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

发现光合生物各种未表征蛋白质中保守的新型重复RNA结合基序

基本信息

批准号：
289469996
负责人：
Privatdozent Dr. Jörg Meurer
金额：
--
依托单位：
Laboratoire dOcéanographie biologique de Banyuls
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 2017-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/289469996?language=en
关键词：
Discovery novel repeated RNA binding

项目摘要

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.

植物特异性因子 HCF145 通过新定义的转录结合基序重复 (TMR) 结构域与其 5 个 UTR、下游 52 个核苷酸的特异性结合，特异性保护质体 psaA-psaB-rps14 mRNA 免受 5 - 3 核酸外切攻击。转录起始位点。 TMR基序在小立碗藓、红藻和绿藻以及蓝藻微鞘藻中的多种、迄今为止尚未表征的蛋白质中是保守的。 PCC 7113。四个谱系中的代表性 TMR 蛋白可以高纯度和高溶解度获得，并以高亲和力和特异性结合异源 ssRNA，表明 TMR 基序扩展了光合生物中出现的 RNA 结合结构域家族的库。值得注意的是，除了 PPR、OPR 和 PUF 蛋白之外，TMR 蛋白代表了一类新颖且非常有趣的 RNA 结合蛋白，具有多个 RNA 识别基序，也被提议形成规则的超螺旋结构。据推测，TMR 基序还提供了用于 RNA 识别的组合氨基酸密码。该项目是关于光合生物中先前未被识别的 TMR 家族代表性成员的 RNA 结合功能的开创性工作。我们将首先通过在转基因生物体中使用串联亲和纯化标记策略以及免疫共沉淀与RNA-seq (RIP-seq)相结合来关注TMR蛋白在体内的功能和精确的RNA靶标。数据将通过突变体分析以及体外 RNA 结合和结构研究来补充。同时，免疫沉淀物将通过质谱法进行分析，以获得有关各自生物体中 TMR 相互作用伙伴的第一手信息。