ABSTRACT

Gemin5 is a multifaceted RNA-binding protein that comprises distinct structural domains, including a WD40 and TPR-like for which the X-ray structure is known. In addition, the protein contains a non-canonical RNA-binding domain (RBS1) towards the C-terminus. To understand the RNA binding features of the RBS1 domain, we have characterized its structural characteristics by solution NMR linked to RNA-binding activity. Here we show that a short version of the RBS1 domain that retains the ability to interact with RNA is predominantly unfolded even in the presence of RNA. Furthermore, an exhaustive mutational analysis indicates the presence of an evolutionarily conserved motif enriched in R, S, W, and H residues, necessary to promote RNA-binding via π-π interactions. The combined results of NMR and RNA-binding on wild-type and mutant proteins highlight the importance of aromatic and arginine residues for RNA recognition by RBS1, revealing that the net charge and the π-amino acid density of this region of Gemin5 are key factors for RNA recognition.

摘要

Gemin5 是一种多方面的 RNA 结合蛋白，包含不同的结构域，包括已知 X 射线结构的 WD40 和 TPR 样。此外，该蛋白质含有一个朝向 C 末端的非经典 RNA 结合域 (RBS1)。为了了解 RBS1 结构域的 RNA 结合特征，我们通过与 RNA 结合活性相关的溶液 NMR 对其结构特征进行了表征。在这里，我们表明即使在存在 RNA 的情况下，保留与 RNA 相互作用能力的 RBS1 结构域的短版本也主要是展开的。此外，详尽的突变分析表明存在富含 R、S、W 和 H 残基的进化保守基序，这是通过 π-π 相互作用促进 RNA 结合所必需的。