The Effects of ERK2-Induced Phosphorylation of METTL3 on N6-methyladenosine (m6A) mRNA Methylation in Melanoma

ERK2 诱导的 METTL3 磷酸化对黑色素瘤中 N6-甲基腺苷 (m6A) mRNA 甲基化的影响

• 批准号: 10078122
• 负责人: Allen C Zhu
• 金额: $ 5.1万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-01-01 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10078122
• 关键词: Affect; Alanine; Apoptotic; BRAF gene; Binding; Biochemical; Biochemistry; Biological Assay; Biological Process; Catalytic Domain; Cell Proliferation; Cell physiology; Cellular biology; Co-Immunoprecipitations; Complex; DNA Damage; DNA Methylation; Data; Deposition; Development; Disease; Embryonic Development; Endometrial Carcinoma; Epigenetic Process; Fractionation; Future; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Genomics; Goals; Growth; Half-Life; Homeostasis; Human; Immune response; Immunofluorescence Immunologic; MAPK1 gene; Malignant - descriptor; Malignant Neoplasms; Malignant neoplasm of lung; Mammals; Mass Spectrum Analysis; Mediating; Melanoma Cell; Memory; Messenger RNA; Methylation; Methyltransferase; Modeling; Modification; Molecular Biology; Monitor; Mus; Mutate; Mutation; Myeloid Leukemia; Nuclear; Nuclear Export; Pathway interactions; Patients; Pharmaceutical Preparations; Phosphorylation; Phosphorylation Site; Phosphotransferases; Play; Post-Transcriptional RNA Processing; Post-Transcriptional Regulation; Process; Protein Kinase; Proteins; Proto-Oncogene Proteins c-akt; RNA; RNA Decay; RNA Splicing; RNA Stability; RNA metabolism; RNA methylation; Reaction; Reader; Regulation; Renal Cell Carcinoma; Research; Role; Signal Pathway; Signal Transduction; Site; Structure; Therapeutic; Transcript; Translations; Tumorigenicity; Untranslated RNA; Virus Diseases; Western Blotting; Work; cancer cell; cancer therapy; differential expression; embryonic stem cell; improved; in vitro Assay; insight; knock-down; leukemia; melanoma; mutant; novel; pluripotency factor; protein activation; response; stem cells; transcription factor; transcriptome; tumor growth

Project Summary The goal of this project is to determine how phosphorylation of METTL3 by ERK2 regulates N6- methyladenosine (m6A) methyltransferase activity to affect the tumorigenicity of melanoma cells. Post- transcriptional RNA modifications are a major component of regulating gene expression. N6-methyladenosine (m6A) is the most abundant internal mRNA and long non-coding RNA modification. Through its effects on RNA stability, structure, export, splicing, and translation, m6A influences many biological processes, including embryonic development, immune response, memory, viral infection, and cancer. m6A is selectively installed on certain mRNA transcripts by the methyltransferase-like 3 (METTL3), which acts as the catalytic subunit of the mammalian m6A methyltransferase “writer” complex, and must be precisely regulated for proper biological function. For example, endometrial cancer often contains reduced METTL3 expression, which leads to reduced m6A methylation. Reduced m6A methylation then affects mRNA metabolism of certain transcripts, which ultimately results in increased activation of the AKT pathway and tumorigenicity. Similar METTL3-dependent phenomena are also observed in leukemia, lung cancer, and embryonic stem cells. While METTL3 can regulate activation of signaling pathways, it is poorly understood how METTL3 is post-translationally regulated and how such regulation affects the methyltransferase activity of METTL3. Preliminary results indicate that activation of ERK2 phosphorylates METTL3, and that lack of METTL3 phosphorylation at these phospho-sites reduces m6A methylation. In murine embryonic stem cells, METTL3 phosphorylation appears to impact pluripotency factor expression. Because dysregulation of kinase signaling, including the ERK pathway, often leads to uncontrolled growth in cancer cells and plays a significant role in tumorigenicity, I aim to determine how ERK2 phosphorylation of METTL3 affects m6A methyltransferase function. I will use melanoma cells as a model because they frequently a contain BRAF V600E mutation that constitutively activates the ERK pathway. With a combination of molecular biology, biochemistry, and genomics, I will accomplish the following aims. (I) First, I will investigate the effects of METTL3 phosphorylation on the m6A methyltransferase complex activity. Specifically, I will study changes in enzymatic activity, m6A “writer” complex association, and subcellular localization. (II) Second, I will determine how METTL3 phosphorylation affects melanoma malignancy. Because m6A-seq data suggests the apoptotic signaling pathway is differentially methylated due to METTL3 phosphorylation, I will validate whether the apoptotic pathway is differentially active. Then I will determine which genes are differentially expressed, and which reader proteins effect those changes in expression. This work will elucidate a novel layer of post-transcriptional regulation in cancer. A better understanding of how RNA methylation is dysregulated by kinase signaling in cancers will be valuable for development of future cancer therapies.

项目摘要 该项目的目标是确定ERK2对METTL3的磷酸化如何调节N6- 甲基腺苷(M6A)甲基转移酶活性影响黑色素瘤细胞的致瘤性。邮寄- 转录RNA修饰是调控基因表达的主要组成部分。N6-甲基腺苷 (M6A)是最丰富的内部mRNA和长的非编码RNA修饰。通过它对RNA的影响 稳定性、结构、输出、剪接和翻译，m6A影响许多生物过程，包括 胚胎发育、免疫反应、记忆、病毒感染和癌症。M6A有选择地安装在 由甲基转移酶样3(METTL3)转录的某些mRNA，它作为甲基转移酶的催化亚单位 哺乳动物m6A甲基转移酶“写入者”复合体，必须受到精确调控才能获得适当的生物学特性 功能。例如，子宫内膜癌通常包含METTL3表达减少，这会导致减少 M6A甲基化。减少的m6A甲基化会影响某些转录物的mRNA代谢，这 最终导致AKT途径的激活和致瘤性的增加。类似的METTL3依赖 在白血病、肺癌和胚胎干细胞中也可以观察到这种现象。而METTL3可以 调节信号通路的激活，对METTL3是如何在翻译后调节的知之甚少 以及这种调控如何影响METTL3的甲基转移酶活性。初步结果表明， ERK2激活使METTL3磷酸化，而在这些磷酸化部位缺乏METTL3磷酸化 减少m6A甲基化。在小鼠胚胎干细胞中，METTL3的磷酸化似乎影响了 多能因子的表达。因为激酶信号的失调，包括ERK通路，通常 会导致癌细胞的不受控制的生长，并在肿瘤发生中发挥重要作用，我的目标是确定 METTL3的ERK2磷酸化对m6A甲基转移酶功能的影响我将使用黑色素瘤细胞作为 这是因为它们经常含有BRAF V600E突变，该突变可结构性地激活ERK途径。 结合分子生物学、生物化学和基因组学，我将实现以下目标。 (I)首先，我将研究METTL3磷酸化对m6A甲基转移酶复合体的影响 活动。具体地说，我将研究酶活性的变化，m6A“编写者”复合体的联系，以及 亚细胞定位。(Ii)第二，我将确定METTL3磷酸化如何影响黑色素瘤 恶毒。因为m6A-seq数据表明，细胞凋亡信号通路存在差异甲基化。 对于METTL3的磷酸化，我将验证凋亡途径是否存在差异活性。那我就会 确定哪些基因是差异表达的，哪些阅读器蛋白会影响这些变化 表情。这项工作将阐明癌症中一个新的转录后调控层。更好的 了解癌症中的RNA甲基化是如何被激酶信号失控的，将对 未来癌症治疗方法的发展。