Piller K J, Rusché L N, Sollner-Webb B
Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
J Biol Chem. 1996 Mar 1;271(9):4613-9. doi: 10.1074/jbc.271.9.4613.
RNA editing in kinetoplastids is the post-transcriptional insertion and deletion of uridylate residues in mitochondrial transcripts, directed by base pairing with guide RNAs. Models for editing propose transesterification or endonuclease plus RNA ligase reactions and may involve a guide RNA-mRNA chimeric intermediate. We have assessed the feasibility of the enzymatic pathway involving chimeras in vitro. Cytochrome b chimeras generated with mitochondrial extract were first found to have junctions primarily at the major endonuclease cleavage sites, supporting the role of endonuclease in chimera formation. Such cytochrome b chimeras are then specifically cleaved by extract endonuclease within the oligo(U) tract at the editing site, and the mRNA cleavage products are then joined by RNA ligase to generate partially edited mRNAs with uridylate residues transferred to an editing site. These in vitro generated partially edited mRNAs mimic partially edited mRNAs generated in vivo. Specific endonuclease cleavage in the editing region of the partially edited RNA demonstrates the potential for further in vitro editing. Finally, sensitivity to various ATP analogs suggests that all editing-like activities reported thus far utilize a mechanism involving RNA ligase.
动质体中的RNA编辑是线粒体转录本中尿苷酸残基的转录后插入和缺失，由与引导RNA的碱基配对指导。编辑模型提出了转酯反应或核酸内切酶加RNA连接酶反应，并且可能涉及引导RNA-mRNA嵌合中间体。我们已经评估了体外涉及嵌合体的酶促途径的可行性。首先发现用线粒体提取物产生的细胞色素b嵌合体主要在主要核酸内切酶切割位点处有连接，这支持了核酸内切酶在嵌合体形成中的作用。然后，这种细胞色素b嵌合体在编辑位点的寡聚（U）区域内被提取物核酸内切酶特异性切割，然后mRNA切割产物通过RNA连接酶连接，以产生具有转移到编辑位点的尿苷酸残基的部分编辑的mRNA。这些体外产生的部分编辑的mRNA模拟体内产生的部分编辑的mRNA。在部分编辑的RNA的编辑区域中的特异性核酸内切酶切割证明了进一步体外编辑的潜力。最后，对各种ATP类似物的敏感性表明，迄今为止报道的所有类似编辑的活性都利用了一种涉及RNA连接酶的机制。
