Characterization of nuclear retained RNA-mediated gene regulatory mechanism

核保留RNA介导的基因调控机制的表征

• 批准号: 8618908
• 负责人: Prasanth Kumar Vijayan Kannanganattu
• 金额: $ 26.54万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-05 至 2016-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8618908
• 关键词: 3' Untranslated Regions; Address; Adenosine; Affect; Biochemical; Biochemistry; Biological Assay; Biological Models; Biomedical Research; Body part; Cell Nucleus; Cell physiology; Cells; Cleaved cell; Code; Cytoplasm; Data; Disease; Dosage Compensation (Genetics); Enzymes; Fractionation; Functional RNA; Future; Gene Expression Regulation; Genes; Genetic Transcription; Genome; Goals; Human; Immune response; In Vitro; Indium; Inosine; Interferons; Intervention; Laboratories; Life; Lipopolysaccharides; Maintenance; Malignant Neoplasms; Mediating; Messenger RNA; Modification; Molecular Biology; Molecular Genetics; Nitric Oxide; Nuclear; Nuclear RNA; Nuclear Structure; Pathway interactions; Play; Process; Proteins; RNA; Regulator Genes; Regulatory Pathway; Research; Role; Signal Transduction; Site; Specific qualifier value; Stress; Structure; Testing; Time; Transcript; Transcription Process; Translating; base; cellular imaging; gene induction; human disease; imprint; innovation; mRNA Precursor; novel; receptor; response

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.

描述(申请人提供)：据估计，真核长的非编码RNA的一部分保留在核(NrRNAs)中。一些研究已经证明了新的nrRNA在重要的细胞过程中扮演着重要的角色，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如何调控重要的基因调控途径。作为一个模型系统，我们专注于CTN-RNA，这是一种定位于副斑点的nrRNA，它调节一氧化氮合成途径中的受体Cat2的表达。CTN-RNA是作为包含Cat2mRNA序列和额外的长3‘非编码区的转录本制成的。CTN-RNA独特的3‘非编码区是其核保持所必需的，并被腺苷修饰为肌苷超编辑。当用干扰素-3和脂多糖处理细胞时，CTN-RNA在其3‘端非编码区被转录后切割，产生成熟的Cat2 mRNA，并迅速从细胞核输出并翻译出来。然而，介导cTN-RNA等nrRNA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，约300个人类基因显示出与CTN-RNA相似的结构特征，这表明这可能是其他基因共同的一种机制，允许快速基因诱导。本提案的目的是确定调控CTN-RNA的核保留及其在应激时的切割的机制/S。根据初步数据，中心假说是CTN-RNA 3‘UTR3’UTR区的转录后修饰和/或序列元件及其与特定因素的相互作用调节核保留和应激诱导的CTN-RNA加工。为了验证这一假说，我们提出了以下具体目标：1)确定导致CTN-RNA核保留的机制；2)确定CTN-RNA与副斑点亚核结构域结合的功能意义；以及3)确定导致应激诱导的CTN-RNA转录后加工和切割的因素。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，这种机制可能被一整类基因共享。这种机制可能是为了在特定信号下加速由长基因编码的蛋白质的快速诱导，否则转录和处理这些蛋白质需要很长时间。这一建议具有创新性，它将分子遗传学与最先进的核内RNA动力学活细胞成像相结合，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是非常独特的，对这个项目至关重要。CTN-RNA调节Cat2的合成，从而调节细胞内NO的水平，NO是参与先天性免疫反应的重要分子。因此，这项拟议的研究具有重要意义，因为了解CTN-RNA介导的基因调控机制在细胞中是如何运作的，将使未来旨在调节这种应激和疾病相关反应的药物干预成为可能。