RNA解旋酶RIG-I参与造血调控的分子机制研究

Retinoic acid inducible gene I (RIG-I) was identified as one of the genes whose expression were highly up-regulated during all-trans retinoic acid (ATRA) induced differentiation of acute promyelocytic leukemia cells. Since RIG-I was subsequently discovered in the innate immunity responses against RNA viruses, it has become the focus of immunology research. However, the biological functions of RIG-I in the absence of viral infection, and especially the molecular mechanisms underlying RIG-I-mediated regulation of leukemic cell differentiation, are poorly understood. In our previous study, we have reported that RIG-I could not only augment STAT1 activation but also inhibit AKT-mTOR signaling pathway to induce differentiation and proliferation inhibition of leukemia cells. In this study, we will (1) explore the endogenous RNAs binding to RIG-I by RNA immunoprecipitation sequencing (RIP-Seq) analysis; (2) further investigate the molecular details of how the endogenous RNA binding to RIG-I through the combination of assays such as CatRAPID and BLI; (3) identify partner proteins interacting with RIG-I by co-immunoprecipitation (Co-IP) and mass spectrometry (MS) analysis; (4) unveil the mechanism underlying the regulation of transcription, translation, etc., of endogenous RIG-I-binding RNA and the effects on cell phenotype by RIG-I using knockdown or overexpression strategies. This may provide novel important evidence for further understanding the detailed molecular mechanism of biological functions such as hematopoietic regulation by the interaction of RIG-I with the endogenous RNAs.

RIG-I是在全反式维甲酸诱导白血病细胞分化过程中上调的一个基因，随后在病毒引发的先天性免疫过程中再次被发现，成为免疫学研究热点。但RIG-I作为RNA解旋酶在非感染条件下的生物学功能，尤其在白血病细胞诱导分化中的分子机制尚未充分阐明。本实验室前期研究发现，RIG-I是可通过促进STAT1活化和抑制AKT-mTOR信号途径，来促进白血病细胞分化、抑制白血病细胞增殖。本课题拟在前期基础上,（1）利用RIP-Seq技术，探索与RIG-I结合的内源RNA；（2）结合CatRAPID、BLI等方法，进一步研究RIG-I与内源RNA结合的方式及亲和力；（3）利用Co-IP/MS等技术寻找RIG-I结合蛋白；（4）通过敲低、过表达等方法，研究RIG-I对内源RNA转录、翻译等调控的机制及对细胞表型的影响。为深入阐明RIG-I通过与内源RNA相互作用进行造血调控等生物学功能的分子机制提供新的理论依据。

维甲酸诱导基因I（RIG-I）是在全反式维甲酸（ATRA）诱导急性早幼粒细胞白血病（APL）细胞分化过程中高表达的一个基因。研究发现RIG-I在抗病毒先天性免疫中发挥重要作用，它能识别病毒特异性的5’三磷酸双链RNA，并激活I型干扰素信号通路。我们推测，在无病毒感染状态下，RIG-I作为RNA解旋酶可能与内源性RNA及相关通路相互作用进而调控细胞分化。因此本项目以蛋白质与RNA相互作用为研究切入点，探索了RIG-I调控髓系分化的分子机制。.首先，应用RNA结合蛋白免疫共沉淀-测序技术（RIP-seq），发现RIG-I在ATRA诱导APL细胞分化过程中可以结合多种内源RNA，其中包括TRIM25 mRNA。TRIM25作为一个泛素/ISG15的E3连接酶，在RIG-I介导的抗病毒反应中起重要作用。通过软件分析、RIP-qPCR、RNA pulldown、亲和力分析等实验，发现RIG-I可通过helicase结构域和CTD结构域与TRIM25 mRNA相互作用，进而增强TRIM25 mRNA的稳定性。接着，通过截短型RIG-I过表达及荧光素酶实验，发现RIG-I还可通过CARD结构域激活STAT1/2等转录因子，STAT1则可进一步结合TRIM25基因的干扰素刺激反应元件（ISRE），进而促进TRIM25及ISGylation通路基因的表达，包括其配体ISG15及负责ISG15与蛋白质底物偶联的几个关键酶。最后，通过差异表达及功能实验，发现敲低TRIM25或者敲低ISG15均可以一定程度抑制细胞分化，表明RIG-I通过促进TRIM25介导的ISGylation在髓系细胞分化过程中起重要作用。该项研究系统地阐明了RIG-I通过与内源性RNA结合及转录调节等方式激活ISGylation通路促进髓系分化的分子机制，为揭示RIG-I-TRIM25-ISGylation信号轴在髓系分化中的作用提供了新思路，也为维甲酸类药物在白血病治疗中的临床应用提供科学依据。发表4篇SCI论著（均标注本项目基金编号）于《PNAS》（2020，IF: 11.205）、《Frontiers in Immunology》（2021，IF：7.561）、《Biomolecules》（2021，IF：4.879）和《BMC Cancer》（2019，IF：3.288）。