Institute for Lung Research, Philipps-University, 35043 Marburg, Germany.
Institute for Immunology, Philipps-University, 35043 Marburg, Germany.
Proc Natl Acad Sci U S A. 2020 Apr 21;117(16):9042-9053. doi: 10.1073/pnas.1920393117. Epub 2020 Apr 2.
RNA has been proposed as an important scaffolding factor in the nucleus, aiding protein complex assembly in the dense intracellular milieu. Architectural contributions of RNA to cytosolic signaling pathways, however, remain largely unknown. Here, we devised a multidimensional gradient approach, which systematically locates RNA components within cellular protein networks. Among a subset of noncoding RNAs (ncRNAs) cosedimenting with the ubiquitin-proteasome system, our approach unveiled ncRNA as a critical structural component of the Toll-like receptor 4 (TLR4) immune signal transduction pathway. RNA affinity antisense purification-mass spectrometry (RAP-MS) revealed binding to optineurin (OPTN), a ubiquitin-adapter platforming TBK1 kinase. binding stabilized OPTN, and consequently, loss of blunted OPTN aggregation, TBK1-dependent IRF3 phosphorylation, and type I interferon (IFN) gene transcription downstream of TLR4. expression was elevated in patients with active pulmonary infection and was highly correlated with IFN levels in bronchoalveolar lavage fluid. Our study uncovers as an integral RNA component of the TLR4-TRIF pathway and predicts further RNAs to be required for assembly and progression of cytosolic signaling networks in mammalian cells.
RNA 被认为是细胞核中一种重要的支架因子，有助于在密集的细胞内环境中组装蛋白质复合物。然而，RNA 对细胞质信号通路的结构贡献在很大程度上仍然未知。在这里，我们设计了一种多维梯度方法，可以系统地定位细胞内蛋白质网络中的 RNA 成分。在与泛素-蛋白酶体系统共沉淀的一组非编码 RNA (ncRNA) 中，我们的方法揭示了 ncRNA 是 Toll 样受体 4 (TLR4) 免疫信号转导途径的关键结构组成部分。RNA 亲和反义纯化-质谱 (RAP-MS) 显示 与泛素接头平台 TBK1 激酶的视神经病变蛋白 (OPTN) 结合。 与 OPTN 的结合稳定了 OPTN，因此， 的缺失削弱了 OPTN 聚集、TBK1 依赖性 IRF3 磷酸化以及 TLR4 下游的 I 型干扰素 (IFN) 基因转录。在患有活动性肺部感染的患者中， 的表达水平升高，并且与支气管肺泡灌洗液中的 IFN 水平高度相关。我们的研究揭示了 是 TLR4-TRIF 途径的一个完整的 RNA 组成部分，并预测进一步的 RNA 是组装和进展哺乳动物细胞中细胞质信号网络所必需的。
