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.

显示植物特异性因子HCF145可特异性地保护质体PSAA-PSAB-RPS14 mRNA，以5-3个通过对新定义的转录本结合基序重复（TMR）结构域的特异性结合到其52 UTR，52 UTR，52 UTR，52核苷酸下降核苷酸下降位置。 TMR基序在Physcomitrella patens，Red和Green藻类以及蓝细菌Microcoleus sp中保守，迄今为止多样化的，未表征的蛋白质。 PCC 7113。可以以高纯度和溶解度获得四个谱系中的代表性TMR蛋白，并结合具有高亲和力和特异性的异源SSRNA，表明TMR基序扩展了光合作用生物体中出现的RNA结构域家族的曲目。值得注意的是，除了PPR，OPR和PUF蛋白外，TMR蛋白还代表了具有多种RNA识别基序的新型且有趣的RNA结合蛋白，也提出形成常规的超螺旋结构。据推测，TMR基序还为RNA识别提供了组合氨基酸代码。该项目是关于光合有机体中先前未识别的TMR家族的代表成员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和与RNA-Seq（RIP-SEQ）结合使用的串联亲和力纯化策略来关注体内TMR蛋白的功能和精确RNA靶标。数据将通过突变分析以及体外RNA结合和结构研究来表达数据。同时，将通过质谱法分析免疫沉淀物，以获得有关各自生物体中TMR相互作用伙伴的首次信息。