Elucidating mechanisms of microRNA pathway deregulation in human cells

项目总结 MicroRNAs是细胞增殖、分化和应激反应的关键调节因子 但人们对它们的生物起源知之甚少。例如，microRNAs的转录速率通常会 与相应的成熟microRNA水平无关，表明后 在这些重要的细胞过程中，转录事件决定了microRNAs的命运。 鉴于microRNAs在人类疾病中被解除管制，获得全面的 MicroRNA生物发生的机制观点可以极大地拓展潜在的治疗 MicroRNAs的机会。 我们最近发现了两种新的机制，在后调控microRNA途径- 转录水平。第一种机制是通过microRNAs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，以抑制其加工成成熟的微RNA。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的RNA修饰 Let-7，参与细胞分化和转化的一个重要的microRNA家族。我们的结果 提示RNA修饰的组合可能协同作用抑制成熟的let-7 生物发生学。第二种机制是通过piwi和microrna之间的串扰。 小路。利用定量蛋白质组学方法研究与依诺沙星相互作用的蛋白质 我们发现了刺激microRNA途径并抑制转化细胞生长的分子。 确定PIWIL3蛋白是该药物的主要靶点。PIWIL3是一种Argavit蛋白 Piwi亚家族，主要在生殖系中表达，并介导RNA干扰 通过piwiRNA。我们的结果揭示了PIWIL3和microRNA之间意想不到的联系 这可能是促进转型的关键途径。 上述过程是新颖的，以前没有描述过。这项建议建立了 根据我们的发现来破译调节细胞外信号转运蛋白表达下调的分子机制 通过RNA修饰和piwi/microRNA生物发生串扰实现microRNA途径。为了澄清 这些机制，我们将采用多方面的方法，利用我们在蜂窝和 分子生物学、RNA生物化学、质谱学和下一代测序。我们 预计从这项提案中获得的发现将为原因和 在人类细胞中解除microRNA途径调控的后果，可以用来改善 人类疾病的诊断和治疗。 1

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