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'端是通过转录后加工产生的。我们已经确定了一些RNA 5'末端有效形成所需的RNA加工因子（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。这些因素中有许多是育性样蛋白的恢复因子，表明细胞质雄性不育系育种过程中5′加工与育性恢复之间存在密切的机制关系。RNA加工因子特异性地与它们的靶RNA相互作用，最有可能将核糖核酸酶招募到切割位点。在靶RNA上可以预测RNA的结合位点，但没有得到这些顺式元件的确切位置和结构的实验数据。此外，实际上对参与5'加工的核糖核酸酶一无所知。在拟建的项目中，我们将重点研究这些尚未解决的植物线粒体转录后5'加工问题。在提议的项目中，我们将描述RPF3和RPF6的顺式元件。这两个因子都是处理来自两种不同线粒体ccmC基因型的ccmC转录本所必需的。这些基因型由66 bp的DNA片段区分，其中包含加工所必需的线粒体序列。66 bp的DNA片段位于5'末端的上游，这在两种线粒体基因型的ccmC转录本中是不同的。利用亲和纯化的重组RPF3和RPF6进行光激活核糖核苷增强交联免疫沉淀（PAR-CLIP）实验和电泳迁移转移实验，将准确地绘制出顺式元件，并鉴定出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）参与至少一些线粒体转录物的5'加工。这些研究包括重组蛋白的RNase活性测试。对表位标记的MNU1和MNU2变体的体内研究将进一步了解这些蛋白及其单个结构域的功能。此外，还将分析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共同参与nad9 mrna的5′加工。该项目将极大地促进我们对植物线粒体转录本5'加工的理解，并将阐明细胞质雄性不育性恢复的相关过程，这是一种经济上重要的线粒体性状，用于生产F1杂交种子。

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.