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

RNA分子中的鸟嘌呤四链体（G-quadruplexes，GQs）最近被描述为基因表达的重要调控元件。作为许多mRNA的5'UTR中的结构基序，它们减少蛋白质合成而不改变mRNA水平。由于对翻译的抑制只是部分的，因此抑制的程度可能受其他细胞因子的调节。本项目的目的是鉴定与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。我们的研究小组首次发现，肿瘤相关基因（zic-1）的5' UTR中的GQ抑制真核细胞的翻译。在进一步的实验中，其将继续与本文所述的方法相互作用，使用两个示例性GQ（在ARPC 2和MMP 16的mRNA中）来鉴定作为相互作用伴侣的细胞蛋白。为了获得更全面的图像，现在将研究NRAS和Bcl-2的mRNA中的GQ。GQ在序列和结构上各不相同，因此可以预期与细胞伴侣的相互作用存在差异。由于NRAS和Bcl-2在细胞增殖和凋亡的调控中起着重要作用，因此本研究旨在探讨蛋白质-GQ相互作用在这些重要细胞过程中调控蛋白质合成的相关性，其中一个核心步骤是通过pull-down assay followed by mass spectrometry（MALDI-TOF）鉴定GQ结合蛋白。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对给定GQ特异的蛋白质。随后通过重组表达产生鉴定的蛋白质，以便通过表面等离子体共振（SPR）光谱测定体外结合常数。在进一步的实验中，其中荧光素酶报告基因将通过体外蛋白质生物合成测定在5' UTR中的GQ-基序的控制下表达，将研究由鉴定的蛋白质介导的阻遏程度。我们将分析结合亲和力和抑制作用之间的假设相关性。在真核细胞中过表达和沉默候选蛋白将用于研究GQ结合蛋白对体内翻译的影响。本项目的最后一部分旨在研究GQ结合蛋白与RNA GQ相互作用的生物学后果。通过稳定或去稳定各自的GQ结构，将评估NRAS和Bcl-2的翻译在调节中枢细胞过程如增殖和凋亡中的调节结果，并寻求通过外部因素影响调节的手段。

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.