Siegall C B, Kumar A
Department of Biochemistry, George Washington University School of Medicine and Health Sciences, Washington D.C. 20037.
Biochem Biophys Res Commun. 1988 Jan 29;150(2):517-25. doi: 10.1016/0006-291x(88)90424-x.
Inhibition of oligonucleotide-directed cleavage of pre-mRNA using exogenously added E. coli RNase H has been utilized as a probe for mRNA-protein interaction. We now show that such an RNase H-like activity is present in splicing competent Hela cell nuclear extract. Using this extract and in vitro transcribed beta-globin pre-mRNA, we have demonstrated that synthetic oligonucleotides, complementary to the splice site sequences, direct preferential cleavage of the 5' splice site. Thus, these experiments using complementary oligonucleotide-directed, endogenous RNase H-like cleavage of pre-mRNA, suggest a useful probe for studying the mRNA-protein complex in vitro.
利用外源添加的大肠杆菌核糖核酸酶H抑制前体mRNA的寡核苷酸定向切割，已被用作mRNA-蛋白质相互作用的探针。我们现在表明，在具有剪接能力的海拉细胞核提取物中存在这种类似核糖核酸酶H的活性。利用这种提取物和体外转录的β-珠蛋白前体mRNA，我们已经证明，与剪接位点序列互补的合成寡核苷酸可引导对5'剪接位点的优先切割。因此，这些使用互补寡核苷酸定向的、内源性前体mRNA的核糖核酸酶H样切割的实验，提示了一种用于体外研究mRNA-蛋白质复合物的有用探针。
