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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HCF 145通过与转录起始位点下游52个核苷酸的5个非翻译区（UTR）特异性结合，特异性保护质体psaA-psaB-rps 14 mRNA免受5 - 3个外切核酸的攻击。TMR基序在小立碗藓、红藻和绿色藻类以及蓝细菌微鞘藻PCC 7113中相当多样的、迄今为止未表征的蛋白质中是保守的。四个谱系中的代表性TMR蛋白可以以高纯度和溶解度获得，并且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，TMR蛋白代表了一类新的和非常有趣的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基序还提供了用于RNA识别的组合氨基酸密码。该项目是对光合生物中以前未被识别的TMR家族的代表成员的RNA结合功能的开创性工作。我们将首先通过在转基因生物中使用串联亲和纯化-标记策略和免疫共沉淀结合RNA-seq（RIP-seq）来关注TMR蛋白的功能和体内精确的RNA靶点。数据将通过突变体分析以及体外RNA结合和结构研究进行补充。同时，将通过质谱法分析免疫沉淀物，以获得有关相应生物体中TMR相互作用伴侣的第一批信息。