Post-transcriptional Gene Expression of the TNF alpha by an FXR1a-associated microRNP

描述（由申请人提供）：通过fxr1a相关的microrna表达TNFα mRNA的转录后基因意义microrna和rna -蛋白复合物（RNPs）是转录后基因表达调控因子，在免疫和癌症中发挥重要作用。静止（G0）细胞在免疫和癌症（如白血病）中很重要，它们抵抗临床治疗并导致复发。G0细胞切换到一种可逆的阻滞状态，由不同的基因表达维持。G0基因的表达及其机制可能为抵抗性癌症提供新的治疗选择。我们发现，在G0中，促进炎症、肿瘤发生和G0的细胞因子TNFα被fxr1a相关的microRNP翻译激活。重要的是，FXR1在G0中增加并导致细胞周期停滞。FXR1a-microRNP、TNFalpha mRNA的募集和翻译机制有待进一步研究，这将有助于进一步了解G0基因的表达。本研究的主要目的是表征G0白血病细胞中导致TNFα mRNA募集和翻译激活的G0 FXR1a-microRNP。具体而言，本研究将研究FXR1a相互作用导致TNFα mRNA被microRNP募集激活，阐明TNFα mRNA的非规范翻译机制，并表征参与TNFα mRNA激活的G0 microRNP。前提是microrna介导的激活需要一个microRNP，包括microRNP效应物AGO2、RNA结合蛋白FXR1a和缺乏抑制物GW182。tnf - α mRNA在细胞核内被microRNP募集。在G0早期（<24小时），观察到抑制；仅在G0晚期（bb0 24小时）观察到激活，表明G0早期和晚期microRNPs明显不同。在G0中，典型聚腺苷化mRNA的翻译减少。与此一致的是，TNFα mRNA的翻译需要PARN死烯基化酶，而FXR1a与翻译因子相互作用，促进非规范翻译。基于这些数据，我们提出特定的mrna在90年代后期由一个独特的FXR1a-microRNP定向进行特化翻译。首先，在G0单核白血病细胞中，FXR1a与导致TNFα mRNA募集的microRNP的核相互作用将以过表达、敲低和与影响相互作用和激活的突变体的互补为特征，同时进行共免疫沉淀和功能分析。其次，通过研究FXR1a与翻译因子的相互作用，以及在G0中parn介导的死蛋白化，将阐明TNFα mRNA翻译激活的机制。第三，通过先前开发的体内交联耦合RNP亲和纯化，分离细胞核和细胞质中与TNFα mRNA相关的早期和晚期G0 microRNPs，以确定调节激活的因素。已确定的因子将分析其在调节激活中的作用。这些G0 microRNPs的功能研究将使我们更好地了解RNPs在临床耐药白血病细胞中G0特异性基因表达中的作用，特别是关键细胞因子的作用。

DESCRIPTION (provided by applicant): Post-Transcriptional Gene Expression of TNFα mRNA by an FXR1a-associated microRNP Significance MicroRNAs and RNA-protein complexes (RNPs) are post-transcriptional gene expression regulators that play essential roles in immunity and cancer. Quiescent (G0) cells are important in immunity and cancers, such as leukemia, where they resist clinical therapy and cause recurrences. G0 cells switch to a reversibly arrested state, maintained by distinct gene expression. G0 gene expression and mechanisms could provide new therapeutic options against resistant cancers. We revealed that in G0, the cytokine, TNFα, that promotes inflammation, tumorigenesis and G0, is translationally activated by an FXR1a-associated microRNP. Importantly, FXR1 increases in G0 and causes cell cycle arrest. FXR1a-microRNP, TNFalpha mRNA recruitment and translation mechanism remain to be characterized and would provide insights into G0 gene expression. Objective The primary goal of this study is to characterize the G0 FXR1a-microRNP in G0 leukemic cells that leads to recruitment and translation activation of TNFα mRNA. Specifically, this study will investigate FXR1a interactions that lead to TNFα mRNA recruitment by the microRNP for activation, elucidate the non-canonical translation mechanism of TNFα mRNA and characterize G0 microRNPs involved in TNFα mRNA activation. Premise MicroRNA-mediated activation requires a microRNP comprising the microRNP effector, AGO2, the RNA binding protein, FXR1a, and lacking the repressor, GW182. TNFα mRNA is recruited by the microRNP in the nucleus. During early G0 (<24hrs), repression is observed; activation is observed only in late G0 (>24hrs), indicating distinct early and late G0 microRNPs. In G0, canonical polyadenylated mRNA translation is reduced. Consistently, PARN deadenylase is required for translation of TNFα mRNA, which is deadenylated, and FXR1a interacts with translation factors, which promote non-canonical translation. Based on these data, we propose that specific mRNAs are directed for specialized translation by a distinct FXR1a-microRNP in late G0. Method First, the nuclear interactions of FXR1a with the microRNP that leads to TNFα mRNA recruitment in G0 monocytic leukemic cells will be characterized by overexpression, knockdown and complementation with mutants that affect interactions and activation, along with co-immunoprecipitations and functional analyses. Second, the mechanism of translation activation of TNFα mRNA will be elucidated by investigating the role of FXR1a interactions with translation factors and of PARN-mediated deadenylation in G0, using previously developed assays. Third, the early and late G0 microRNPs associated with TNFα mRNA in the nucleus and cytoplasm will be isolated by previously developed in vivo crosslinking coupled RNP affinity purification, to identify factors that regulate activation. Identified factors will be analyzed for their role in regulating activation. Outcome These functional studies of G0 microRNPs will provide a greater understanding of the role of RNPs in specific gene expression in G0, in particular, of critical cytokines, in clinically resistant leukemia cells.