Rusché L N, Huang C E, Piller K J, Hemann M, Wirtz E, Sollner-Webb B
Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Mol Cell Biol. 2001 Feb;21(4):979-89. doi: 10.1128/MCB.21.4.979-989.2001.
Kinetoplastid RNA editing is a posttranscriptional insertion and deletion of U residues in mitochondrial transcripts that involves RNA ligase. A complex of seven different polypeptides purified from Trypanosoma brucei mitochondria that catalyzes accurate RNA editing contains RNA ligases of approximately 57 kDa (band IV) and approximately 50 kDa (band V). From a partial amino acid sequence, cDNA and genomic clones of band IV were isolated, making it the first cloned component of the minimal RNA editing complex. It is indeed an RNA ligase, for when expressed in Escherichia coli, the protein autoadenylylates and catalyzes RNA joining. Overexpression studies revealed that T. brucei can regulate of total band IV protein at the level of translation or protein stability, even upon massively increased mRNA levels. The protein's mitochondrial targeting was confirmed by its location, size when expressed in T. brucei and E. coli, and N-terminal sequence. Importantly, genetic knockout studies demonstrated that the gene for band IV is essential in procyclic trypanosomes. The band IV and band V RNA ligases of the RNA editing complex therefore serve different functions. We also identified the gene for band V RNA ligase, a protein much more homologous to band IV than to other known ligases.
动质体RNA编辑是一种在线粒体转录本中对U残基进行转录后插入和缺失的过程，该过程涉及RNA连接酶。从布氏锥虫线粒体中纯化得到的由七种不同多肽组成的复合物可催化精确的RNA编辑，其中包含分子量约为57 kDa（条带IV）和约50 kDa（条带V）的RNA连接酶。根据部分氨基酸序列，分离出了条带IV的cDNA和基因组克隆，使其成为最小RNA编辑复合物中第一个被克隆的组分。它确实是一种RNA连接酶，因为当在大肠杆菌中表达时，该蛋白会自动腺苷酸化并催化RNA连接。过表达研究表明，即使在mRNA水平大幅增加的情况下，布氏锥虫也能在翻译或蛋白质稳定性水平上调节条带IV蛋白的总量。该蛋白的线粒体靶向性通过其在布氏锥虫和大肠杆菌中表达时的位置、大小以及N端序列得以证实。重要的是，基因敲除研究表明，条带IV的基因在原循环锥虫中是必需的。因此，RNA编辑复合物中的条带IV和条带V RNA连接酶发挥着不同的功能。我们还鉴定出了条带V RNA连接酶的基因，该蛋白与条带IV的同源性远高于与其他已知连接酶的同源性。
