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

mRNA 稳定性、结构和命运的顺式和反式作用决定因素（续）

• 批准号: 404540462
• 负责人: Dr. Andreas Schlundt
• 金额: --
• 依托单位: Institut für Molekulare Biowissenschaften
• 依托单位国家: 德国
• 项目类别: Independent Junior Research Groups
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/404540462?language=en
• 关键词: Cis; trans; acting; determinants; mRNA

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.

转录后调控调控基因的表达，例如，在发育和免疫反应期间。MRNAs在其3‘-非翻译区(UTR)中含有结构化和非结构化顺式调控元件，由具有特定结构域(RBD)的反式作用RNA结合蛋白(RBPs)识别，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中心(MRNPs)中的作用的详细信息。此外，mRNPs的3D排列往往在总体或相关细节上逃避结构分析。在本提案之前的工作中，我们解决了由多个线性或结构化顺式调控元件组成的RNA调控中心在被多域限制性商业惯例IMP3和Roquin读取时对靶mRNAs命运的作用。这两种蛋白质都显著影响特异性的mRNA监控：Roquin通过识别mRNA茎环进行免疫调节；IMP3，一种致癌的RBP，通过与多个短线性mRNA顺式元件的保护性接触而与癌症进展相关。扩展单一顺式-反式，即RNA基序-RBD相互作用的静态图像，我们发现在两个系统特定的mRNA识别和处理中都存在顺式-反式网络。重点是多顺式元件组成在序列中的作用与它们在mRNPs中的三维排列的比较，并推导出决定mRNPs命运的一般概念。我们的工作，以及来自其他实验室的新颖见解，引发了与上述相关的新问题的纠缠。这包括由蛋白质-蛋白质(PPI)和RNA-RNA相互作用介导的RNP化学计量学的作用，以及特别是m6A-甲基化影响的RNA结构在形成复合体方面的作用。作为先前工作的后续继续，本提案将在剩余和新出现的目标中处理蛋白质及其靶RNA。基于所获得的Roquin ROQ结构域与T细胞共受体OX40的3‘UTR形成络合物的知识，我们将使用X射线结晶学和Cryo-EM来获得具有多个结构顺式元件的高分辨结构。此外，我们将借助核磁共振和自然质谱学来研究Roquin-和目标RNA低聚体在RNP形成中的作用。两者共同有助于通过集成结构生物学确定具有组合靶标RNA基序的Roquin N-末端的高分辨率结构。为了用多结构域RBP IMP3识别癌基因-mRNAs中聚集的ssRNA元件，我们将利用系统的RNP生化和SAXS技术重点研究Kh3-4介导的PPI对RNA亲和力和特异性的作用。这将涉及使用质谱学方法在全长背景下扩展IMP3 RBD-RBD和RBD-RNA接口的工作。最后，一个基于核磁共振和辅助生物物理方法的项目部分将解决IMP3的串联RBDS与研究充分的M6A阅读器IMP1相比的差异M6A-RNA识别问题。