.
Sensitivity of splice sites to antisense oligonucleotides in vivo.
复制标题
体内剪接位点对反义寡核苷酸的敏感性。
DOI：
10.1017/s135583829998130x
复制
发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Kole,R
中科院分区：
文献类型：
--
作者：
Sierakowska,H;Sambade,MJ;Schümperli,D;Kole,R
已开发出一系列稳定表达β-珠蛋白前体mRNAs的HeLa细胞系，该细胞系携带内含子2的654、705或745位点突变。这些突变产生了异常的5‘剪接位点，并激活了上游常见的3’隐蔽剪接位点，导致了异常剪接的β-珠蛋白mRNA。反义寡核苷酸在体内阻断了异常剪接位点并恢复了前mRNA的正确剪接，显示了这些位点对反义探针的敏感性存在重大差异。尽管靶向的前mRNAs只有单点突变不同，但纠正剪接所需的寡核苷酸的有效浓度差异高达750倍。异常5‘剪接位点之间的差异影响了5’和3‘剪接位点的敏感性，尤其是通过将异常5’剪接位点修饰为共同序列，这两个剪接位点的敏感性都严重降低。这些结果表明，在体内非常相似的前mRNAs的剪接有很大的差异。它们还表明，反义寡核苷酸可能为研究剪接机制与前-mRNA的相互作用提供有用的工具。
A series of HeLa cell lines which stably express beta-globin pre-mRNAs carrying point mutations at nt 654, 705, or 745 of intron 2 has been developed. The mutations generate aberrant 5' splice sites and activate a common 3' cryptic splice site upstream leading to aberrantly spliced beta-globin mRNA. Antisense oligonucleotides, which in vivo blocked aberrant splice sites and restored correct splicing of the pre-mRNA, revealed major differences in the sensitivity of these sites to antisense probes. Although the targeted pre-mRNAs differed only by single point mutations, the effective concentrations of the oligonucleotides required for correction of splicing varied up to 750-fold. The differences among the aberrant 5' splice sites affected sensitivity of both the 5' and 3' splice sites; in particular, sensitivity of both splice sites was severely reduced by modification of the aberrant 5' splice sites to the consensus sequence. These results suggest large differences in splicing of very similar pre-mRNAs in vivo. They also indicate that antisense oligonucleotides may provide useful tools for studying the interactions of splicing machinery with pre-mRNA.