Structure of human IFIT1 with capped RNA reveals adaptable mRNA binding and mechanisms for sensing N1 and N2 ribose 2′-O methylations
Structure of human IFIT1 with capped RNA reveals adaptable mRNA binding and mechanisms for sensing N1 and N2 ribose 2′-O methylations
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DOI：
10.1073/pnas.1612444114
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发表时间：
2017-03-14
影响因子：
11.1
通讯作者：
Nagar, Bhushan
中科院分区：
文献类型：
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作者：
Abbas, Yazan M.;Laudenbach, Beatrice Theres;Nagar, Bhushan
IFIT1(干扰素诱导蛋白与四肽重复序列-1)是宿主先天免疫抗病毒反应的效应物，通过选择性地抑制病毒mRNA的翻译来阻止病毒感染的传播。它依赖于与翻译起始因子eIF4F竞争的能力来特异性识别外源封顶的mRNAs，而对宿主mRNAs保持不活跃，该mRNAs在第一帽-近端核苷酸(N1)处以核糖2‘-O甲基化为标志。我们在这里报道了几种与RNA结合的人Ifit1的晶体结构，包括一个1.6埃的带帽RNA的复合体。IFT1形成一个充满水的、带正电荷的RNA结合隧道，带有一个单独的疏水延伸部分，意外地以多种构象(syn和anti)与帽结合，从而产生一种相对可塑性和非特异性的结合模式，这与eIF4E形成了鲜明的对比。被隧道包围的帽-近端核苷酸提供了与eIF4F竞争的亲和力，同时允许IFIT1选择对抗N1甲基化的mRNA。凝胶位移结合分析证实，N1甲基化干扰IFIT 1结合，但干扰方式依赖于RNA，而翻译分析表明，仅N1甲基化不足以阻止在高浓度IFIT 1下的mRNA识别。结构和功能分析表明，N_2位的2‘-O甲基化，另一个丰富的mRNA修饰，也不利于RNA的结合，从而揭示了它在自我和非自我mRNA识别中的潜在协同作用。最后，结构导向突变分析证实了RNA结合对于缺乏病毒N1甲基化的人类冠状病毒突变株的Ifit1限制的重要性。我们的结构和生化分析为IFIT1对封顶病毒RNA的翻译抑制提供了新的分子基础。
IFIT1 (IFN-induced protein with tetratricopeptide repeats-1) is an effector of the host innate immune antiviral response that prevents propagation of virus infection by selectively inhibiting translation of viral mRNA. It relies on its ability to compete with the translation initiation factor eIF4F to specifically recognize foreign capped mRNAs, while remaining inactive against host mRNAs marked by ribose 2'-O methylation at the first cap-proximal nucleotide (N1). We report here several crystal structures of RNA-bound human IFIT1, including a 1.6-angstrom complex with capped RNA. IFIT1 forms a water-filled, positively charged RNA-binding tunnel with a separate hydrophobic extension that unexpectedly engages the cap in multiple conformations (syn and anti) giving rise to a relatively plastic and nonspecific mode of binding, in stark contrast to eIF4E. Cap-proximal nucleotides encircled by the tunnel provide affinity to compete with eIF4F while allowing IFIT1 to select against N1 methylated mRNA. Gel-shift binding assays confirm that N1 methylation interferes with IFIT1 binding, but in an RNA-dependent manner, whereas translation assays reveal that N1 methylation alone is not sufficient to prevent mRNA recognition at high IFIT1 concentrations. Structural and functional analysis show that 2'-O methylation at N2, another abundant mRNA modification, is also detrimental for RNA binding, thus revealing a potentially synergistic role for it in self-versus nonself-mRNA discernment. Finally, structure-guided mutational analysis confirms the importance of RNA binding for IFIT1 restriction of a human coronavirus mutant lacking viral N1 methylation. Our structural and biochemical analysis sheds new light on the molecular basis for IFIT1 translational inhibition of capped viral RNA.