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

适当的免疫反应需要CD4+ T细胞做出深刻的细胞命运决定，这些决定受到转录后控制。在这些决定中起关键作用的是rna结合蛋白（RBP） Roquin。虽然Roquin与其共识衰变元件的原型相互作用以及随后的死基化和靶mRNA的脱帽已被很好地理解，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。它不仅涉及与额外的辅因子和不同的转录后效应物的相互作用，导致不同的转录后调控模式，而且还涉及与mrna和复合结合位点的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的抑制。我们和同事发现的其他共同调节因子包括内切酶regase -1和死化因子Cnot1。因此，在第二个资助期，我们将表征和量化Roquin与Nufip2、Regnase-1和cont1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、微电子衍射和核磁共振对它们的共配合物进行结构分析。我们将利用这些信息来产生特异性地消除特定相互作用的突变，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，我们在Icos mRNA的2.6 kb长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，我们将对这一诱变筛选的结果进行深入分析。在筛选中发现的顺式作用元件将使用CRISPR/Cas9技术在T细胞中重新评估其调节功能的可重复性，并了解其特定的调节模式。此外，我们的目标是通过使用rna连接的接近连接方法来鉴定与这些顺式元件结合的蛋白质。通过这种方法鉴定的rna结合蛋白和辅助因子，将评估它们与相应的顺式作用元件以及Roquin， Regnase-1和Nufip2的结合。在T细胞中，最相关因子的重要性和特定功能将通过敲除进行评估，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助因子合作选择特定的翻译抑制模式，我们旨在确定Roquin-1依赖性靶mrna的组合转录后基因调控原理。

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.