RNAs Containing Modified Nucleotides Fail To Trigger RIG-I Conformational Changes for Innate Immune Signaling.
RNAs Containing Modified Nucleotides Fail To Trigger RIG-I Conformational Changes for Innate Immune Signaling.
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DOI：
10.1128/mbio.00833-16
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发表时间：
2016-09-20
期刊：
影响因子：
6.4
通讯作者：
Gehrke L
中科院分区：
文献类型：
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作者：
Durbin AF;Wang C;Marcotrigiano J;Gehrke L
入侵的病原体核酸被细胞质（视黄酸诱导基因I [RIG-I]样）和膜结合（toll样）模式识别受体识别并结合，激活先天免疫信号。当修饰过的核苷酸存在于RNA分子中时，会降低这些信号反应的强度。然而，解释信号钝化的机制尚未阐明。在这项研究中，我们使用了几种独立的生物学试验，包括抑制病毒复制、RIG-I:RNA结合试验和限制胰蛋白酶对RIG-I:RNA复合物的消化，以开始了解含有修饰核苷酸的RNA如何避免或抑制先天免疫信号。实验是基于一个模型的先天免疫激活RNA分子，即丙型肝炎病毒的polyU/UC RNA结构域，该结构域在体外用典型核苷酸或八个修饰核苷酸中的一个进行转录。该方法揭示了与单个修饰核苷酸或修饰核苷酸类别相关的特征分析反应。例如，虽然n -6-甲基腺苷（m6A）和假尿嘧啶核苷酸都与信号传导减弱相关，但含有m6A修饰的RNA与RIG-I结合较差，而含有假尿嘧啶的RNA与RIG-I结合的亲和力较高，但无法触发与信号传导相关的典型RIG-I构象变化。这些数据促进了对RNA介导的先天免疫信号的理解，对于将核苷酸修饰应用于RNA治疗具有额外的相关性。先天免疫系统提供了对病毒感染的第一反应，必须区分宿主和病原体的核酸，以在不激活自身免疫反应的情况下建立保护性免疫反应。虽然已知RNA中核苷酸修饰的存在与先天免疫信号的减少有关，但其潜在机制尚未探索。这里报告的数据对于定义机制细节来解释含有修饰核苷酸的rna的信号抑制是重要的。结果表明，含有修饰核苷酸的rna在rig - i样先天免疫激活途径的早期步骤中断信号传导，并且具有相似化学结构的核苷酸修饰可以被组织成抑制或逃避先天免疫信号传导步骤的类别。这些数据有助于确定含有修饰核苷酸的rna对先天免疫信号抑制的分子基础。该结果对设计逃避先天免疫检测的治疗性rna具有重要意义。
Invading pathogen nucleic acids are recognized and bound by cytoplasmic (retinoic acid-inducible gene I [RIG-I]-like) and membrane-bound (Toll-like) pattern recognition receptors to activate innate immune signaling. Modified nucleotides, when present in RNA molecules, diminish the magnitude of these signaling responses. However, mechanisms explaining the blunted signaling have not been elucidated. In this study, we used several independent biological assays, including inhibition of virus replication, RIG-I:RNA binding assays, and limited trypsin digestion of RIG-I:RNA complexes, to begin to understand how RNAs containing modified nucleotides avoid or suppress innate immune signaling. The experiments were based on a model innate immune activating RNA molecule, the polyU/UC RNA domain of hepatitis C virus, which was transcribed in vitro with canonical nucleotides or with one of eight modified nucleotides. The approach revealed signature assay responses associated with individual modified nucleotides or classes of modified nucleotides. For example, while both N-6-methyladenosine (m6A) and pseudouridine nucleotides correlate with diminished signaling, RNA containing m6A modifications bound RIG-I poorly, while RNA containing pseudouridine bound RIG-I with high affinity but failed to trigger the canonical RIG-I conformational changes associated with robust signaling. These data advance understanding of RNA-mediated innate immune signaling, with additional relevance for applying nucleotide modifications to RNA therapeutics. The innate immune system provides the first response to virus infections and must distinguish between host and pathogen nucleic acids to mount a protective immune response without activating autoimmune responses. While the presence of nucleotide modifications in RNA is known to correlate with diminished innate immune signaling, the underlying mechanisms have not been explored. The data reported here are important for defining mechanistic details to explain signaling suppression by RNAs containing modified nucleotides. The results suggest that RNAs containing modified nucleotides interrupt signaling at early steps of the RIG-I-like innate immune activation pathway and also that nucleotide modifications with similar chemical structures can be organized into classes that suppress or evade innate immune signaling steps. These data contribute to defining the molecular basis for innate immune signaling suppression by RNAs containing modified nucleotides. The results have important implications for designing therapeutic RNAs that evade innate immune detection.