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 本项目的目标是确定ERK 2对胃L3的磷酸化如何调节N6- 甲基腺苷（m6 A）甲基转移酶活性影响黑色素瘤细胞的致瘤性。后 转录RNA修饰是调节基因表达的主要成分。N6-甲基腺苷 (m6A)是最丰富的内部mRNA和长非编码RNA修饰。通过对RNA的影响 稳定性、结构、输出、剪接和翻译，m6 A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。m6 A选择性地安装在 甲基转移酶样3（methyltransferase-like 3，简称L3）是一种转录因子，它是转录因子的催化亚基。 哺乳动物m6 A甲基转移酶“作家”复合物，必须精确调控，以获得适当的生物活性。 功能例如，子宫内膜癌通常含有减少的胃L3表达，这导致减少的胃蛋白酶活性。 m6 A甲基化。降低m6 A甲基化然后影响某些转录物的mRNA代谢， 最终导致AKT途径的活化和致瘤性增加。相似的L3依赖性胃溃疡 在白血病、肺癌和胚胎干细胞中也观察到这种现象。而L3可以 调节信号通路的激活，但对胃L3是如何在免疫后调节的知之甚少。 以及这种调节如何影响胃L3的甲基转移酶活性。初步结果表明 ERK 2的激活使胃L3磷酸化，而在这些磷酸化位点缺乏胃L3磷酸化 降低m6 A甲基化。在小鼠胚胎干细胞中，胃L3磷酸化似乎影响 多能性因子表达。由于激酶信号传导（包括ERK通路）的失调， 导致癌细胞不受控制的生长，并在致瘤性中发挥重要作用，我的目的是确定 胃L3的ERK 2磷酸化如何影响m6 A甲基转移酶功能。我会用黑色素瘤细胞 因为它们经常含有BRAF V600 E突变，该突变组成性激活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，我将研究胃L3磷酸化对m6 A甲基转移酶复合物的影响 活动具体来说，我将研究酶活性的变化，m6 A“作家”复合体协会， 亚细胞定位(II)其次，我将确定胃L3磷酸化如何影响黑色素瘤 恶性肿瘤因为m6 A-seq数据表明凋亡信号通路由于以下原因而被差异甲基化： 对于胃L3磷酸化，我将验证凋亡途径是否具有差异活性。那我就 确定哪些基因差异表达，以及哪些阅读器蛋白影响这些变化， 表情这项工作将阐明癌症中转录后调控的新层。更好的 了解RNA甲基化是如何通过癌症中的激酶信号传导失调的， 未来癌症治疗的发展。