Differential protein- and RNA-interactions of Roquin specify alternative modes of post-transcriptional gene regulation

Roquin 的差异蛋白和 RNA 相互作用指定了转录后基因调控的替代模式

• 批准号: 313381103
• 负责人: Professor Dr. Vigo Heissmeyer
• 金额: --
• 依托单位: Forschungsbereich Immunologie
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/313381103?language=en
• 关键词: Differential; protein; RNA; interactions; Roquin

Appropriate immune responses require CD4+ T cells to make profound cell fate decisions, which are placed under post-transcriptional control. A key player in these decisions is the RNA-binding protein (RBP) Roquin. While the prototypic interaction of Roquin with its consensus decay element and subsequent deadenylation and decapping of the target mRNA is well understood, many observations imply a much higher complexity of Roquin-mediated gene regulation. It not only involves interactions with additional cofactors and different post-transcriptional effectors causing different modes of post-transcriptional regulation but also interactions with different recognition elements in mRNAs and composite binding sites. In the first funding period, we studied the post-transcriptional regulation of Icos mRNA by the RNA-binding protein Roquin-1. We could show that Roquin-1 directly interacts with its cofactor Nufip2 and thereby increases Icos mRNA repression. Additional co-regulators identified by us and colleagues include the endonuclease Regnase-1 and the deadenylation factor Cnot1. In the second funding period we will therefore characterize and quantify the interaction of Roquin with Nufip2, Regnase-1, and Cnot1, map their interaction surfaces in case of direct interactions, and perform structural analyses of their co-complexes by X-ray crystallography, micro electron diffraction, and NMR. We will use this information to generate mutations that specifically abolish a particular interaction, verify their loss of binding, and characterize in T cells their global effects on gene regulation. In the first funding period, we performed a saturating mutagenesis screen with over 10,000 distinct mutations in the 2.6 kb long 3‘-UTR of Icos mRNA and used next-generation sequencing to quantify the effects of mutations on Icos mRNA regulation. In the second funding period we will perform an in-depth analysis of the results of this mutagenesis screen. Cis-acting elements identified in the screen will be reassessed in T cells using the CRISPR/Cas9 technology for reproducibility of their regulatory function and to understand their specific modes of regulation. Furthermore, we aim to identify proteins binding close to these cis-elements by using a RNA-tethered proximity ligation approach. RNA-binding proteins and co-factors identified this way, will be assessed for their binding to the corresponding cis-acting element, as well as to Roquin, Regnase-1 and Nufip2. The importance and specific functions of the most relevant factors will be assessed in T cells by knock-out, followed by target gene analysis. In summary, by elucidating how Roquin-1 cooperates with different trans-acting co-factors to select specific modes of translational inhibition we aim at identifying principles of combinatorial post-transcriptional gene regulation of Roquin-dependent target mRNAs.

适当的免疫反应需要 CD4+ T 细胞做出深刻的细胞命运决定，这些决定置于转录后控制之下。这些决定中的一个关键角色是 RNA 结合蛋白 (RBP) Roquin。虽然 Roquin 与其共有衰变元件以及随后的目标 mRNA 的脱腺苷化和脱帽的原型相互作用已被充分了解，但许多观察结果表明 Roquin 介导的基因调控的复杂性要高得多。它不仅涉及与其他辅因子和不同转录后效应子的相互作用，导致不同的转录后调节模式，还涉及与 mRNA 和复合结合位点中不同识别元件的相互作用。在第一个资助期，我们研究了RNA结合蛋白Roquin-1对Icos mRNA的转录后调控。我们可以证明 Roquin-1 直接与其辅因子 Nufip2 相互作用，从而增加 Icos mRNA 抑制。我们和同事发现的其他协同调节因子包括核酸内切酶 Regnase-1 和去腺苷酸化因子 Cnot1。因此，在第二个资助期，我们将表征和量化 Roquin 与 Nufip2、Regnase-1 和 Cnot1 的相互作用，绘制直接相互作用情况下的相互作用表面，并通过 X 射线晶体学、微电子衍射和 NMR 对其共配合物进行结构分析。我们将利用这些信息来生成突变，专门消除特定的相互作用，验证它们的结合丧失，并在 T 细胞中表征它们对基因调控的整体影响。在第一个资助期，我们对 Icos mRNA 2.6 kb 长的 3'-UTR 中超过 10,000 个不同的突变进行了饱和诱变筛选，并使用下一代测序来量化突变对 Icos mRNA 调节的影响。在第二个资助期，我们将对诱变筛选的结果进行深入分析。筛选中鉴定出的顺式作用元件将使用 CRISPR/Cas9 技术在 T 细胞中重新评估，以重现其调节功能并了解其特定的调节模式。此外，我们的目标是通过使用 RNA 系留的邻近连接方法来识别与这些顺式元件结合的蛋白质。通过这种方式鉴定的 RNA 结合蛋白和辅因子将评估其与相应顺式作用元件以及 Roquin、Regnase-1 和 Nufip2 的结合。最相关因子的重要性和特定功能将通过敲除在 T 细胞中进行评估，然后进行靶基因分析。总之，通过阐明 Roquin-1 如何与不同的反式作用辅因子合作来选择特定的翻译抑制模式，我们的目标是确定 Roquin 依赖性靶 mRNA 的组合转录后基因调控原理。