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: 9902462
• 负责人: Blerta Xhemalce
• 金额: $ 32.34万
• 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/9902462
• 关键词: Affect; Biochemical; Biogenesis; Cell Differentiation process; Cell Proliferation; Cell physiology; Cells; Code; Contact Inhibition; Data; Diabetes Mellitus; Diagnosis; Disease; Down-Regulation; Drug Targeting; Enzymes; Estrogen receptor positive; 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; 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; outcome forecast; 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

项目摘要 microRNA是细胞增殖、分化和应激反应的关键调节因子 但对它们的生物起源知之甚少。例如，microRNA的转录速率通常 与相应的成熟microRNA水平无关，这表明， 在这些重要的细胞过程中，转录事件决定了microRNA的命运。 鉴于microRNAs在人类疾病中的失调，获得一个全面的 microRNA生物发生的机制观点可以极大地扩展潜在的治疗方法， microRNA的机会 我们最近发现了两种新的机制，在后- 转录水平。第一种机制是通过microRNA的转录后修饰。 我们确定BCDIN 3D RNA甲基转移酶甲基化特定的microRNA， 前体，以抑制其通过Dicer加工成成熟的microRNA。我们进一步鉴定了RNA 和BCDIN 3D的蛋白质伴侣，揭示了BCDIN 3D特异性前体的新RNA修饰。 let-7是参与细胞分化和转化的关键microRNA家族。我们的结果 提示RNA修饰组合可能协同作用以抑制成熟let-7 生物起源第二种机制是通过piwi和microRNA之间的串扰 途径。利用与依诺沙星相互作用的蛋白质的定量蛋白质组学， 分子显示刺激microRNA途径并抑制转化细胞的生长，我们 鉴定PIWIL 3蛋白为该药物的主要靶点。PIWIL 3是一种Argonaut蛋白， PIWI亚家族，主要在生殖系中表达并介导RNA干扰 通过piwiRNA。我们的研究结果揭示了PIWIL 3和microRNA之间的意外联系。 这可能是促进转型的关键途径。 上述方法是新颖的并且先前未被表征。这一建议建立 根据我们的研究结果，破译了介导下调节的分子机制， 通过RNA修饰和piwi/microRNA生物发生串扰的microRNA途径。阐明 这些机制，我们将采用多方面的方法，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 我希望从这项提案中获得的调查结果能为原因提供重要的新见解， 人类细胞中microRNA通路失调的后果，可以利用这些后果来改善 人类疾病的诊断和治疗。 1