PDF(Pubmed)
Abstract：
Microscale thermophoresis (MST) is a well-established method to quantify protein-RNA interactions. In this study, we employed MST to analyze the RNA binding properties of glycine-rich RNA binding protein 7 (GRP7), which is known to have multiple biological functions related to its ability to bind different types of RNA. However, the exact mechanism of GRP7\'s RNA binding is not fully understood. While the RNA-recognition motif of GRP7 is known to be involved in RNA binding, the glycine-rich region (known as arginine-glycine-glycine-domain or RGG-domain) also influences this interaction. To investigate to which extend the RGG-domain of GRP7 is involved in RNA binding, mutation studies on putative RNA interacting or modulating sites were performed. In addition to MST experiments, we examined liquid-liquid phase separation of GRP7 and its mutants, both with and without RNA. Furthermore, we systemically investigated factors that might affect RNA binding selectivity of GRP7 by testing RNAs of different sizes, structures, and modifications. Consequently, our study revealed that GRP7 exhibits a high affinity for a variety of RNAs, indicating a lack of pronounced selectivity. Moreover, we established that the RGG-domain plays a crucial role in binding longer RNAs and promoting phase separation.
摘要：
微尺度热电泳（MST）是一种公认的定量蛋白质-RNA相互作用的方法。在这项研究中,我们使用MST分析富含甘氨酸的RNA结合蛋白7（GRP7）的RNA结合特性，已知其具有与其结合不同类型RNA的能力相关的多种生物学功能。然而,GRP7的RNA结合的确切机制尚不完全清楚。虽然已知GRP7的RNA识别基序参与RNA结合，富含甘氨酸的区域(称为精氨酸-甘氨酸-甘氨酸-结构域或RGG-结构域)也影响这种相互作用。为了研究GRP7的RGG结构域与RNA结合有关的延伸，对推定的RNA相互作用或调节位点进行突变研究。除了MST实验，我们检查了GRP7及其突变体的液-液相分离，有和没有RNA。此外，我们通过测试不同大小的RNA，系统地研究了可能影响GRP7RNA结合选择性的因素，结构,和修改。因此,我们的研究表明，GRP7对多种RNA表现出很高的亲和力，表明缺乏明显的选择性。此外，我们确定RGG结构域在结合较长的RNA和促进相分离中起着至关重要的作用。
