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

Moss DYW 型五肽重复 (PPR) 蛋白作为各种遗传系统中可修饰的 C 至 U RNA 编辑因子。

• 批准号: 265715627
• 负责人: Professor Dr. Volker Knoop, since 12/2022
• 金额: --
• 依托单位: Abteilung Molekulare Evolution
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/265715627?language=en
• 关键词: Moss; DYW; type; pentatricopeptide; repeat

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.

陆地植物线粒体和叶绿体转录本中特异的五肽重复序列(PPR)蛋白介导胞苷到尿苷(C-to-U)的RNA编辑。Dyw型PPR蛋白是植物型C-U RNA编辑的关键因子。它们识别特定的RNA靶标以定义要编辑的C，并进行胞苷去氨基以产生尿氨酸。尽管关键的PPR-RNA结合密码已经被阐明，C-末端的Dyw结构域被确定为酶结构域，但具体的胞苷转化是如何完成的仍然是未知的。短生苔藓的RNA编辑系统是陆地植物界中最简单的系统之一，它的细胞器转录中只有9个dyw型PPR蛋白分配到13个编辑位点。在其他植物中，如模式植物拟南芥，编辑系统已经进化到更复杂的程度，有450多个要编辑的位点和识别和脱氨因子的各种组合，加上额外的辅助编辑因子，对于许多站点的有效转换非常重要。因此，我们将研究重点放在苔藓编辑系统上。通过插入单个苔藓dyw型PPR基因及其靶标，我们能够将C-to-U RNA编辑转移到大肠杆菌中。这种设置为快速研究蛋白质因子和RNA靶侧的修饰提供了独特的可能性。我们在这里的目的是进一步阐明总共五个不同的dyw型PPR蛋白的功能。通过修改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很容易地在细菌系统中进行测试，然后再转移到所选择的植物系统(这里是Physcomitrella patens和Arabiopsis thaliana)。虽然到目前为止，线粒体基因组的遗传修饰在植物中是不可行的，但Dyw型PPR蛋白作为可工程的线粒体RNA编辑将为基础和应用细胞器研究开辟新的途径。在这个项目中，我们的目标是分析通过插入工程PPR蛋白而产生的突变，以更深入地了解线粒体的能量代谢。同时，我们将研究Dyw型PPR蛋白是否也能够编辑植物细胞质中的转录本。最终，我们也许能够在细胞器和核转录中引入特定的C-U变化，从而设计出不同植物系统的功能。