Dissecting the binding behaviour of the multi-RRM protein Rrm4 during endosomal mRNA transport in Ustilago maydis

MRNAs的运输对于确定蛋白质在细胞中表达的地点和时间至关重要。玉米病原菌Ustilago maydis侵染菌丝内体mRNA沿微管运输是一种研究较多的转位机制。关键因素是Rrm4，它包含三个用于RNA结合的RNA识别基序(RRM)结构域。最近，我们发现富含甘氨酸的小RNA结合蛋白Grp1是穿梭内体mRNPs的一个新的相互作用伙伴和成分。通过对两个共同定位的限制性商业惯例的比较研究，我们首次从转录组的角度看待内体mRNA的运输。这表明这两种蛋白质都有一大组约2000个共同的靶向mRNA。两种限制性商业惯例都优先结合在邻近的3‘非编码区，这表明这是一种联合作用。对于Rrm4，序列UAUG被鉴定为一个确定的结合基序，主要出现在开放阅读框中的结合位点，并与Rrm4‘S第三个RRM结合。有趣的是，Rrm4专门识别翻译里程碑位点，如开始和停止密码子，这表明运输和目标mRNAs的翻译调控之间存在密切联系。在第二个资金阶段，我们将讨论(I)Rrm4如何通过其三个RRM结构域实现不同的运输策略，以及(Ii)与翻译标志性位点结合对编码蛋白的时空表达有什么分子后果。为此，我们将与FOR2333的合作伙伴合作，并结合全球方法，如iCLIP和核糖体图谱。因此，这项工作将为理解具有多个RNA结合域和不同相互作用伙伴的限制性商业惯例在细胞过程中如何协调其功能奠定基础。

The transport of mRNAs is crucial to determine where and when proteins are expressed in the cell. A well-studied translocation mechanism is endosomal mRNA transport along microtubules in infectious hyphae of the corn pathogen Ustilago maydis. The key factor is Rrm4, containing three RNA recognition motif (RRM) domains for RNA binding. Recently, we identified the small glycine-rich RNA-binding protein (RBP) Grp1 as a novel interaction partner and component of shuttling endosomal mRNPs. Using a comparative study of the two co-localising RBPs, we cast the first transcriptome-wide view of endosomal mRNA transport. This revealed that both proteins share a large set of about 2,000 common target mRNAs. Both RBPs bind preferentially in the 3´ UTR in close vicinity, suggesting a conjoint function. In case of Rrm4, the sequence UAUG was identified as a defined binding motif that occurs mostly in binding sites in open reading frames and is bound by Rrm4’s third RRM. Interestingly, Rrm4 specifically recognises translational landmark sites such as start and stop codons, suggesting a close link between transport and translational regulation of target mRNAs. Thus, the endosomal transport machinery appears to use tailor-made transport strategies for distinct sets of its cargo mRNAs.In the second funding period, we will address (i) how the different transport strategies are achieved by Rrm4 via its three RRM domains, and (ii) what are the molecular consequences of binding to translational landmark sites for the spatio-temporal expression of the encoded proteins. To this end, we will team up with partners of the FOR2333 and combine global approaches, such as iCLIP and ribosome profiling. Thus, this work will lay the foundation to understand how RBPs with multiple RNA-binding domains and various interaction partners orchestrate their function during cellular processes such as mRNA transport.