The Human antigen R protein (HuR) is an RNA-binding protein that recognizes U/AU-rich elements in diverse RNAs through two RNA-recognition motifs, RRM1 and RRM2, and post-transcriptionally regulates the fate of target RNAs. The natural product dihydrotanshinone-I (DHTS) prevents the association of HuR and target RNAs in vitro and in cultured cells by interfering with the binding of HuR to RNA. Here, we report the structural determinants of the interaction between DHTS and HuR and the impact of DHTS on HuR binding to target mRNAs transcriptome-wide. NMR titration and Molecular Dynamics simulation identified the residues within RRM1 and RRM2 responsible for the interaction between DHTS and HuR. RNA Electromobility Shifts and Alpha Screen Assays showed that DHTS interacts with HuR through the same binding regions as target RNAs, stabilizing HuR in a locked conformation that hampers RNA binding competitively. HuR ribonucleoprotein immunoprecipitation followed by microarray (RIP-chip) analysis showed that DHTS treatment of HeLa cells paradoxically enriched HuR binding to mRNAs with longer 3′UTR and with higher density of U/AU-rich elements, suggesting that DHTS inhibits the association of HuR to weaker target mRNAs. In vivo, DHTS potently inhibited xenograft tumor growth in a HuR-dependent model without systemic toxicity.

中文翻译：

---

二氢丹参酮-I对HuR结构和功能的调节

人类抗原R蛋白（HuR）是一种RNA结合蛋白，可通过两个RNA识别基序RRM1和RRM2识别各种RNA中富含U / AU的元素，并在转录后调节靶RNA的命运。天然产物二氢丹参酮-I（DHTS）可在体外阻止HuR与靶RNA的缔合并通过干扰HuR与RNA的结合来培养细胞。在这里，我们报告了DHTS与HuR之间的相互作用的结构决定因素以及DHTS对HuR结合至整个转录组的目标mRNA的影响。NMR滴定和分子动力学模拟确定了RRM1和RRM2中负责DHTS和HuR之间相互作用的残基。RNA电迁移率变化和Alpha筛选分析表明，DHTS通过与靶RNA相同的结合区与HuR相互作用，从而使HuR稳定在锁定构象中，从而阻碍了RNA竞争性结合。HuR核糖蛋白免疫沉淀后进行微阵列（RIP芯片）分析表明，DHTS处理HeLa细胞矛盾地富集了HuR与mRNA的结合，具有更长的3'UTR和更高的U / AU富集元素密度，在体内，DHTS在HuR依赖性模型中有效抑制异种移植瘤的生长，而没有全身毒性。