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

mRNA的转运对于确定蛋白质在细胞中何时何地表达至关重要。玉米黑粉菌侵染性菌丝中内体mRNA沿着微管转运是一个研究充分的易位机制。关键因子是Rrm4，其含有用于RNA结合的三个RNA识别基序（RRM）结构域。最近，我们确定了小甘氨酸丰富的RNA结合蛋白（RBP）Grp1作为一种新的相互作用的合作伙伴和组成部分穿梭内体mRNP。使用两个共定位的RBP的比较研究，我们投的第一个转录组范围内的内体mRNA运输的观点。这表明这两种蛋白质共享约2，000个共同靶mRNA的大集合。这两种RBP优先结合在3 ′ UTR附近，表明结合功能。在Rrm4的情况下，序列UAUG被鉴定为限定的结合基序，其主要出现在开放阅读框中的结合位点中，并被Rrm4的第三RRM结合。有趣的是，Rrm4特异性识别翻译标志性位点，如起始和终止密码子，这表明靶mRNA的转运和翻译调控之间存在密切联系。因此，内体转运机制似乎使用量身定制的运输策略，其货物mRNA.In第二个资金周期，我们将解决（i）不同的运输策略是如何通过RRM 4通过其三个RRM结构域实现的，以及（ii）什么是结合到翻译的地标网站的编码蛋白质的时空表达的分子后果。为此，我们将与FOR 2333的合作伙伴合作，并结合联合收割机的全球方法，如iCLIP和核糖体分析。因此，这项工作将为理解具有多个RNA结合结构域和各种相互作用伙伴的RBP如何在mRNA转运等细胞过程中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.