Identification and characterization of RNA cis-elements and nucleases required for 5' processing of mitochondrial RNAs in Arabidopsis thaliana

在拟南芥中，大多数线粒体RNA的5‘端是由转录后加工产生的。我们已经确定了有效形成几个RNA的5‘末端所需的一些RNA处理因子(RPF)。这些因子是五肽重复(PPR)蛋白，由典型的P基序组成，缺乏酶活性。这些因子中有许多是类育性蛋白的恢复者，表明在细胞质雄性不育系育种中，5‘处理与育性恢复之间存在密切的机制关系。RNA加工因子与它们的目标RNA特异地相互作用，最有可能的是将核糖核酸酶招募到裂解位点。可以预测目标RNA上的RNA结合位点，但还没有关于这些顺式元件的确切位置和结构的实验数据。此外，对参与5‘加工的假定核糖核酸酶几乎一无所知。在拟议的项目中，我们将重点关注这些尚未解决的植物线粒体转录后5‘处理的问题。在拟议的项目中，我们将表征RPF3和RPF6的顺式元件。这两个因素都是处理来自两种不同线粒体CCMC基因的CCMC转录本所必需的。这些基因类型通过66个碱基对的DNA片段进行区分，该片段包含加工所必需的线粒体序列。66bpDNA片段位于CCMC转录本的5‘端上游，CCMC转录本来自这两种线粒体。用亲和纯化的重组RPF3和RPF6进行光活化核糖核苷增强的交联链免疫沉淀(PAR-CLIP)实验和凝胶迁移率改变分析，可以准确地定位顺式元件并识别参与这些因子结合的核苷酸。第二个主题将集中在两个潜在的线粒体核酸酶(MNU1和MNU2)，涉及至少一些线粒体转录本的5‘处理。这些研究包括用重组蛋白进行核糖核酸酶活性测试。对MNU1和MNU2的表位标记变体的活体研究将提供关于这些蛋白质及其个别结构域的功能的进一步信息。此外，还将分析这两种蛋白质之间的相互作用以及MNUs与其他蛋白质的相互作用。最后，PAR-CLIP实验将确定MNU1、MNU2和RPF2的RNA结合位点。RPF2与MNU1和MNU2合作进行nad9 mRNA的5‘处理。该项目将有助于我们理解植物线粒体转录本的5’处理，也将阐明细胞质雄性不育育性恢复的相关过程，细胞质雄性不育是用于生产F1杂交种子的一种重要的线粒体经济性状。

In Arabidopsis thaliana, the 5' ends of most mitochondrial RNAs are generated by post-transcriptional processing. We have identified a number of RNA PROCESSING FACTORS (RPF) required for the efficient formation of the 5' termini of several RNAs. These factors are pentatricopeptide repeat (PPR) proteins, which consist of canonical P motifs and lack enzymatic activity. Many of these factors are RESTORER of FERTILITY-LIKE proteins indicating the close mechanistic relationship between 5' processing and fertility restoration during breeding with cytoplasmic male sterile lines. RNA processing factors specifically interact with their target RNAs most likely recruiting a ribonuclease to the cleavage sites. The RNA binding sites can be predicted on the target RNA, but no experimental data have been obtained on the exact location and the structure of these cis-elements. In addition, virtually nothing is known about the postulated ribonuclease participating in 5' processing. In the proposed project, we will focus on these unsolved issues of post-transcriptional 5' processing in plant mitochondria.In the proposed project we will characterize the cis-elements for RPF3 and RPF6. Both factors are required for processing of ccmC transcripts derived from two different mitochondrial ccmC genotypes. These genotypes are differentiated by a 66 bp DNA segment, which contains mitochondrial sequences essential for processing. The 66 bp DNA segment is located upstream of the 5' termini that differ between ccmC transcripts derived from the two mitochondrial genotypes. Photoactivatable-ribonucleoside-enhanced crosslink-immunoprecipitation (PAR-CLIP) experiments and electrophoretic mobility shift assays with affinity purified recombinant RPF3 and RPF6 will exactly map the cis-elements and identify nucleotides involved in binding of these factors. The second topic will focus on two potential mitochondrial nucleases (MNU1 and MNU2) involved in 5' processing of at least some of the mitochondrial transcripts. These studies include RNase activity tests with recombinant proteins. In vivo investigations with epitope-tagged variants of MNU1 and MNU2 will give further information about the function of these proteins and individual domains thereof. Furthermore, the interaction of both proteins with each other and the interaction of MNUs with other proteins will be analyzed. Finally PAR-CLIP experiments will identify the RNA binding sites for MNU1, MNU2 and RPF2. RPF2 cooperates with MNU1 and MNU2 in the 5' processing of nad9 mRNAs.The proposed project will substantially contribute to our understanding of 5' processing of plant mitochondrial transcripts and will also shed light on the related processes involved in fertility restoration of cytoplasmic male sterility, an economically important mitochondrial trait used for the production of F1 hybrid seeds.