Moss DYW-type pentatricopeptide repeat (PPR) proteins as modifiable C-to-U RNA editing factors in diverse genetic systems.

特定的五肽重复序列（PPR）蛋白介导陆生植物线粒体和叶绿体转录物中的胞苷到尿苷（C-to-U）RNA编辑。DYW型PPR蛋白是植物C-to-U RNA编辑的关键因子。它们识别特定的RNA靶点以定义待编辑的C，并进行胞苷脱氨基以产生尿苷。尽管关键的PPR-RNA结合密码已被阐明，并且C末端DYW结构域现已被确定为酶结构域，但具体如何完成特定胞苷的转化仍然难以捉摸。苔藓小立碗藓的RNA编辑系统是陆生植物界中最简单的系统之一，其细胞器转录组中只有9个DYW型PPR蛋白分配给13个编辑位点。在其他植物如模式开花植物拟南芥中，编辑系统已经朝着更复杂的方向发展，具有超过450个待编辑的位点以及识别和脱氨基因子的各种组合，加上对许多位点的有效转化很重要的额外辅助编辑因子。因此，我们把我们的研究重点放在苔藓编辑系统。通过插入单一苔藓DYW型PPR基因及其靶基因，我们能够将C到U RNA编辑转移到细菌大肠杆菌中。这种设置提供了一种独特的可能性，可以快速研究蛋白质因子和RNA靶侧的修饰。我们在这里的目的是进一步阐明共五个不同的DYW型PPR蛋白的功能。通过修饰结合位置或蛋白质基序来重定向这五个编辑因子，现在可以在转移到选择的植物系统（这里是小立碗藓和拟南芥）之前在细菌系统中容易地进行测试。虽然线粒体基因组的遗传修饰是不可行的植物到目前为止，DYW型PPR蛋白作为工程线粒体RNA编辑器将开辟新的途径，为基础和应用细胞器的研究。在这个项目中，我们的目标是分析通过插入工程PPR蛋白产生的突变体，以更深入地了解线粒体能量代谢。同时，我们将研究DYW型PPR蛋白是否也能够在植物细胞质中编辑转录本。最终，我们可能能够在细胞器和核转录物中引入特定的C到U变化，从而设计各种植物系统的功能。

Specific pentatricopeptide repeat (PPR) proteins mediate cytidine-to-uridine (C-to-U) RNA editing in mitochondrial and chloroplast transcripts of land plants. DYW-type PPR proteins are the key factors of plant-type C-to-U RNA editing. They recognize specific RNA targets to define the C to be edited and perform cytidine deamination to create uridines. How exactly the conversion of specific cytidines is accomplished, is still elusive, although the crucial PPR-RNA binding code is elucidated and the C-terminal DYW domain is identified as the enzymatic domain by now. The RNA editing system of the moss Physcomitrella patens with only nine DYW-type PPR proteins assigned to 13 editing sites in its organellar transcriptomes is one of the simplest in the land plant kingdom. In other plants like the model flowering plant Arabidopsis thaliana the editing system has evolved towards much more complexity with more than 450 sites to be edited and various combinations of recognition- and deamination factors plus additional helper editing factors important for efficient conversion of many sites. We therefore focused our research on the moss editing system. By insertion of single moss DYW-type PPR genes and their targets, we were able to transfer C-to-U RNA editing into the bacterium Escherichia coli. This setup offers a unique possibility to quickly investigate modifications both on the protein factor and the RNA target side. We here aim to further elucidate the functionality of altogether five different DYW-type PPR proteins. Re-direction of these five editing factors by modification of binding positions or protein motifs can now easily be tested in the bacterial system prior transfer into the plant system of choice (here Physcomitrella patens and Arabidopsis thaliana). While genetic modification of the mitochondrial genome is not feasible in plants up to date, DYW-type PPR proteins as engineerable mitochondrial RNA editors would open new ways for basic and applied organellar research. Within this project, we aim to analyze the mutants generated via insertion of engineered PPR proteins to get deeper insights into the mitochondrial energy metabolism. In parallel, we will investigate whether DYW-type PPR proteins are also capable to edit transcripts in the plant cytosol. Ultimately, we might be able to introduce specific C-to-U changes in organellar and nuclear transcripts and thereby engineer functions of diverse plant systems of choice.