Involvement of a site-specific trans-acting factor and a common RNA-binding protein in the editing of chloroplast mRNAs: development of a chloroplast in vitro RNA editing system

RNA editing in higher plant chloroplasts involves C-->U conversion at approximately 30 specific sites. An in vitro system supporting accurate editing has been developed from tobacco chloroplasts. Mutational analysis of substrate mRNAs derived from tobacco chloroplast psbL and ndhB mRNAs confirmed the participation of cis-acting elements that had previously been identified in vivo. Competition analysis revealed the existence of site-specific trans-acting factors interacting with the corresponding upstream cis-elements. A chloroplast protein of 25 kDa was found to be specifically associated with the cis-element involved in psbL mRNA editing. Immunological analyses revealed that an additional factor, the chloroplast RNA-binding protein cp31, is also required for RNA editing at multiple sites. This combination of site-specific and common RNA-binding proteins recognizes editing sites in chloroplasts.     

高等植物叶绿体RNA编辑研究进展

RNA编辑普遍存在于陆生植物中,在高等植物叶绿体中以C→U的替换为主,可能是叶绿体产生功能蛋白的重要方式。近年来,使用体外分析、叶绿体转化和紫外交联等技术,使叶绿体RNA编辑机制的研究取得较大进展。本文对这些新的进展进行了概述,并对高等植物叶绿体RNA编辑研究中有待解决的问题进行了展望。     

The evolution of chloroplast RNA editing

RNA editing alters the nucleotide sequence of an RNA molecule so that it deviates from the sequence of its DNA template. Different RNA-editing systems are found in the major eukaryotic lineages, and these systems are thought to have evolved independently. In this study, we provide a detailed analysis of data on C-to-U editing sites in land plant chloroplasts and propose a model for the evolution of RNA editing in land plants. First, our data suggest that the limited RNA-editing system of seed plants and the much more extensive systems found in hornworts and ferns are of monophyletic origin. Further, although some eukaryotic editing systems appear to have evolved to regulate gene expression, or at least are now involved in gene regulation, there is no evidence that RNA editing plays a role in gene regulation in land plant chloroplasts. Instead, our results suggest that land plant chloroplast C-to-U RNA editing originated as a mechanism to generate variation at the RNA level, which could complement variation at the DNA level. Under this model, many of the original sites, particularly in seed plants, have been subsequently lost due to mutation at the DNA level, and the function of extant sites is merely to conserve certain codons. This is the first comprehensive model for the evolution of the chloroplast RNA-editing system of land plants and may also be applicable to the evolution of RNA editing in plant mitochondria.     

High‐throughput quantification of chloroplast RNA editing extent using multiplex RT‐PCR mass spectrometry

RNA editing in plants, animals, and humans modifies genomically encoded cytidine or adenosine nucleotides to uridine or inosine, respectively, in mRNAs. We customized the MassARRAY System (Sequenom Inc., San Diego, CA, USA, www.sequenom.com ) to assay multiplex PCR‐amplified single‐stranded cDNAs and easily analyse and display the captured data. By using appropriate oligonucleotide probes, the method can be tailored to any organism and gene where RNA editing occurs. Editing extent of up to 40 different nucleotides in each of either 94 or 382 different samples (3760 or 15 280 editing targets, respectively) can be examined by assaying a single plate and by performing one repetition. We have established this mass spectrometric method as a dependable, cost‐effective and time‐saving technique to examine the RNA editing efficiency at 37 Arabidopsis thaliana chloroplast editing sites at a high level of multiplexing. The high‐throughput editing assay, named Multiplex RT‐PCR Mass Spectrometry (MRMS), is ideal for large‐scale experiments such as identifying population variation, examining tissue‐specific changes in editing extent, or screening a mutant or transgenic collection. Moreover, the required amount of starting material is so low that RNA from fewer than 50 cells can be examined without amplification. We demonstrate the use of the method to identify natural variation in editing extent of chloroplast C targets in a collection of Arabidopsis accessions.