Indentification of RNA G-quadruplex binding proteins and analysis of the biological relevance of the protein-RNA G-quadruplex interaction

近年来，RNA分子中的鸟嘌呤四链(G-四链，GQs)被认为是基因表达的重要调控元件。作为大量mRNAs 5‘UTRs中的结构基序，它们在不改变mRNA水平的情况下减少蛋白质的合成。由于对翻译的抑制只是部分的，抑制的程度可能会受到额外的细胞因素的调节。该项目旨在识别与GQ结合的蛋白质，并研究蛋白质-GQ相互作用对细胞过程的影响。我们的团队首次发现肿瘤相关基因(ZIC-1)5‘非编码区的GQ抑制真核细胞中的翻译。在进一步的实验中，将继续与这里描述的方法交互作用，两个示例性GQ(在ARPC2和MMP16的mRNAs中)被用于识别细胞蛋白质作为相互作用伙伴。为了获得更全面的图像，现在将对NRAS和Bcl2的mRNAs中的GQ进行研究。GQ在序列和结构上各不相同，因此可以预期与细胞伙伴的交互会有所不同。最重要的是，NRAS和Bcl2在细胞增殖和凋亡的调控中发挥着重要的作用，因此我们旨在研究在这些重要的细胞过程中，蛋白质-Gq-相互作用在调节蛋白质合成方面的相关性。该项目的一个中心步骤是通过下拉分析和质谱仪(MALDI-TOF)鉴定Gq结合蛋白。对几种GQ结构的分析将允许区分具有与GQ结构结合的一般性质的蛋白质和特定于给定GQ的蛋白质。其他与RNA有普遍亲和力的蛋白质将被排除在适当的对照之外。随后将通过重组表达产生识别的蛋白质，以便通过表面等离子体共振(SPR)光谱确定体外结合常数。在进一步的实验中，荧光素酶报告将通过体外蛋白质生物合成试验在5‘UTR中的GQ基序控制下表达，将研究所识别的蛋白质介导的抑制程度。我们将分析结合亲和力和抑制效应之间的假设相关性。候选蛋白在真核细胞中的过表达和沉默将被用来研究GQ结合蛋白对活体翻译的影响。拟议项目的最后一部分旨在研究GQ结合蛋白与RNA GQs相互作用的生物学后果。通过稳定或破坏各自的GQ结构，将评估NRAS和Bcl2的翻译在调节中枢细胞过程中的后果，如增殖和凋亡，并寻求通过外部因素影响调节的方法。

Guanine-quadruplexes (G-quadruplexes, GQs) in RNA molecules have recently been described as important regulatory elements of gene expression. As structural motifs in the 5' UTRs of numerous mRNAs, they reduce protein synthesis without altering the mRNA level. Since the inhibition of translation is only partial, the extent of repression is likely to be regulated by additional cellular factors. The proposed project aims at identifying proteins that bind to the GQs and at investigating the influence of the protein-GQ interaction on cellular processes.Our group showed for the first time that a GQ in the 5' UTR of a tumor-relevant gene (zic-1) inhibits translation in eukaryotic cells. In further experiments, which will be continued in interaction with the approach described here, two exemplary GQs (in the mRNAs of ARPC2 and MMP16) were used to identify cellular proteins as interaction partners. In order to obtain a more comprehensive picture, the GQs in the mRNAs of NRAS and Bcl-2 will now be investigated. The GQs vary in sequence and structure, so that differences in the interactions with cellular partners can be expected. Most importantly, NRAS and Bcl-2 play an important role in the regulation of cell proliferation and apoptosis, so that we aim at investigating the relevance of protein-GQ-interactions in regulating protein synthesis in these important cellular processes.A central step of the proposed project is the identification of the GQ-binding proteins by pull-down assays followed by mass spectrometry (MALDI-TOF). The analysis of several GQ-structures will allow distinguishing between proteins with the general property to bind to GQ-structures and proteins that are specific for a given GQ. Further proteins with a general affinity to RNA will be excluded with suitable controls.The identified proteins will subsequently be produced by recombinant expression, in order to determine in vitro binding constants by Surface Plasmon Resonance (SPR) spectroscopy. In further experiments, in which a luciferase reporter will be expressed by in vitro protein biosynthesis assays under control of a GQ-motif in the 5' UTR, the extent of repression mediated by the identified proteins will be investigated. We will analyze a hypothesized correlation between binding affinity and the inhibitory effect. Overexpression and silencing of the candidate proteins in eukaryotic cells will then be used to study the effects of GQ-binding proteins on translation in vivo.The last part of the proposed project aims at investigating the biological consequences of the interaction between the GQ-binding proteins and RNA GQs. By stabilizing or destabilizing the respective GQ-structures, the consequences of the modulation of translation of NRAS and Bcl-2 in modulating central cellular processes such as proliferation and apoptosis will be assessed and a means to influence the modulation through external factors sought.