Yu W, Sánchez H, Schuster W
Institute for Applied Genetics, Free University of Berlin, Germany.
Methods. 1998 May;15(1):63-74. doi: 10.1006/meth.1998.0606.
In plant organelles transcripts are modified posttranscriptionally by RNA editing. This modification process changes almost every protein-coding RNA at specific cytidine and uridine positions. Therefore, mitochondrially encoded protein sequences differ from the genomically fixed information and show, after editing, a higher conservation. To investigate this unusual processing step in plant mitochondria, several assays have been developed. However, compared with the progress made in other RNA editing fields, knowledge about the factors involved in plant mitochondrial editing is limited. One reason for this is the lack of a reliable in vitro system for mitochondria. To reveal the biochemical nature of the RNA editing reaction in plant mitochondria, we developed an in vitro system by which we were able to show that cytidine is specifically modified to uridine by a deamination or transamination process. Here we describe the development of a pea in vitro system and discuss assays to follow the editing process.
在植物细胞器中，转录本在转录后会通过RNA编辑进行修饰。这种修饰过程几乎会在特定的胞嘧啶和尿嘧啶位置改变每一个蛋白质编码RNA。因此，线粒体编码的蛋白质序列与基因组固定的信息不同，并且在编辑后显示出更高的保守性。为了研究植物线粒体中这一不同寻常的加工步骤，已经开发了几种检测方法。然而，与其他RNA编辑领域取得的进展相比，关于植物线粒体编辑所涉及的因子的知识仍然有限。造成这种情况的一个原因是缺乏可靠的线粒体体外系统。为了揭示植物线粒体中RNA编辑反应的生化本质，我们开发了一种体外系统，通过该系统我们能够证明胞嘧啶通过脱氨或转氨过程被特异性地修饰为尿嘧啶。在此，我们描述豌豆体外系统的开发，并讨论跟踪编辑过程的检测方法。
