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: 9910872
• 负责人: Allen C Zhu
• 金额: $ 5.05万
• 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/9910872
• 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; 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 该项目的目的是确定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肿瘤性。邮政- 转录RNA修饰是调节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。通过其对RNA的影响 稳定性，结构，出口，拼接和翻译，M6A影响许多生物学过程，包括 胚胎发育，免疫响应，记忆，病毒感染和癌症。 M6A有选择地安装在 通过甲基转移酶样3（mettl3）的某些mRNA转录本，作为催化亚基 哺乳动物M6A甲基转移酶“作者”复合物，必须精确调节适当的生物学 功能。例如，子宫内膜癌通常含有降低的Mettl3表达，从而导致降低 M6a甲基化。然后，降低的M6a甲基化会影响某些转录本的mRNA代谢，这 最终导致AKT途径和肿瘤性的激活增加。类似的METTL3依赖性 在白血病，肺癌和胚胎干细胞中也观察到现象。而mettl3可以 调节信号通路的激活，众所周知，METTL3在翻译后如何受到调节 以及这种调节如何影响mettl3的甲基转移酶活性。初步结果表明 ERK2的激活磷酸化磷酸化，并且在这些磷酸化位置缺乏Mettl3磷酸化 减少M6A甲基化。在鼠类胚胎干细胞中，Mettl3磷酸化似乎会影响 多能因子表达。因为激酶信号的失调，包括ERK途径，通常 导致癌细胞中不受控制的生长并在肿瘤性中起重要作用，我旨在确定 METTL3的ERK2磷酸化如何影响M6A甲基转移酶功能。我将使用黑色素瘤细胞作为 模型是因为它们经常包含BRAF V600E突变，该突变可组成性地激活ERK途径。 结合了分子生物学，生物化学和基因组学，我将完成以下目标。 （i）首先，我将研究Mettl3磷酸化对M6A甲基转移酶复合酶络合物复合物的影响 活动。特别是，我将研究酶活性的变化，M6A“作者”复杂关联和 亚细胞定位。 （ii）第二，我将确定Mettl3磷酸化如何影响黑色素瘤 恶性。因为M6A-Seq数据表明凋亡信号通路是差异化的 对于Mettl3磷酸化，我将验证凋亡途径是否具有差异性活性。那我会的 确定哪些基因的表达不同，哪些读取器蛋白会影响这些变化 表达。这项工作将阐明癌症转录后调节的新层。更好 了解癌症中激酶信号传导失调的RNA甲基化将是有价值的 未来癌症疗法的发展。