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

描述(申请人提供)：由FXR1a相关的microRNP显著的转录后基因表达TNFa mRNA，microRNAs和RNA-蛋白质复合体(RNPs)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。静止期(G0)细胞在免疫和癌症(如白血病)中很重要，在这些癌症中，它们抵制临床治疗并导致复发。G0细胞切换到可逆的停滞状态，由不同的基因表达维持。G0基因的表达及其机制可能为耐药肿瘤的治疗提供新的选择。我们发现在G0中，促进炎症、肿瘤发生和G0的细胞因子肿瘤坏死因子�是由FXR1a相关的microRNP翻译激活的。重要的是，FXR1在G0中增加并导致细胞周期停滞。FXR1a-�、TFXR1a-microRNP、TFXR1a-microRNP的基因募集和翻译机制仍有待研究，将为G0基因的表达提供新的线索。目的研究G0白血病细胞中G0 FXR1a-microRNP对肿瘤坏死因子α(TNFa)mRNA的募集和翻译激活作用。具体地说，本研究将研究FXR1a相互作用导致微RNP募集TNFa mRNA进行激活，阐明TFFRNAm RNA的非规范翻译机制，并表征参与TFFF1a m RNA激活的G0微RNPs。前提microRNA介导的激活需要一个microRNP，它由microRNP效应器AGO2、RNA结合蛋白FXR1a组成，缺乏阻遏因子GW182。核内的微小�募集了肿瘤坏死因子RNP的基因。在G0早期(&lt；24小时)，观察到抑制；只有在G0晚期(&gt；24小时)才观察到激活，表明明显的早期和晚期G0 microRNP。在G0中，规范的多腺苷化的mRNA翻译减少。一直以来，肿瘤坏死因子FXR1a的翻译都需要PARN Deadenylase来完成，而�与翻译因子相互作用，从而促进非规范翻译。基于这些数据，我们认为在G0后期，特定的mRNAs被一个不同的FXR1a-microRNP指导进行专业翻译。方法首先，FXR1a与微小�的核相互作用导致G0单核白血病细胞中肿瘤坏死因子RNP的重新募集，其特征是过表达、敲除和与影响相互作用和激活的突变体互补，以及免疫共沉淀和功能分析。其次，通过研究FXR1a与翻译因子的相互作用以及在G0中PARN介导的去烯化作用，将利用先前开发的方法来阐明TFXR1a基因翻译激活的机制。第三，早期和晚期G0microRNP与胞核和胞浆中的肿瘤坏死因子rnp相关的microRNPs将通过先前开发的体内交联型�亲和纯化来分离，以 确定调节激活的因素。将分析已确定的因素，以确定它们在调节激活方面的作用。结果这些对G0microRNPs的功能研究将使我们更好地了解RNPs在G0特定基因表达中的作用，特别是在临床耐药白血病细胞中关键细胞因子的表达中。

DESCRIPTION (provided by applicant): Post-Transcriptional Gene Expression of TNFa 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, TNF�, 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, TNF� 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 TNFa mRNA. Specifically, this study will investigate FXR1a interactions that lead to TNFa mRNA recruitment by the microRNP for activation, elucidate the non-canonical translation mechanism of TNF� mRNA and characterize G0 microRNPs involved in TNF� mRNA activation. Premise MicroRNA-mediated activation requires a microRNP comprising the microRNP effector, AGO2, the RNA binding protein, FXR1a, and lacking the repressor, GW182. TNF� 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 TNF� 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 TNF� 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 TNF� 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 TNF� 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.