Elucidating mechanisms of microRNA pathway deregulation in human cells

阐明人类细胞中 microRNA 通路失调的机制

• 批准号: 10392141
• 负责人: Blerta Xhemalce
• 金额: $ 5.38万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-05-01 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10392141
• 关键词: Affect; Biochemical; Biogenesis; Cell Differentiation process; Cell Proliferation; Cell physiology; Cells; Code; Data; Diabetes Mellitus; Diagnosis; Disease; Down-Regulation; Drug Targeting; Enzymes; Event; Family; Functional disorder; Genetic Transcription; Goals; Growth; Heart Diseases; Human; Human Pathology; Knowledge; Lead; Link; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Messenger RNA; Methods; Methylation; Methyltransferase; MicroRNAs; Mission; Modification; Molecular; Molecular and Cellular Biology; Nerve Degeneration; Pathology; Pathway interactions; Post-Transcriptional RNA Processing; Process; Prognosis; Proteins; Proteomics; RNA; RNA Biochemistry; RNA Interference; Regulation; Regulatory Pathway; Research; Sorting - Cell Movement; Stress; Structure; Testing; Therapeutic; Transcript; Transcription Process; Translations; United States National Institutes of Health; Untranslated RNA; Virus Diseases; Work; base; cell transformation; disease diagnosis; epitranscriptomics; exosome; extracellular vesicles; genome-wide; human disease; improved; innovation; insight; next generation sequencing; novel; piRNA; response; small molecule; trait

PROJECT SUMMARY microRNAs are critical regulators of cellular proliferation, differentiation and response to stress but their biogenesis is poorly understood. For example, transcription rates of microRNAs often do not correlate with the level of the corresponding mature microRNA levels, suggesting that post- transcriptional events determine the fate of microRNAs during these important cellular processes. Given that microRNAs are deregulated in human diseases, obtaining a comprehensive mechanistic view of microRNA biogenesis could greatly expand the potential therapeutic opportunities of microRNAs. We recently discovered two new mechanisms regulating the microRNA pathway at the post- transcriptional level. The first mechanism is through post-transcriptional modification of microRNAs. We determined that the BCDIN3D RNA methyltransferase methylates specific microRNA precursors to inhibit their processing into mature microRNAs by Dicer. We further identified RNA and protein partners of BCDIN3D, which revealed new RNA modifications of specific precursors of let-7, a crucial microRNA family involved in cell differentiation and transformation. Our results suggest that a combination of RNA modifications may act in concert to suppress mature let-7 biogenesis. The second mechanism is through a crosstalk between the piwi and micro RNA pathways. Using quantitative proteomics of the proteins interacting with Enoxacin, a small molecule shown to stimulate the microRNA pathway and inhibit growth of transformed cells, we identified the PIWIL3 protein as a primary target of this drug. PIWIL3 is an Argonaut protein of the PIWI subfamily that is mainly expressed in the germline and that mediates RNA interference through piwiRNAs. Our results reveal an unexpected link between PIWIL3 and the microRNA pathway that could be crucial for promoting transformation. The above-described processes are novel and previously uncharacterized. This proposal builds upon our findings to decipher the molecular mechanisms that mediate down-regulation of the microRNA pathway by RNA modifications and piwi/microRNA biogenesis crosstalk. To elucidate these mechanisms, we will employ a multifaceted approach using our expertise in cellular and molecular biology, RNA biochemistry, mass spectrometry and next generation sequencing. We expect the findings acquired from this proposal to provide critical new insights into the causes and consequences of microRNA pathway deregulation in human cells that can be leveraged to improve the diagnosis and treatment of human diseases. 1

项目概要 microRNA 是细胞增殖、分化和应激反应的关键调节因子 但它们的生物起源却知之甚少。例如，microRNA 的转录率通常会影响 与相应成熟 microRNA 水平不相关，表明后 转录事件决定了 microRNA 在这些重要细胞过程中的命运。 鉴于 microRNA 在人类疾病中不受管制，获得全面的 microRNA生物发生的机制观点可以极大地扩展潜在的治疗 microRNA 的机会。 我们最近发现了两种调节 microRNA 通路的新机制 转录水平。第一个机制是通过 microRNA 的转录后修饰。 我们确定 BCDIN3D RNA 甲基转移酶甲基化特定的 microRNA 前体抑制 Dicer 将其加工成成熟的 microRNA。我们进一步鉴定了RNA 和 BCDIN3D 的蛋白质伙伴，揭示了特定前体的新 RNA 修饰 let-7，参与细胞分化和转化的重要 microRNA 家族。我们的成果 表明 RNA 修饰的组合可能协同作用来抑制成熟的 let-7 生物发生。第二种机制是通过 piwi 和 micro RNA 之间的串扰 途径。使用与依诺沙星相互作用的蛋白质的定量蛋白质组学，一种小 分子被证明可以刺激 microRNA 途径并抑制转化细胞的生长，我们 确定 PIWIL3 蛋白是该药物的主要靶点。 PIWIL3 是一种 Argonaut 蛋白 PIWI 亚家族主要在种系中表达并介导 RNA 干扰 通过 piwiRNA。我们的结果揭示了 PIWIL3 和 microRNA 之间的意外联系 路径可能对促进转型至关重要。 上述过程是新颖的并且是以前未表征的。该提案构建 根据我们的发现来破译介导下调的分子机制 通过 RNA 修饰和 piwi/microRNA 生物发生串扰的 microRNA 途径。阐明 这些机制，我们将利用我们在细胞和 分子生物学、RNA 生物化学、质谱和下一代测序。我们 期望从该提案中获得的调查结果能够为原因和问题提供重要的新见解 人类细胞中 microRNA 通路失调的后果可用于改善 人类疾病的诊断和治疗。 1