Identification and characterization of factors for chloroplast RNA editing in Nicotiana tabacum

叶绿体RNA编辑对于叶绿体基因的正确表达至关重要，从而对叶绿体的生物发生也是至关重要的。RNA编辑发展迅速。这导致了编辑站点的物种特定模式。例如，烟草拥有40个叶绿体编辑点，只有19个与拟南芥共享。总共在种子植物的叶绿体中发现了大约100个不同的编辑位点。烟草很早就被用于编辑研究。它过去是，现在也是定义编辑位点的顺式作用元件作图和突变的首选模式生物，因为它具有以高效和定向的方式操纵叶绿体基因组的技术能力。然而，由于其基因组序列的可用性和遗传筛选的传导性，在拟南芥中鉴定出了进行叶绿体RNA编辑的核编码因子，这一研究计划将使烟草重新成为研究高等植物叶绿体RNA编辑的主要模式生物。为了促进这一点，我们组装了迄今为止未知的烟草可能编辑因子的基因序列。此外，还建立了22种被子植物编辑因子/编辑位点关系预测的生物信息学框架。这些进展与叶绿体转化相结合，使我们能够同时操纵同一物种中的顺式元件和编辑因子基因，从而系统地剖析叶绿体RNA编辑的机制。在这项研究计划中，生物信息预测的叶绿体RNA编辑因子将通过反向遗传学在烟草中得到确认，选定的因子将通过最先进的分子生物学方法进行表征。叶绿体编辑小体的新成分的候选已经并将被RNA亲和层析分离出来，并对它们在RNA编辑中的作用进行了表征。这一体系将得到高效的体外编辑系统的有力支持。所获得的结果将使我们能够设计出高精度的实验，旨在通过未来两个相互作用伙伴的操纵来研究反式因子与其RNA靶标之间的相互作用。这也包括来自其他物种的异源编辑因子/编辑位点对。同样，基于所获得的结果，将有可能利用叶绿体转化来阐明RNA编辑与剪接和翻译的相互作用。最后，将建立可诱导或组织特异性地产生翻译起始密码子的方法，以便通过利用异源RNA编辑位点及其同源编辑因子来控制叶绿体(反式)基因的表达。

Chloroplast RNA editing is essential for the proper expression of chloroplast genes and, thus, for chloroplast biogenesis. RNA editing evolves rapidly. This results in species-specific patterns of editing sites. Nicotiana tabacum (tobacco), for instance, possesses 40 chloroplast editing sites and only 19 are shared with Arabidopsis thaliana. In total, about 100 different editing sites have been found in the chloroplasts of seed plants. Tobacco has been early on employed in editing research. It was and is the model organism of choice for the mapping and mutagenesis of the cis-acting elements that define editing sites, because of the technical ability to manipulate the chloroplast genome in an efficient and directed manner. However, the nuclear encoded factors that carry out chloroplast RNA editing were identified in Arabidopsis, because of the availability its genomic sequence and the conductibility of genetic screens.This research program will revive tobacco as prime model organism for studying chloroplast RNA editing in higher plants. To facilitate this, the hitherto unknown gene sequences of the putative editing factors of tobacco were assembled. Furthermore, a bioinformatic framework for the prediction of editing factor/editing site relationships for 22 angiosperm species has been established. These advancements in combination with chloroplast transformation allow us to simultaneously manipulate both cis-elements and editing factor genes in the same species and thus, to systematically dissect the mechanism of chloroplast RNA editing. Within this research program, bioinformatically predicted chloroplast RNA editing factors will be confirmed by reverse genetics in tobacco and selected factors will be characterized by state-of-the-art molecular biological methods. Candidates for novel components of the chloroplast editosomes have been and will be isolated by RNA-affinity chromatography and characterized for their role in RNA editing. This set-up will be strongly supported by an efficient in vitro editing system.The obtained results will enable to design highly precise experiments aimed to study the interaction between trans-factors and their RNA targets by the manipulation of both interaction partners in the future. This includes also heterologous editing factor/editing site couples from other species. Likewise, based on the obtained results, it will be possible to employ chloroplast transformation to elucidate the interplay of RNA editing with splicing and translation. Finally, approaches will be established for the inducible or tissue-specific generation of translational start codons in order to control the expression of chloroplast (trans-)genes by employing heterologous RNA editing sites and their cognate editing factors.