Discovery of a novel repeated RNA-binding motif conserved in diverse uncharacterized proteins of photosynthetic organisms

植物特异性因子HCF145通过将新定义的转录结合基序重复（TMR）结构域特异性结合到转录起始位点下游52个核苷酸的5个UTR上，特异性地保护质粒psaa - psb -rps14 mRNA免受5 - 3外切核攻击。TMR基元在相当多样的蛋白质中都是保守的，在小立小藻、红藻和绿藻以及微藻蓝藻PCC 7113中都是如此。在这4个谱系中均可获得具有代表性的高纯度、高溶解度的TMR蛋白，并能以高亲和力和特异性结合异源ssRNA，这表明TMR基元扩展了光合生物中出现的rna结合结构域家族。值得注意的是，除了PPR、OPR和PUF蛋白外，TMR蛋白是一类新颖而有趣的RNA结合蛋白，具有多个RNA识别基序，也被提出形成规则的超螺旋结构。据推测，TMR基序也为RNA识别提供了组合氨基酸代码。本项目是研究光合生物中以前未被识别的TMR家族代表成员rna结合功能的先驱工作。我们将首先通过在转基因生物中使用串联亲和纯化-标记策略以及结合RNA-seq （RIP-seq）的共免疫沉淀技术来研究TMR蛋白在体内的功能和精确的RNA靶点。数据将通过突变分析以及体外rna结合和结构研究来补充。同时，免疫沉淀物将通过质谱分析，以获得有关各自生物体中TMR相互作用伙伴的第一手信息。

The plant-specific factor HCF145 was shown to specifically protect the plastid psaA-psaB-rps14 mRNA against 5 - 3 exonucleolytic attack via specific binding of the newly defined transcript-binding motif repeat (TMR) domains to its 5 UTR, 52 nucleotides downstream of the transcription start site. TMR motifs are conserved in quite diverse, so far uncharacterized proteins in Physcomitrella patens, red and green algae as well as in the cyanobacterium Microcoleus sp. PCC 7113. Representative TMR proteins in the four lineages could be obtained in high purity and solubility and bind heterologous ssRNA with high affinity and specificity, indicating that TMR motifs extend the repertoire of RNA-binding domain families that emerged in photosynthetic organisms. Remarkably, besides PPR, OPR and PUF proteins, TMR proteins represent a novel and quite interesting class of RNA-binding proteins with multiple RNA recognition motifs, also proposed to form a regular superhelical structure. Presumably, TMR motifs also provide a combinatorial amino acid code for RNA recognition. This project is a pioneer work on the RNA-binding function of representative members of the previous unrecognized TMR family in photosynthetic organisms. We will first focus on the function and the precise RNA targets of TMR proteins in vivo by using tandem affinity purification-tagging strategies in transgenic organisms and co-immunoprecipitation in combination with RNA-seq (RIP-seq). Data will be complememted by mutant analysis as well as in vitro RNA-binding and structural studies. In parallel, immunoprecipitates will be analysed by mass spectrometry to gain first informations about TMR interaction partners in the respective organisms.