Characterization of nuclear retained RNA-mediated gene regulatory mechanism

描述（由申请人提供）：据估计，一部分真核长非编码RNA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，并且它们的异常表达与包括癌症在内的各种疾病有关。我实验室的长期目标是了解哺乳动物nrRNAs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，一个paraspeckle-localized nrRNA，调节Cat 2的表达，一氧化氮合成途径中的受体。CTN-RNA被制成含有Cat 2 mRNA序列加上另外的长3' UTR的转录物。CTN-RNA的独特3 'UTR是其核保留所需的，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处被转录后切割以产生成熟的Cat 2 mRNA，其快速输出到细胞核外并被翻译。然而，介导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，约300个人类基因显示出与CTN-RNA相似的结构特征，表明这可能是其他基因共享的共同机制，以允许快速基因诱导。本提案的目的是确定调节CTN-RNA的核保留及其在应激时的裂解的机制。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CTN-RNA的核保留和应激诱导的加工。为了验证这一假设，我们提出了以下具体目标：1）确定负责CTN-RNA核保留的机制; 2）确定CTN-RNA与旁斑亚核结构域的关联的功能意义;以及3）确定负责应激诱导的转录后加工和CTN-RNA切割的因子。我们的方法是创新的，因为我们将描述CTN-RNA所利用的新基因调控机制，这可能是整个一类基因所共有的。这种机制可能已经进化到加速由长基因编码的蛋白质在特定信号下的快速诱导，否则需要很长时间才能转录和处理。该提案在其分子遗传学与核内RNA动力学的最新活细胞成像的结合方面具有创新性，以解决CTN-RNA和CAT 2基因调控的动力学。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Cat 2的合成，从而调节NO的细胞水平，NO是参与先天免疫应答的重要分子。因此，拟议的研究是重要的，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，旨在调节这种压力和疾病相关的反应。 公共卫生相关性：我实验室的长期重点是了解哺乳动物核保留RNA（nrRNAs）在基因调控中的作用。在本提案中，我们的目标是研究机制，使核保留的nrRNA; CTN-RNA和表征的组件，促进其应力诱导的加工。由于CTN-RNA调节细胞水平可诱导的一氧化氮（NO）（身体先天免疫反应的一部分），因此了解CTN-RNA介导的基因调节将对生物医学研究产生直接影响。

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.