In-depth functional characterization of chloroplast-localized mTERF proteins

细胞体基因表达（Organellar gene expression， OGE）在植物发育中起着至关重要的作用，但其调控机制在很大程度上仍是一个谜。因此，OGE需要各种核编码蛋白来促进细胞器rna的转录、剪接、修剪和编辑，并调节其翻译。所谓的线粒体转录终止因子（mTERF）家族成员存在于后生动物和植物中，并在不同水平上调节OGE。然而，我们才刚刚开始了解它们在这些过程中具体功能的机械细节。在前期和前期的工作计划中，我们重点鉴定和选择了10种可能在叶绿体中起作用的mTERF蛋白。对另一个mTERF mTERF6进行了详细描述。mTERF6蛋白定位于叶绿体和线粒体，敲除该蛋白可导致幼苗致死。我们发现了mTERF6与叶绿体异亮氨酸tRNA基因（trni - 2）中的一个RNA序列之间的特异性相互作用。然而，mTERF6也可以与相同序列的DNA结合。在体外，重组mTERF6结合到其质体DNA靶位点可以终止转录。在体内，mTERF6作为促进叶绿体tRNAIle(GAU) （trni - 2）成熟的因子。我们项目的主要目标是：(i)详细了解mTERF6的分子功能，（ii）揭示剩余尚未研究的叶绿体定位mTERF蛋白的分子功能。更具体地说，我们想研究泄漏的mterf6-1突变体中更多细胞器trna的氨基酰化状态，我们想确定trnI的转录后修饰。2采用RNA质谱法。在第二步中，我们将通过下拉和关联内疚方法的组合来确定mTERF6交互伙伴。为了揭示mTERF6可能参与其他转录过程，RNA-Seq将被应用。此外，将对10个选定的mTERF蛋白进行更详细的分析；部分原因是招募了成功建立的表征mTERF6蛋白的方法。为此，我们将应用广泛的技术，包括生物化学，突变分析，RNAi和蛋白质过表达，GFP定位研究，以及胁迫和驯化处理。对于最有前途的mterf， RNA质谱分析、RIP和DIP-Seq分析将导致对其分子功能的深入表征。

Organellar gene expression (OGE) is crucial for plant development, but the mechanisms that control it are still largely elusive. Thus, OGE requires various nucleus-encoded proteins that promote transcription, splicing, trimming and editing of organellar RNAs, and regulate their translation. Members of the so-called mitochondrial transcription termination factor (mTERF) family are found in metazoans and plants and regulate OGE at different levels. However, we are just beginning to understand the mechanistic details of their specific functions in these processes. In our preceding and preliminary work program we focused on the identification and selection for 10 mTERF proteins presumably functioning in the chloroplast. One additional mTERF, mTERF6, was characterized in detail. The mTERF6 protein is localized in chloroplasts and mitochondria and its knockout results in seedling lethality. We revealed a specific interaction between mTERF6 and a RNA sequence in the chloroplast isoleucine tRNA gene (trnI.2). However, mTERF6 can also bind to DNA of the same sequence. In vitro, recombinant mTERF6 bound to its plastid DNA target site can terminate transcription. In vivo, mTERF6 acts as a factor promoting chloroplast tRNAIle(GAU) (trnI.2) maturation. The major goals of our project are (i) to understand the molecular function of mTERF6 in detail, and (ii) to reveal the molecular functions of the remaining not yet investigated putative chloroplast-localized mTERF proteins. More specifically, we want to investigate the aminoacylation status of further organelle tRNAs in the leaky mterf6-1 mutant, and we want to determine the post-transcriptional modifications of trnI.2 with RNA mass spectrometry. In a second step, we will identify mTERF6 interaction partners by a combination of pull-down and guilt-by-association approaches. To reveal a possible involvement of mTERF6 in other transcriptional processes, RNA-Seq will be applied. Furthermore, the 10 selected mTERF proteins will be analyzed in more detail; in part by recruiting the methods successfully established to characterize the mTERF6 protein. To this end, we will apply a wide range of techniques that include biochemistry, mutant analysis, RNAi and protein over-expression, GFP localization studies, and stress and acclimation treatments. For the most promising mTERFs, RNA mass spectrometry, and RIP- and DIP-Seq analyses will lead to an in-depth characterization of their molecular functions.