Target mRNAs and binding sites of the key RNA-binding protein Rrm4 during microtubule-dependent mRNA transport in the plant pathogen Ustilago maydis

微管依赖的mRNA转运对于确定正确的时空蛋白表达是重要的。这个过程中的关键参与者是RNA结合蛋白（RBP），它识别靶mRNA中的RNA拉链。为了理解潜在的生物学机制，全转录组的观点具有重要价值。在植物病原真菌玉米黑粉菌中，微管依赖的mRNA运输在感染性丝状体的有效生长过程中是必不可少的。最近的研究发现了这一转运过程的关键RNA结合蛋白。Rrm 4属于含有三个RNA识别基序（RRM 1 -3）的ELAV型RBP组。Rrm 4的功能涉及识别细胞定位相关的靶mRNA。mRNA的潜在长距离运输与内体穿梭耦合，内体穿梭分别由负端和正端定向马达分裂Dyn 1/2和Kin 3介导。在这里，我们计划使用新的iCLIP方法（单个核苷酸交联和免疫沉淀）在转录组范围内识别Rrm 4的靶mRNA及其单核苷酸分辨率的结合位点。结合来自不同突变体和相关同源蛋白的数据将允许精确定位串联RRM 1/2和RRM 3结构域的共有结合位点，其显示功能差异。在一个互补的方法中，我们将建立一个通用的研究策略，解决新的蛋白质的功能，其时空表达受微管依赖的mRNA运输。为此，我们将分析各自的缺失突变体，以及对编码蛋白质的亚细胞定位的目标mRNA运输的影响。接下来，我们将使用遗传和蛋白质相互作用的方法来获得这些新蛋白质功能的更具体的信息。所描述的方法将大大提高我们对细胞极性，膜运输，分泌，感染和生物技术的理解。

Microtubule-dependent mRNA transport is important to determine the correct spatio-temporal protein expression. Crucial players in this process are RNA-binding proteins (RBPs) that recognize RNA zipcodes in target mRNAs. In order to understand the underlying biological mechanisms a transcriptome-wide view is of great value. In the phytopathogenic fungus Ustilago maydis, microtubule-dependent mRNA transport is essential during efficient growth of infectious filaments. Recent studies uncovered the key RNA-binding protein of this transport process. Rrm4 belongs to the group of ELAV-type RBPs containing three RNA recognition motives (RRM1-3). Rrm4 function involves recognition of cytotopically related target mRNAs. The underlying long distance transport of mRNAs is coupled to endosomal shuttling mediated by the minus-end and plus-end directed motors split Dyn1/2 and Kin3, respectively. Here, we plan to use new iCLIP method (individual nucleotide cross-linking and immune precipitation) for the transcriptome-wide identification of target mRNAs of Rrm4 as well as its binding sites at single nucleotide resolution. Combining data from different mutants and related homologous proteins will allow pinpointing the consensus binding sites for tandem RRM1/2 and RRM3 domains, which show functional differences. In a complementary approach, we will establish a universal research strategy that addresses the functions of novel proteins whose spatio-termporal expression is regulated by microtubule-dependent mRNA transport. To this end we will analyse respective deletion mutants as well as the influence of target mRNA transport on subcellular localization of encoded proteins. Next, we will use genetic and protein interaction approaches to gain more specific information on the function of these novel proteins. The described approaches will considerably improve our understanding of cell polarity, membrane trafficking, secretion, infection and biotechnology.