Mechanisms and functions of N6-methyladenosine (m6A) in cancer

摘要 基因表达调控的一个重要机制是动态调节的，而且可能是可逆的， 信使核糖核酸的修饰。这些修饰可以对mRNA的稳定性产生显著影响， 翻译，以及信使核糖核酸代谢的其他方面。我们在这个领域发挥了奠基性的作用，开发了 N6-甲基腺苷(M6A)全转录组作图技术。我们的地图研究提供了 首次有证据表明，m6A可以被动态调节，并有可能在mRNA中赋予新的功能。我们 最近发现急性髓系白血病(AML)细胞表现出METTL3和METTL14水平升高， 作为形成m6A的甲基转移酶的异源二聚体。我们发现M6A能促进自我更新 在AML和CD34干细胞中，m6A的缺失会触发分化程序。因此，M6A具有关键的 在特定发育阶段的造血分化中的作用，这一过程在 AML。因此，在转录组水平上精确描述m6A的这些阶段特异性模式 是理解m6A如何影响发育转变的关键。开发新的方法来定位M6A 与造血和AML相关的稀有细胞群将有助于揭示这种表位转录是如何 修饰对于急性髓系白血病以及可能的其他疾病的分化的调节和解除调节至关重要 癌症。此外，m6A的作用在很大程度上被认为反映了特定的“阅读器”蛋白的作用， 它与mRNA中的m6A结合，影响其在细胞中的命运。主要读者是位于核心的YTHDC1，以及 YTHDF家族位于细胞质中，由三个几乎相同的对偶组成，可能有 冗余功能。为了显著提高我们对m6A在急性髓系白血病中的作用的理解， 这项建议的具体目标是：(1)以细胞类型特定的方式可视化和定位m6A在mRNA中的表达。 在这里，我们描述了以特定细胞类型的方式检测和定位m6A的方法的发展 以及它们在了解M6A在造血和AML中的动力学方面的应用。(2)界定功能 正常血细胞和急性髓系白血病对m6A阅读器YTHDC1的要求。基于全基因组的 屏幕和我们的初步数据，YTHDC1是可能调解主要方面的读者的有力候选者 M6A在急性髓系白血病中的作用。在这里，我们评估了YTHDC1在正常和恶性中的功能作用 利用人脐血细胞、急性髓系白血病细胞系和原发急性髓系白血病患者进行造血。(三)确定 YTHDF胞浆m6A阅读器对信使核糖核酸命运的作用和调控YTHDF蛋白似乎 是胞质中m6A mRNAs的主要调节者。我们将确定YTHDF蛋白是如何被调节到 介导其m6A-mRNA的不稳定作用，以及YTHDF蛋白是否影响细胞分化和 癌细胞系和急性髓系白血病中的增殖。总体而言，我们的项目将开发新的使能技术 研究癌症中的m6A，并测试m6A阅读器促进癌症进展的机制。

SUMMARY An important mechanism of gene expression regulation is dynamically regulated, and possibly reversible, nucleotide modifications in mRNA. These modifications can have marked effects on mRNA stability, translation, and other aspects of mRNA metabolism. We had a founding role in this field by developing the technology for transcriptome-wide mapping of N6-methyladenosine (m6A). Our mapping study provided the first evidence that m6A could be dynamically regulated, and potentially impart new functions in mRNA. We recently showed that acute myeloid leukemia (AML) cells exhibit elevated levels of METTL3 and METTL14, the heterodimer that acts as the m6A-forming methyltransferase. We found that m6A promotes self-renewal in AML and in CD34+ stem cells, and depletion of m6A triggers a differentiation program. Thus, m6A has critical roles in hematopoietic differentiation at specific stages of development, and this process is deregulated in AML. Therefore, precise characterization of these stage-specific patterns of m6A at a transcriptome-wide level is critical to understand how m6A affects developmental transitions. Developing new methods to map m6A in the rare cell populations relevant to hematopoiesis and AML would help to reveal how this epitranscriptomic modification is critical for the regulation and deregulation of differentiation seen in AML, and possibly other cancers. Additionally, the effects of m6A are largely thought to reflect the actions of specific “reader” proteins, which bind m6A in mRNA to affect its fate in cells. The major readers are YTHDC1 in the nucleus, and the YTHDF family in the cytoplasm, which comprise three nearly identical paralogs, and which may have redundant functions. In order to significantly advance our understanding of the role of m6A in AML, the specific aims of this proposal are: (1) To visualize and map m6A in mRNA in a cell-type specific manner. Here we describe the development of methods for detecting and mapping m6A in a cell type-specific manner and their application to understand m6A dynamics in hematopoiesis and AML. (2) To define the functional requirement for the m6A reader YTHDC1 in normal blood cells and in AML. Based on a genome-wide screen and our preliminary data, YTHDC1 is a strong candidate for the reader that may mediate major aspects of the effect of m6A in AML. Here we assess the functional role for YTHDC1 in both normal and malignant hematopoiesis using human cord blood cells, AML cell lines and primary AML patients. (3) To determine the roles and regulation of the YTHDF cytosolic m6A readers on mRNA fate. The YTHDF proteins appear to be the major regulators of m6A mRNAs in the cytosol. We will determine how YTHDF proteins are regulated to mediate their m6A-mRNA destabilizing effects and if YTHDF proteins influence cellular differentiation and proliferation in cancer cell lines and in AML. Overall, our project will develop new enabling technologies for studying m6A in cancer and test mechanisms by which the m6A readers contribute to cancer progression.