Cheng S C, Abelson J
Proc Natl Acad Sci U S A. 1986 Apr;83(8):2387-91. doi: 10.1073/pnas.83.8.2387.
We have fractionated a yeast whole cell extract that can accurately splice synthetic actin and CYH2 pre-mRNAs. Three fractions, designated I, II, and III, have been separated by use of ammonium sulfate fractionation and chromatography on heparin agarose. Each fraction alone has no splicing activity. Fractions I and II allow the first step of the splicing reaction to proceed, giving rise to the splicing intermediates, free exon 1, and intron-exon 2. Addition of fraction III completes the reaction. Micrococcal nuclease treatment of the whole cell extract or of either fraction I or II abolished splicing activity, indicating that fractions I and II have RNA moieties that are required in the splicing reaction. The nature of the RNAs was examined using antibodies directed against the trimethylated cap structure unique to small nuclear RNAs. Preincubation of the whole cell extract with protein A-Sepharose coupled to trimethylated cap antibody abolished splicing activity. This indicates that at least one essential RNA component contains a trimethyl cap. Thus, in yeast as in mammalian systems, small nuclear RNAs are involved in mRNA splicing.
我们对一种能够精确剪接合成的肌动蛋白和CYH2前体mRNA的酵母全细胞提取物进行了分级分离。通过硫酸铵分级分离和肝素琼脂糖柱层析，分离出了三个组分，分别命名为I、II和III。单独的每个组分都没有剪接活性。组分I和II能使剪接反应的第一步进行，产生剪接中间体、游离的外显子1和内含子-外显子2。加入组分III则完成反应。用微球菌核酸酶处理全细胞提取物或组分I或II中的任何一个，都会消除剪接活性，这表明组分I和II含有剪接反应所需的RNA部分。使用针对小核RNA特有的三甲基化帽结构的抗体来研究RNA的性质。将全细胞提取物与偶联有三甲基化帽抗体的蛋白A-琼脂糖预先孵育会消除剪接活性。这表明至少一种必需的RNA组分含有三甲基帽。因此，与哺乳动物系统一样，酵母中的小核RNA也参与mRNA剪接。
