Characterization of nuclear retained RNA-mediated gene regulatory mechanism

描述（由申请人提供）：据估计，真核生物长链非编码rna的一部分保留在细胞核中（nrrna）。一些研究已经证明了新型nrrna在重要细胞过程中发挥的关键作用，它们的异常表达与包括癌症在内的各种疾病有关。我的实验室的长期目标是了解哺乳动物nrrna如何调节重要的基因调控途径。作为一个模型系统，我们专注于CTN-RNA，一种副斑定位的nrRNA，调节Cat2的表达，Cat2是一氧化氮合成途径中的一种受体。CTN-RNA是由包含Cat2 mRNA序列和一个额外的长3' UTR的转录本制成的。CTN-RNA独特的3'UTR是其核保留所必需的，并被腺苷修饰为肌苷超编辑。在用干扰素-3和脂多糖处理细胞后，CTN-RNA在其3'UTR上进行转录后切割，产生成熟的Cat2 mRNA，该mRNA迅速输出到细胞核外并被翻译。然而，介导诸如CTN-RNA等nrrna的核保留和应激诱导的转录后切割的机制仍有待阐明。有趣的是，大约有300个人类基因表现出与CTN-RNA相似的结构特征，这表明这可能是其他基因共享的共同机制，从而允许快速基因诱导。本研究的目的是确定调节CTN-RNA的核保留及其在应激下的裂解的机制。基于初步数据的中心假设是，CTN-RNA的3'UTR中的转录后修饰和/或序列元件及其与特定因子的相互作用调节了核保留和应力诱导的CTN-RNA加工。为了验证这一假设，我们提出以下具体目标：1)确定CTN-RNA核保留的机制；2)确定CTN-RNA与副斑子亚核结构域关联的功能意义；3)确定应激诱导的CTN-RNA转录后加工和裂解的相关因素。我们的方法是创新的，因为我们将描述CTN-RNA利用的一种新的基因调控机制，这可能是由整个基因类共享的。这种机制可能是为了加速长基因编码的蛋白质在特定信号上的快速诱导，否则需要很长时间来转录和处理。该提案的创新之处在于将分子遗传学与核内RNA动力学的活细胞成像技术相结合，以解决CTN-RNA和CAT2基因调控的动力学问题。我在RNA分子生物学/生物化学和活细胞成像方面的专业知识对这个项目来说是非常独特和必不可少的。CTN-RNA调节Cat2合成，从而调节NO的细胞水平，NO是参与先天免疫反应的重要分子。因此，这项研究具有重要意义，因为了解CTN-RNA介导的基因调控机制如何在细胞中运作，将使未来的药理学干预能够调节这种应激和疾病相关反应。

DESCRIPTION (provided by applicant): It is estimated that a fraction of the eukaryotic long non-coding RNAs are retained in the nucleus (nrRNAs). Several studies already have demonstrated crucial roles played by novel nrRNAs in vital cellular processes and their abnormal expression has been implicated in various diseases including cancer. The long-term goal of my laboratory is to understand how mammalian nrRNAs regulates vital gene regulatory pathways. As a model system, we are focusing on CTN-RNA, a paraspeckle-localized nrRNA that regulates the expression of Cat2, a receptor in the Nitric Oxide synthesis pathway. CTN-RNA is made as a transcript containing the Cat2 mRNA sequence plus an additional, long 3' UTR. The unique 3'UTR of CTN-RNA is required for its nuclear-retention and is modified by adenosine to inosine hyper-editing. Upon treating cells with Interferon-3 and lipopolysaccharide, CTN-RNA is post-transcriptionally cleaved at its 3'UTR to produce a mature Cat2 mRNA that is rapidly exported out of the nucleus and translated. However, the mechanisms those mediate the nuclear retention of nrRNAs like CTN-RNA and stress-induced post-transcriptional cleavage remain to be elucidated. Interestingly, ~300 human genes show similar structural features to CTN-RNA indicating that this may be a common mechanism shared by other genes to allow rapid gene induction. The objective of the present proposal is to determine the mechanism/s that regulate nuclear retention of CTN-RNA and its cleavage upon stress. The central hypothesis, based on preliminary data, is that post-transcriptional modifications and/or sequence elements in the 3'UTR of CTN-RNA and their interaction with specific factors regulate nuclear- retention and stress-induced processing of CTN-RNA. To test this hypothesis, we propose the following specific aims: 1) Identify the mechanism responsible for CTN-RNA nuclear retention; 2) Determine the functional significance of the CTN-RNA association with paraspeckle sub-nuclear domains; and 3) Identify the factors responsible for stress-induced post-transcriptional processing and cleavage of CTN-RNA. Our approach is innovative because we will characterize a new gene regulatory mechanism utilized by CTN-RNA, which is likely shared by an entire class of genes. This mechanism may have evolved to accelerate the rapid induction of proteins encoded by long genes upon specific signals, which otherwise would take a long time to transcribe and process. This proposal is innovative in its combination of molecular genetics with state of the art live cell imaging of intranuclear RNA dynamics to address the dynamics of CTN-RNA and CAT2 gene regulation. My expertise in both RNA molecular biology / biochemistry and live cell imaging is quite unique and essential to this project. CTN-RNA regulates Cat2 synthesis, thereby modulates the cellular levels of NO, an important molecule involved in innate immune response. Thus, the proposed research is significant because understanding how CTN-RNA mediated gene regulatory mechanism operates in the cell would enable future pharmacological interventions aimed at modulating such stress and disease related response. PUBLIC HEALTH RELEVANCE: The long-term focus of my laboratory is to understand the roles played by mammalian nuclear-retained RNAs (nrRNAs) in gene regulation. In the present proposal we aim to study the mechanism that allows the nuclear-retention of nrRNA; CTN-RNA and characterize the components that facilitate its stress induced processing. Since CTN-RNA regulates the cellular levels inducible Nitric oxide (NO), part of the bodies innate immune response, understanding CTN-RNA mediated gene regulation would have direct implications for biomedical research.