Krämer A, Frick M, Keller W
Division of Molecular Biology, German Cancer Research Center, Heidelberg.
J Biol Chem. 1987 Dec 25;262(36):17630-40.
Components essential for nuclear pre-messenger RNA splicing have been partially purified from HeLa cell nuclear extracts by chromatography on DEAE-Sepharose, heparin-Sepharose, Mono Q, and Mono S. We have obtained six fractions which, when combined, efficiently splice a synthetic adenovirus 2 major late RNA substrate in vitro. All fractions contain components that support the formation of splicing intermediates (the cleaved 5' exon and the intron-exon 2 lariat). At least one of the fractions also contains an activity that is essential for the second step in the splicing reaction, namely cleavage at the 3' splice site and exon ligation. Two of the fractions are enriched in the major small nuclear ribonucleoprotein particles U1, U2, U4/U6, and U5. They participate in the formation of the splicing complexes which precedes the cleavage and ligation reactions. The remaining four fractions appear to contain protein factors, as suggested by their resistance to micrococcal nuclease.
通过在二乙氨基乙基琼脂糖（DEAE - Sepharose）、肝素琼脂糖（heparin - Sepharose）、Mono Q和Mono S上进行层析，已从HeLa细胞核提取物中部分纯化了核前体信使RNA剪接所需的成分。我们获得了六个组分，将它们组合后能在体外高效剪接合成的腺病毒2主要晚期RNA底物。所有组分都含有支持剪接中间体（切割后的5'外显子和内含子 - 外显子2套索结构）形成的成分。至少有一个组分还含有剪接反应第二步所必需的活性，即3'剪接位点的切割和外显子连接。其中两个组分富含主要的小核核糖核蛋白颗粒U1、U2、U4/U6和U5。它们参与在切割和连接反应之前的剪接复合体的形成。其余四个组分似乎含有蛋白质因子，这从它们对微球菌核酸酶的抗性可以看出。
