Identification of the Plant RNA Editosome

智力优势：植物细胞器在转录后将mRNA中的某些胞苷转化为尿苷，这一过程被称为RNA编辑。这个过程对于许多叶绿体和线粒体基因的正确表达至关重要，这些基因已经积累了T到C突变。如果不进行编辑，这些基因将产生非功能性蛋白质，这将对光合作用和呼吸作用等重要过程产生有害影响。植物RNA编辑蛋白复合物，即植物编辑体的完整组成尚不清楚。将C转化为U的选择是由Pentatricopeptide Repeat-Motif (PPR) RNA结合蛋白完成的，该蛋白识别靠近C靶点的RNA上的序列。除了大的PPR蛋白家族外，最近还发现了另外两个植物蛋白家族的成员，RIP和ORRM家族，它们是细胞器中RNA编辑所必需的。与位点特异性PPR蛋白不同的是，ORRM和RIP蛋白对叶绿体和/或线粒体中大多数编辑过的Cs的编辑效率至关重要，因此可能是大多数编辑体的组成部分。其他ORRM家族成员的作用将通过突变分析和基因沉默结合深度测序编辑试验进行检测。ORRM和RIP蛋白将通过串联亲和纯化标签在转基因植物中表达，分离编辑体，然后用质谱方法鉴定编辑体的组成蛋白。更广泛的影响：C-to-U RNA编辑不仅可以改变编码的氨基酸，还可以创建起始和停止密码子，深刻影响蛋白质的积累和功能。了解特定位点的植物RNA编辑机制将为未来通过对密码子进行转录后修饰的植物和其他生物的基因工程铺平道路。该项目将为研究生和本科生提供培训机会，包括参加暑期实习项目的其他学院的学生，该项目提供研究经验和正式讲座。PI将继续努力促进女性在STEM领域的招聘、保留和提升。coPI将传播一份详细的深度测序协议，该协议不仅对RNA编辑研究人员有价值，而且对在实验生物体中研究RNA转录组子集的科学家更有价值。

Intellectual Merit: Plant organelles convert certain cytidines to uridines in mRNA post-transcriptionally in a process known as RNA editing. This process is critical for proper expression of many chloroplast and mitochondrial genes that have accumulated T to C mutations. If not edited, these genes would produce non-functional proteins that would have a detrimental effect on important processes such as photosynthesis and respiration. The complete composition of the plant RNA editing protein complexes, the plant editosomes, is unknown. The selection of the C to be converted to U is performed by Pentatricopeptide Repeat-Motif (PPR) RNA-binding proteins, which recognize sequences on the RNA in close proximity to the C target. In addition to the large PPR protein family, members of two additional plant protein families, the RIP and ORRM families, have recently been found to be required for RNA editing in organelles. Unlike the site-specific PPR proteins, which are needed for editing of only one or a few individuals Cs, the ORRM and RIP proteins are essential for editing efficiency of the majority of edited Cs in chloroplasts and/or mitochondria and thus likely are components of most editosomes. The role of additional ORRM family members will be examined by mutant analysis and gene silencing in conjunction with a deep sequencing editing assay. ORRM and RIP proteins will be expressed with tandem affinity purification tags in transgenic plants in order to isolate editosomes, whose component proteins will then be identified by mass spectrometric methods.Broader Impacts: C-to-U RNA editing is able not only to change the encoded amino acid, but can also create start and stop codons, profoundly affecting the accumulation and function of proteins. Understanding the site-specific plant RNA editing mechanism will pave the way for future genetic engineering of plants and other organisms through deliberate post-transcriptional modification of codons. The project will provide training opportunities for both graduate and undergraduate students, including ones from other colleges who are participating in a summer internship program that offers research experience and formal lectures. The PI will continue her efforts to promote the recruitment, retention, and advancement of women in STEM fields. The coPI will disseminate a detailed deep sequencing protocol that is valuable not only for RNA editing researchers, but more generally for scientists studying a subset of the RNA transcriptomes in their experimental organisms.