Molecular Mechanism of Chloroplast RNA Editing

RNA编辑是RNA加工的一种形式，发生在各种生物体中。编辑对于基因的正常功能和/或调控通常是必不可少的。在维管植物中，线粒体和叶绿体rna通过C-to-U碱基修饰进行修饰。编辑有时会为植物细胞器蛋白质创建起始密码子，并且需要恢复指定高度保守氨基酸的密码子。植物细胞器中的RNA编辑似乎是一种改善T-to-C突变的纠正机制，否则T-to-C突变会阻止基因表达或导致无功能蛋白质。叶绿体中RNA编辑的C靶标周围的序列被认为是被蛋白质特异性识别的，可能会招募其他蛋白质参与C到u的酶修饰。该项目的目标是鉴定叶绿体RNA编辑装置的未知成分。虽然已鉴定出编辑过的C上游的顺式元件，但对识别正确的C并将其转化为u所需的反式因子知之甚少。将努力分离玉米叶绿体提取物，对编辑装置进行部分纯化，这将受到质谱法进行蛋白质鉴定。候选编辑复合体成员将进行功能分析。一个高通量的分析，编辑所有叶绿体编辑位点在一个物种将被开发。将确定结合编辑因子所需的RNA序列。编辑因子蛋白的抗血清将用于纯化方案，以识别编辑复合物的成分。生物化学和分子遗传学方法的结合应该允许解剖维管植物叶绿体中将Cs转化为Us所需的分子装置。RNA编辑是对基因表达产生深远影响的基本生物学过程。了解植物细胞器如何调节这一过程可能最终导致通过提高叶绿体和线粒体的能量获取和利用效率来提高农业生产力的应用。这个项目将为一些研究生和本科生提供生物化学、分子生物学和遗传学方面的培训。将制作用于植物细胞结构和显微镜实验练习的材料。

RNA editing is a form of RNA processing that occurs in diverse organisms. Editing is often essential for proper gene function and/or regulation. In vascular plants, mitochondrial and chloroplast RNAs are modified by C-to-U base modification. Editing sometimes creates start codons for plant organelle proteins, and is required to restore codons that specify highly conserved amino acids. RNA editing in plant organelles appears to be a correction mechanism to ameliorate T-to-C mutations that would otherwise prevent gene expression or result in non-functional proteins. Sequences surrounding C targets of RNA editing in chloroplasts are thought to be specifically recognized by proteins, perhaps recruiting other proteins involved in the enzymatic modification of C to U. The goal of this project is to identify unknown components of the chloroplast RNA editing apparatus. While cis-elements upstream of edited Cs have been identified, little is known about the trans-factors required to recognize the correct C and convert it to U. Efforts will be made to fractionate maize chloroplast extracts for partial purification of the editing apparatus, which will be subjected to mass spectrometry for protein identification. Candidate editing complex members will undergo functional analysis. A high-throughput assay for editing of all chloroplast editing sites in a species will be developed. RNA sequence requirements for binding of editing factors will be determined. Antisera to an editing factor protein will be used in purification schemes to identify components of the editing complex. A combination of biochemical and molecular genetic approaches should allow dissection of the molecular apparatus needed to convert Cs to Us in vascular plant chloroplasts. RNA editing is a fundamental biological process with profound effects on gene expression. Understanding how organelles in plants regulate this process may eventually result in applications that could improve agricultural productivity by increasing efficiency of energy acquisition and utilization by chloroplasts and mitochondria. This project will provide training in biochemistry, molecular biology, and genetics for several graduate students and undergraduates. Materials useful for laboratory exercises in plant cell structure and microscopy will be produced.