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

描述（由申请人提供）：FXR 1a相关microRNP对TNFα mRNA的转录后基因表达意义MicroRNA和RNA-蛋白质复合物（RNP）是转录后基因表达调节因子，在免疫和癌症中发挥重要作用。静止（G 0）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。G 0细胞通过不同的基因表达而转换到可逆的停滞状态。G 0基因的表达和机制可以为耐药癌症提供新的治疗选择。我们发现，在G 0期，促进炎症、肿瘤发生和G 0期的细胞因子TNFα被FXR 1a相关的microRNP激活。重要的是，FXR 1在G 0期增加并导致细胞周期停滞。FXR 1a-microRNP、TNF α mRNA募集和翻译机制仍有待表征，并将为G 0基因表达提供见解。目的研究G 0期白血病细胞中FXR 1a-microRNP对肿瘤坏死因子α（TNFα）mRNA募集和翻译激活的作用。具体而言，本研究将研究导致microRNP募集TNFα mRNA以激活的FXR 1a相互作用，阐明TNFα mRNA的非经典翻译机制，并表征参与TNFα mRNA激活的G 0 microRNP。Preventive MicroRNA介导的激活需要包含microRNP效应子AGO 2、RNA结合蛋白FXR 1a且缺乏阻遏物GW 182的microRNP。TNFα mRNA由细胞核中的microRNP募集。在G 0早期（<24小时），观察到抑制;仅在G 0晚期（> 24小时）观察到激活，表明不同的G 0早期和晚期microRNP。在G 0，典型的多聚腺苷酸化mRNA翻译减少。因此，PARN去腺苷酶是翻译去腺苷化的TNFα mRNA所必需的，而FXR 1a与翻译因子相互作用，促进非经典翻译。基于这些数据，我们提出，特定的mRNA是针对专门的翻译由一个独特的FXR 1a-microRNP在晚G 0。方法首先，FXR 1a与microRNP的核相互作用导致G 0单核细胞白血病细胞中TNFα mRNA的募集，其特征在于过表达，敲低和与影响相互作用和激活的突变体互补，沿着免疫共沉淀和功能分析。其次，将使用先前开发的测定法，通过研究FXR 1a与翻译因子的相互作用以及G 0期PARN介导的去腺苷化的作用，阐明TNFα mRNA翻译激活的机制。第三，将通过先前开发的体内交联偶联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G 0 microRNP，以鉴定调节活化的因子。将分析确定的因子在调节激活中的作用。结果这些功能的研究G 0 microRNP将提供一个更好的理解RNP在特定的基因表达在G 0，特别是关键的细胞因子，在临床耐药白血病细胞的作用。

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.