Allen T E, Heidmann S, Reed R, Myler P J, Göringer H U, Stuart K D
Seattle Biomedical Research Institute, Seattle, Washington, 98109-1651, USA.
Mol Cell Biol. 1998 Oct;18(10):6014-22. doi: 10.1128/MCB.18.10.6014.
RNA editing in Trypanosoma brucei mitochondria produces mature mRNAs by a series of enzyme-catalyzed reactions that specifically insert or delete uridylates in association with a macromolecular complex. Using a mitochondrial fraction enriched for in vitro RNA editing activity, we produced several monoclonal antibodies that are specific for a 21-kDa guide RNA (gRNA) binding protein initially identified by UV cross-linking. Immunofluorescence studies localize the protein to the mitochondrion, with a preference for the kinetoplast. The antibodies cause a supershift of previously identified gRNA-specific ribonucleoprotein complexes and immunoprecipitate in vitro RNA editing activities that insert and delete uridylates. The immunoprecipitated material also contains gRNA-specific endoribonuclease, terminal uridylyltransferase, and RNA ligase activities as well as gRNA and both edited and unedited mRNA. The immunoprecipitate contains numerous proteins, of which the 21-kDa protein, a 90-kDa protein, and novel 55- and 16-kDa proteins can be UV cross-linked to gRNA. These studies indicate that the 21-kDa protein associates with the ribonucleoprotein complex (or complexes) that catalyze RNA editing.
布氏锥虫线粒体中的RNA编辑通过一系列酶催化反应产生成熟的mRNA，这些反应与一个大分子复合物相关，特异性地插入或删除尿苷酸。利用富含体外RNA编辑活性的线粒体组分，我们制备了几种单克隆抗体，它们对一种最初通过紫外线交联鉴定的21 kDa引导RNA（gRNA）结合蛋白具有特异性。免疫荧光研究将该蛋白定位到线粒体，尤其定位于动质体。这些抗体导致先前鉴定的gRNA特异性核糖核蛋白复合物超迁移，并免疫沉淀插入和删除尿苷酸的体外RNA编辑活性。免疫沉淀的物质还含有gRNA特异性核糖核酸酶、末端尿苷酰转移酶和RNA连接酶活性，以及gRNA和编辑及未编辑的mRNA。免疫沉淀物包含许多蛋白质，其中21 kDa蛋白、90 kDa蛋白以及新的55 kDa和16 kDa蛋白可通过紫外线与gRNA交联。这些研究表明，21 kDa蛋白与催化RNA编辑的核糖核蛋白复合物相关。
