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

适当的免疫应答需要CD 4 + 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参与者是RNA结合蛋白（RBP）Roquin。虽然Roquin与其共有衰变元件的原型相互作用以及随后的靶mRNA的去腺苷化和去帽是很好理解的，但许多观察结果暗示Roquin介导的基因调控的复杂性要高得多。它不仅涉及与额外的辅因子和不同的转录后效应子的相互作用，导致不同的转录后调节模式，而且还涉及与mRNA中的不同识别元件和复合结合位点的相互作用。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Nufip 2相互作用，从而增加Icos mRNA的抑制。我们和同事鉴定的其他共调节因子包括核酸内切酶Regnase-1和去腺苷化因子Cnot 1。因此，在第二个资助期内，我们将表征和量化Roquin与Nufip 2，Regnase-1和Cnot 1的相互作用，在直接相互作用的情况下绘制它们的相互作用表面，并通过X射线晶体学，微电子衍射和NMR对其共复合物进行结构分析。我们将利用这些信息来产生突变，专门消除一个特定的相互作用，验证他们的结合损失，并在T细胞中表征其对基因调控的全局影响。在第一个资助期内，我们在Icos mRNA的2.6 kb长的3 '-UTR中进行了超过10，000个不同突变的饱和诱变筛选，并使用下一代测序来量化突变对Icos mRNA调控的影响。在第二个资助期内，我们将对诱变筛选的结果进行深入分析。将使用CRISPR/Cas9技术在T细胞中重新评估筛选中鉴定的顺式作用元件，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栓系邻近连接方法来鉴定与这些顺式元件结合的蛋白质。将评估以这种方式鉴定的RNA结合蛋白和辅因子与相应的顺式作用元件以及Roquin、Regnase-1和Nufip 2的结合。最相关因子的重要性和特异性功能将在T细胞中通过敲除进行评估，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以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的原理。

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.