Tschudi C, Ullu E
Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06510.
Cell. 1990 May 4;61(3):459-66. doi: 10.1016/0092-8674(90)90527-l.
We have used permeable cells of Trypanosoma brucei to analyze the role of snRNAs in the trans splicing process. Degradation of U2, U4, or U6 snRNA by site-directed cleavage with complementary deoxyoligonucleotides and RNAase H inhibits trans splicing of the spliced leader (SL) RNA and newly synthesized alpha-tubulin pre-mRNAs. Cleavage of U snRNAs abolishes the appearance of putative trans splicing reaction intermediates and products, namely, linear branched molecules consisting of the SL intron joined to high molecular weight RNA (Y structures) and free SL intron. This indicates that U snRNAs are required for an early step in trans splicing. alpha-tubulin transcripts synthesized in the absence of trans splicing are unstable, suggesting that the addition of the SL sequence stabilizes pre-mRNAs against degradation. Our results provide direct evidence for the participation of U2 and U4/U6 snRNPs in trans splicing.
我们利用布氏锥虫的通透细胞来分析小核仁核糖核酸（snRNAs）在反式剪接过程中的作用。通过用互补脱氧寡核苷酸和核糖核酸酶H进行定点切割来降解U2、U4或U6 snRNA，会抑制剪接前导序列（SL）RNA和新合成的α-微管蛋白前体信使核糖核酸（pre-mRNAs）的反式剪接。U snRNAs的切割消除了假定的反式剪接反应中间体和产物的出现，即由与高分子量RNA连接的SL内含子组成的线性分支分子（Y结构）和游离的SL内含子。这表明U snRNAs是反式剪接早期步骤所必需的。在没有反式剪接的情况下合成的α-微管蛋白转录本不稳定，这表明SL序列的添加使前体信使核糖核酸稳定以免于降解。我们的结果为U2和U4/U6小核核糖核蛋白颗粒（snRNPs）参与反式剪接提供了直接证据。
