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In-depth functional characterization of chloroplast-localized mTERF proteins

叶绿体定位 mTERF 蛋白的深入功能表征

基本信息

批准号：
310039167
负责人：
Privatdozentin Dr. Tatjana Kleine
金额：
--
依托单位：
Lehrstuhl für Molekularbiologie der Pflanzen
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2016
资助国家：
德国
起止时间：
2015-12-31 至 2021-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/310039167?language=en
关键词：
depth functional characterization chloroplast localized

项目摘要

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.

细胞器基因表达（OGE）对于植物发育至关重要，但控制它的机制仍然很大程度上难以捉摸。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，并调节它们的翻译。所谓的线粒体转录终止因子 (mTERF) 家族的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的特定功能的机制细节。在我们之前的初步工作计划中，我们重点关注了 10 种可能在叶绿体中发挥作用的 mTERF 蛋白的鉴定和选择。另一种 mTERF，即 mTERF6，得到了详细表征。 mTERF6 蛋白位于叶绿体和线粒体中，其敲除会导致幼苗死亡。我们揭示了 mTERF6 与叶绿体异亮氨酸 tRNA 基因 (trnI.2) 中的 RNA 序列之间的特异性相互作用。然而，mTERF6 也可以结合相同序列的 DNA。在体外，重组 mTERF6 与其质体 DNA 靶位点结合可以终止转录。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，以及 (ii) 揭示其余尚未研究的假定叶绿体定位 mTERF 蛋白的分子功能。更具体地说，我们想要研究渗漏 mterf6-1 突变体中进一步细胞器 tRNA 的氨酰化状态，并且我们想要通过 RNA 质谱法确定 trnI.2 的转录后修饰。第二步，我们将通过下拉和关联内疚方法的组合来识别 mTERF6 相互作用伙伴。为了揭示 mTERF6 可能参与其他转录过程，将应用 RNA-Seq。此外，还将对选定的 10 种 mTERF 蛋白进行更详细的分析；部分是通过招募已成功建立的方法来表征 mTERF6 蛋白。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 定位研究以及应激和驯化治疗。对于最有前途的 mTERF，RNA 质谱分析以及 RIP 和 DIP-Seq 分析将对其分子功能进行深入表征。