The role of m6A RNA modification as modulator of dsRNA induced cell-intrinsic innate immune responses in hematopoiesis

项目摘要 N6-甲基腺苷(M6A)是最丰富的RNA修饰，在RNA代谢中起着关键作用。M6A 作者和读者在造血干和祖细胞中高表达，并在 骨髓发育不良和白血病。 我们发现，在造血干细胞和祖细胞(HSPC)中，METTL3的缺失导致了AN的上调 抗病毒先天免疫反应特征以及明显的增殖和分化缺陷。在……里面 特别是，我们首次报道了m6A RNA修饰的丢失导致dsRNA的异常形成， 导致MDA5-RIG-I、PKR-eIF2A和OAS-核糖核酸酶L通路的激活。按差分 从METTL3KO和WT细胞中免疫沉淀dsRNAs，我们鉴定了正常情况下Long的一个特定亚群 高度m6A修饰的转录本具有低折叠能，意味着有形成广泛的二级RNA的倾向 结构。通过MAV的缺失或RNASEL的敲除部分挽救了先天免疫信号的中断 体外集落形成和体内竞争性移植试验。这些数据表明 M6A RNA修饰的作用之一是维持内源RNA的单链，从而 它们将RNA标记为“自身”，并将它们与外源病原体衍生的dsRNA区分开来。 在这个SHARE II应用中，我们建议剖析m6A RNA修饰在HSPC中的作用，特别是它 与其作为异常的先天免疫信号的抑制者的作用有关。我们将通过以下方式确定机制 哪种METTL3介导的m6A RNA修饰可以防止dsRNA的异常形成和随后的诱导 关于干扰素信号，我们将剖析m6a缺失如何导致有害的免疫反应，从而扰乱 HSPC的功能，我们将评估这一现象在造血系中的普遍性。

Project Summary N6-methyladenosine (m6A) is the most abundant RNA modification and plays key roles in RNA metabolism. m6A writers and readers are highly expressed in hematopoietic stem and progenitor cells and dysregulated in myelodysplasia and leukemia. We show that loss of Mettl3 in hematopoietic stem and progenitor cells (HSPCs) resulted in upregulation of an anti-viral innate immune response signature as well as marked proliferation and differentiation defects. In particular, we are the first to report that loss of m6A RNA modification resulted in aberrant dsRNA formation, leading to activation of the MDA5-RIG-I, PKR-eIF2A, and OAS-RNAse L pathways. By differential immunoprecipitation of dsRNAs from Mettl3 KO versus WT cells, we identified a specific subset of long, normally highly m6A modified transcripts with low folding energies, signifying propensity to form extensive RNA secondary structures. Disruption of innate immune signaling via deletion of Mavs or knockdown of Rnasel in part rescued colony formation in vitro and engraftment in competitive transplantation assays in vivo. These data suggest that one of the roles of m6A RNA modifications is to maintain single-strandedness of endogenous RNAs whereby they mark RNAs as “self” and distinguish them from exogenous pathogen-derived dsRNAs. In this SHINE II application we propose to dissect the role of m6A RNA modification in HSPCs specifically as it pertains to its role as suppressor of aberrant innate immune signaling. We will determine the mechanism by which METTL3-mediated m6A RNA modification prevents abnormal dsRNA formation and consequent induction of interferon signaling, we will dissect how m6A loss results in a deleterious immune response that disrupts HSPC function, and we will assess the universality this phenomenon across hematopoietic lineages.