Characterization of nuclear-retained RNA-mediated gene regulatory mechanisms

核保留RNA介导的基因调控机制的表征

• 批准号: 10379740
• 负责人: Prasanth Kumar Vijayan Kannanganattu
• 金额: $ 12.5万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-15 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10379740
• 关键词: Alternative Splicing; Binding; Biological Assay; Biological Models; Breast Cancer Cell; Breast Cancer Treatment; Cell physiology; Cells; Chromosome Mapping; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Data; Defect; Disease; Experimental Models; Family; Family member; Future; Genes; Genetic Transcription; Human Genome; Hypoxia; Image; Knowledge; MALAT1 gene; Malignant Neoplasms; Mammary Neoplasms; Mediating; Mission; Molecular; Neoplasm Metastasis; Nuclear; Outcome; Pathway interactions; Prognostic Marker; Proteins; Public Health; RNA; RNA Splicing; Regulator Genes; Research; Resolution; Role; Signal Transduction; Spliced Genes; Techniques; Testing; United States National Institutes of Health; genome-wide; imaging study; in vivo; innovation; insight; mRNA Precursor; malignant breast neoplasm; member; mouse model; new therapeutic target; prevent; recruit; response; tumor

Human genome encodes a large number of non-protein coding RNA (ncRNA) genes, including thousands long ncRNA (lncRNA) genes. MALAT1 is an abundant, conserved and nuclear speckle localized lncRNA that promotes breast tumor metastasis. MALAT1 is induced several folds during hypoxia, and influences the pre-mRNA alternative splicing (AS) of genes controlling hypoxia response. However, the molecular mechanisms by which MALAT1 controls AS during hypoxia signaling remain to be elucidated. Genome- wide RNA mapping analyses reveal that MALAT1 interacts with transcriptionally active genes and their pre- mRNA. Further, MALAT1 interacts with several members of the SR-family of pre-mRNA splicing factors (SRSFs), and cells with deregulated expression of MALAT1 show defects in SRSF-mediated AS. The objective of the present proposal is to delineate the molecular function of MALAT1 in SRSF-mediated AS, by utilizing hypoxia response as an experimental model system. The central hypothesis is that MALAT1 by enriching SRSFs in nuclear speckles, controls the binding of SRSFs with their target pre-mRNAs and other SRSF interactors. Guided by strong preliminary data, this hypothesis will be tested in the following specific aims: 1) Determine how MALAT1 regulates SRSF-mediated alternative splicing (AS). 2) Determine the significance of nuclear speckle enrichment of MALAT1 in pre-mRNA processing. In the first aim, PI will determine how MALAT1 regulates the binding and recruitment of SRSF1 (a prototypical member of SRSF proteins) to their target pre-mRNAs in hypoxic breast cancer cells. PI will also determine the involvement of MALAT1 in AS during in vivo hypoxia response in tumor mouse models. Under the second aim, PI, by using super-resolution and live imaging studies will determine the involvement of MALAT1 in the, 1) spatial organization of speckle components, including SRSFs, and 2) regulated localization of genes in speckle proximity. The approach is technically innovative, because it employs state of the art techniques, including super-resolution imaging and CRISPR/dCasRx-mediated RNA tethering assays. The proposed research is significant because deciphering the role of MALAT1 in regulating the expression of hypoxia responsive genes will have broad translational significance in the context of breast cancer treatment. Ultimately, this knowledge will pave way to future studies utilizing MALAT1 as a novel therapeutic target against cancer.

人类基因组编码大量的非蛋白质编码RNA（ncRNA）基因，包括数千个 长ncRNA（lncRNA）基因。MALAT 1是一种丰富、保守的核斑点定位lncRNA 促进乳腺癌转移的基因MALAT 1在缺氧期间被诱导数倍，并影响细胞的生长。 前mRNA选择性剪接（AS）基因控制缺氧反应。然而，分子 MALAT 1在缺氧信号传导期间控制AS的机制仍有待阐明。基因组- 广泛的RNA图谱分析表明，MALAT 1与转录活性基因及其前体相互作用， mRNA。此外，MALAT 1与前mRNA剪接因子的SR家族的几个成员相互作用 （SRSFs），MALAT 1表达失调的细胞显示SRSFs介导的AS缺陷。的 本发明的目的是描述MALAT 1在SRSF介导的AS中的分子功能， 通过利用缺氧反应作为实验模型系统。核心假设是MALAT 1通过 在核斑点中富集SRSF，控制SRSF与其靶前mRNA的结合，以及其他 SRSF互动者。在强有力的初步数据的指导下，这一假设将在以下具体情况下得到检验。 目的：1）研究MALAT 1对SRSF介导的可变剪接（AS）的调控作用。2)确定 MALAT 1核斑点富集在前体mRNA加工中的意义。在第一个目标中，PI将 确定MALAT 1如何调节SRSF 1（SRSF的原型成员）的结合和募集 蛋白质）与其靶前体mRNA在缺氧乳腺癌细胞中的相互作用。PI还将确定参与 肿瘤小鼠模型体内缺氧反应期间AS中的MALAT 1。在第二个目标下，PI，通过使用 超分辨率和实时成像研究将确定MALAT 1参与，1）空间 斑点成分的组织，包括SRSFs，和2）斑点中基因的调节定位 接近这种方法在技术上是创新的，因为它采用了最先进的技术，包括 超分辨率成像和CRISPR/dCasRx介导的RNA系链测定。拟议的研究是 重要的是，因为破译MALAT 1在调节低氧应答基因表达中的作用， 基因在乳腺癌治疗中具有广泛的翻译意义。最终这 这些知识将为利用MALAT 1作为抗癌新治疗靶点的未来研究铺平道路。