RIG-I是先天免疫模式识别受体家族中的重要成员，长期来，研究均集中于它作为受体识别外源病毒RNA的功能及机理。本项目拟在哺乳动物及我们前期研究的基础上，首次在大黄鱼中探索RIG-I识别内源性microRNA（miRNA）调节中性粒细胞分化发育的机制。通过野生型和突变型RIG-I与miRNA136/145互作效应的比较分析，揭示RIG-I的3个关键功能域识别和结合miRNA136/145的活性差异，阐明RIG-I识别miRNA的分子结构基础与作用规律。利用慢病毒介导的siRNA基因沉默技术，研究RIG-I-miRNA136/145在中性粒细胞分化发育中的功能以及IRF3/7-IFN和/或IKK-NF-κB介导的信号调节机制。通过项目实施，发现内源性miRNA活化胞质模式识别受体调控免疫细胞发育的新功能，拓展RIG-I的功能内涵，丰富鱼类免疫学内容，为鱼类病害免疫防治提供新的科学依据。

As a cytosolic pattern recognition receptor, RIG-I (retinoic acid induced gene I) plays a significant role in the sensing of pathogen-associated molecular patterns (PAMPs) within viral RNA. It is commonly believed that the agonist for RIG-I is short double-stranded blunt-ended 5'ppp RNA. However, some other RNA structures, like polyuridine motifs that contain interspersed C nucleotides (known as poly-U/UC), the 5'pppAU-RNA polymers generated by RNA polymerase III transcription from exogenous dAdT DNA templates, etc., can also activate RIG-I, indicating a broad recognition spectrum of RIG-I. Here in this project, we will elucidate whether the endogenous non-coding microRNA can also be the ligand of RIG-I and its physiological significance. We focus on the non-coding RNA-microRNA136/145 mediated RIG-I activation and its role on neutrophil differentiation in Larimichthys crocea. The structural features, distribution patterns and subcellular localizations of RIG-I are to be elucidated. To confirm the interaction between RIG-I and microRNA136/145, the in vivo and in vitro pull down assays will be conducted and the interaction domains of RIG-I with microRNA136/145 are to be addressed. By using the lentivirus mediated knockdown system, we will study the roles of RIG-I and/or microRNA136/145 knockdown on neutrophil development and function. Furthermore, we also attempt to elaborate the signaling pathways underneath this phenomenon. We will focus on which of the signaling pathways, IRF3/7-IFN, IKK-NF-κB or both of them, involves in the downstream signal transduction. We hope that our project will improve the traditional cognition of RIG-I as a cytoplasm PRR (pattern recognition receptor), as it may also involve in many other biological processes, including neutrophil differentiation. Besides, our project confirms that along with the exogenous RNA component, the endogenous non-coding RNA can also activate RIG-I in Larimichthys crocea. Furthermore, the evaluation of microRNA136/145 in the protection of Larimichthys crocea upon pathogen infection will also brought new perspectives in disease prevention of the aquaculture industry.