Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA.
Genes Dev. 2013 Apr 15;27(8):928-40. doi: 10.1101/gad.216531.113.
Mammalian Quaking (QKI) and its Caenorhabditis elegans homolog, GLD-1 (defective in germ line development), are evolutionarily conserved RNA-binding proteins, which post-transcriptionally regulate target genes essential for developmental processes and myelination. We present X-ray structures of the STAR (signal transduction and activation of RNA) domain, composed of Qua1, K homology (KH), and Qua2 motifs of QKI and GLD-1 bound to high-affinity in vivo RNA targets containing YUAAY RNA recognition elements (RREs). The KH and Qua2 motifs of the STAR domain synergize to specifically interact with bases and sugar-phosphate backbones of the bound RRE. Qua1-mediated homodimerization generates a scaffold that enables concurrent recognition of two RREs, thereby plausibly targeting tandem RREs present in many QKI-targeted transcripts. Structure-guided mutations reduced QKI RNA-binding affinity in vitro and in vivo, and expression of QKI mutants in human embryonic kidney cells (HEK293) significantly decreased the abundance of QKI target mRNAs. Overall, our studies define principles underlying RNA target selection by STAR homodimers and provide insights into the post-transcriptional regulatory function of mammalian QKI proteins.
哺乳动物 Quaking（QKI）及其秀丽隐杆线虫同源物 GLD-1（生殖系发育缺陷）是进化上保守的 RNA 结合蛋白，它们在后转录水平上调节对发育过程和髓鞘形成至关重要的靶基因。我们展示了由 Qua1、KH 和 Qua2 结构域组成的 STAR（信号转导和 RNA 激活）结构域的 X 射线结构，该结构域与包含 YUAAY RNA 识别元件（RRE）的高亲和力体内 RNA 靶标结合。STAR 结构域的 KH 和 Qua2 结构域协同作用，特异性地与结合 RRE 的碱基和糖磷酸骨架相互作用。Qua1 介导的同源二聚化生成一个支架，能够同时识别两个 RRE，从而可能靶向许多 QKI 靶标转录本中存在的串联 RRE。结构导向突变降低了 QKI 在体外和体内的 RNA 结合亲和力，并且在人胚肾细胞（HEK293）中表达 QKI 突变体显著降低了 QKI 靶标 mRNA 的丰度。总体而言，我们的研究定义了 STAR 同源二聚体选择 RNA 靶标的原则，并为哺乳动物 QKI 蛋白的转录后调节功能提供了见解。
