Sierakowska H, Sambade M J, Schümperli D, Kole R
Lineberger Comprehensive Cancer Center and Department of Pharmacology, University of North Carolina, Chapel Hill 27599, USA.
RNA. 1999 Mar;5(3):369-77. doi: 10.1017/s135583829998130x.
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.
已经构建了一系列稳定表达β-珠蛋白前体mRNA的HeLa细胞系，这些前体mRNA在第2内含子的第654、705或745位核苷酸处携带点突变。这些突变产生异常的5'剪接位点，并激活上游一个共同的隐蔽3'剪接位点，导致β-珠蛋白mRNA剪接异常。反义寡核苷酸在体内阻断了异常剪接位点并恢复了前体mRNA的正确剪接，揭示了这些位点对反义探针敏感性的主要差异。尽管靶向的前体mRNA仅相差单点突变，但校正剪接所需的寡核苷酸有效浓度相差高达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；特别是，通过将异常5'剪接位点修饰为共有序列，两个剪接位点的敏感性都严重降低。这些结果表明，体内非常相似的前体mRNA剪接存在很大差异。它们还表明，反义寡核苷酸可能为研究剪接机制与前体mRNA的相互作用提供有用的工具。
