Cis- and trans-acting determinants of mRNA stability, structure and fate (as a continuation)

转录后调节操纵基因表达，例如，在发育和免疫反应中。mRNA在其3 '-非翻译区（UTR）中含有结构化和非结构化顺式调节元件，其被具有特定结构域（RBD）的反式作用RNA结合蛋白（RBP）识别，从而介导mRNA的命运。我们经常缺乏关于RNA序列，结构和修饰在调节蛋白-mRNA枢纽（mRNP）中的作用的细节。此外，mRNP的3D排列通常在总体或相关细节上逃避结构分析。在此提议之前的工作中，我们已经解决了由多个线性或结构化顺式调控元件组成的RNA调控中心在被多结构域RBP IMP 3和Roquin读取时对靶mRNA命运的作用。这两种蛋白质各自显著影响特定的mRNA监视：Roquin通过识别mRNA茎环参与免疫调节; IMP 3，一种致癌RBP，通过与多个短线性mRNA顺式元件的保护性接合与癌症进展相关。扩展单一的顺-反，即RNA基序RBD相互作用的静态图片，我们已经发现了特定的mRNA识别和处理的两个系统的顺-反网络。一个重点是多顺式元件组合物在序列中的作用相比，它们在mRNP的三维排列，并得出mRNA的命运决定的一般概念。我们的工作，以及其他实验室的新见解，引发了与上述相关的新问题。这包括由蛋白质-蛋白质（PPI）和RNA-RNA相互作用介导的RNP化学计量的作用，以及特别是受m6 A-甲基化影响的RNA结构对复合物形成的作用。作为先前工作的后续延续，该提案将在剩余和新出现的目标中解决蛋白质及其靶RNA。基于在Roquin ROQ结构域与T细胞共受体Ox 40的3 'UTR的复合物形成中获得的知识，我们将使用X射线晶体学和Cryo-EM来获得具有多个结构化顺式元件的高分辨率结构。此外，我们将研究Roquin-和目标RNA寡聚体的作用与NMR和天然质谱的帮助下RNP形成。两者一起有助于通过整合结构生物学确定Roquin N-末端与组合靶RNA基序的高分辨率结构。为了识别多结构域RBP IMP 3在癌基因mRNA中的成簇ssRNA元件，我们将使用系统的RNP生物化学和SAXS关注KH 3 -4介导的PPI对RNA亲和力和特异性的作用。这将与使用质谱方法在全长背景下扩展IMP3 RBD-RBD和RBD-RNA界面的工作有关。最后，基于NMR和辅助生物物理方法的项目部分将解决IMP 3的串联RBD与充分研究的m6 A读取器IMP 1相比的差异m6 A-RNA识别。

Posttranscriptional regulation steers gene expression e.g., during development and immune response. mRNAs contain structured and unstructured cis-regulatory elements in their 3'-untranslated regions (UTR), recognized by trans-acting RNA-binding proteins (RBPs) with specified domains (RBDs) mediating the mRNA’s fate. We often lack details about the role of RNA sequence, structure and modification in regulatory protein-mRNA hubs (mRNPs). Also, the 3D-arrangement of mRNPs often escapes structural analysis, in total or relevant details. In the work prior to this proposal, we have tackled the role of RNA-regulatory hubs composed of either multiple linear or structured cis-regulatory elements for the fate of target mRNAs when read by the multi-domain RBPs IMP3 and Roquin. The two proteins each significantly affect specific mRNA surveillance: Roquin in immune regulation by recognition of mRNA stem-loops; IMP3, an oncogenic RBP relevant in cancer progression through protective engagement with multiple short linear mRNA cis elements. Extending the static picture of single cis-trans, i.e. RNA motif-RBD interactions, we have discovered cis-trans networks in specific mRNA recognition and processing for both systems. A focus was on the role of multiple-cis-element composition in sequence as compared to their 3D-arrangement in mRNPs and to derive general concepts of mRNA fate-decision. Our work, and novel insights from other labs, have provoked pestering new questions to address, related to the above. This includes the role of RNP stoichiometries mediated by protein-protein (PPI) and RNA-RNA interactions as well as, specifically, the role of RNA structure influenced by m6A-methylation for complex formation. As a consequent continuation of the prior work, this proposal will address both proteins and their target RNAs in the remaining and newly occurring aims. Based on the obtained knowledge in complex formation of the Roquin ROQ domain with the 3’UTR of T cell co-receptor Ox40, we will use X-ray crystallography and Cryo-EM to obtain high-resolution structures with multiple structured cis elements. Further, we will investigate the role of Roquin- and target RNA oligomers for RNP formation with the help of NMR and native mass spectrometry. Both together shall aid the determination of a high-resolution structure of the Roquin N-terminus with a combined target RNA motif via integrated structural biology. For the recognition of clustered ssRNA elements in oncogene-mRNAs by the multi-domain RBP IMP3 we will focus on the role of KH3-4-mediated PPI for RNA-affinity and -specificity using systematic RNP biochemistry and SAXS. This will engage with work on expanded IMP3 RBD-RBD and RBD-RNA interfaces in full-length contexts using mass spectrometry approaches. Finally, a project part on the basis of NMR and accessory biophysical approaches will address the differential m6A-RNA recognition by tandem RBDs of IMP3 in comparison to the well-studied m6A-reader IMP1.