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

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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.