Deciphering the molecular code of protein-RNA binding during mRNA localisation

破译 mRNA 定位过程中蛋白质-RNA 结合的分子密码

• 批准号: 283166754
• 负责人: Dr. Katharina Zarnack
• 金额: --
• 依托单位: Buchmann Institute for Molecular Life Sciences
• 依托单位国家: 德国
• 项目类别: Research Units
• 财政年份: 2015
• 资助国家: 德国
• 起止时间: 2014-12-31 至 2019-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/283166754?language=en
• 关键词: Deciphering; molecular; code; protein; RNA

mRNA localisation and local translation are regulated by RNA-binding proteins (RBPs) that constitute critical regulators of eukaryotic gene expression. Target mRNAs are recognised via cis-acting elements in the RNA sequence, so-called zipcodes or localisation elements, which are composed of characteristic sequence motifs and/or structural elements. RBPs recruit the target mRNAs into large transport particles which most often move actively along cytoskeletal tracks in the cytoplasm. Recently, evidence is accumulating that co-trafficking of mRNAs with membrane structures is another frequently occurring mode of intracellular mRNA transport. In order to address the prevalence and possible functions of this new transport route, we will perform a comprehensive survey of membrane-associated RBPs in fungi and mammals in collaboration with several groups of the FOR2333.Over the last years, new ribonomic techniques have been introduced that allow to study RBP binding and its regulatory outcome on a genomic scale, including UV crosslinking and immunoprecipitation (CLIP) and related methods to map RBP binding sites in vivo. However, the analysis and interpretation of these data is still in its infancy. In order to overcome these limitations and reliably map and quantify RBP binding, we will benchmark existing tools based on publicly available iCLIP datasets for different RBPs. Moreover, we will develop robust quantification strategies that correct for differences in transcript abundance, among others. The resulting RBP binding sites will be characterised in terms of motif occurrence, binding site conformations and RNA secondary structure formation. By interrelating these features we will obtain new insights into the molecular code that determines RBP binding. In collaboration with several groups of the FOR2333, we will exploit the established analysis workflows to learn more about the RNA binding mode of the She mRNA transport complex in yeast, the target recognition of the translational regulator Pumilio2 in mammalian neurons and the functional impact of the exon-junction complex on mRNA localisation in fruit flies.

mRNA定位和局部翻译由RNA结合蛋白（RBP）调节，这些蛋白构成真核基因表达的关键调节因子。靶mRNA通过RNA序列中的顺式作用元件（所谓的邮政编码或定位元件）识别，其由特征性序列基序和/或结构元件组成。RBP将靶mRNA募集到大的转运颗粒中，这些转运颗粒通常沿着细胞质中的细胞骨架轨道活跃地移动。最近，越来越多的证据表明，与膜结构的mRNA的共同运输是细胞内mRNA运输的另一种常见模式。为了解决这种新的运输途径的流行和可能的功能，我们将与FOR 2333的几个小组合作，对真菌和哺乳动物中的膜相关RBP进行全面调查。在过去的几年里，新的核糖组学技术已经引入，可以在基因组水平上研究RBP结合及其调控结果，包括UV交联和免疫沉淀（CLIP）以及相关方法来绘制体内RBP结合位点。然而，对这些数据的分析和解释仍处于起步阶段。为了克服这些局限性并可靠地映射和量化RBP结合，我们将基于公开可用的iCLIP数据集对现有工具进行基准测试。此外，我们将开发强大的量化策略，以纠正转录丰度的差异等。所得的RBP结合位点将在基序发生，结合位点构象和RNA二级结构形成方面进行表征。通过将这些特征相互关联，我们将获得对决定RBP结合的分子密码的新见解。在与FOR 2333的几个小组的合作中，我们将利用已建立的分析工作流程来更多地了解酵母中She mRNA转运复合物的RNA结合模式，哺乳动物神经元中翻译调节因子Pumilio 2的靶向识别以及果蝇中外显子连接复合物对mRNA定位的功能影响。