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

基本信息

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
项目类别：
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

项目摘要

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.

项目概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。转录 RNA 修饰是调节基因表达的主要组成部分。 (m6A) 是最丰富的内部 mRNA 和长非编码 RNA 通过其对 RNA 的影响。 m6A 的稳定性、结构、输出、剪接和翻译影响许多生物过程，包括 m6A 选择性地安装在胚胎发育、免疫反应、记忆、病毒感染和癌症上。甲基转移酶样 3 (METTL3) 负责某些 mRNA 转录，该酶充当 mRNA 的催化亚基哺乳动物 m6A 甲基转移酶“编写器”复合体，必须精确调节以实现适当的生物学功能例如，子宫内膜癌通常含有减少的METTL3表达，从而导致减少。 m6A 甲基化减少会影响某些转录本的 mRNA 代谢。最终导致 AKT 通路激活增加和类似 METTL3 依赖性。在白血病、肺癌和胚胎干细胞中也观察到这种现象，而 METTL3 可以。调节信号通路的激活，但人们对 METTL3 的翻译后调节机制知之甚少以及这种调节如何影响 METTL3 的甲基转移酶活性。 ERK2 的激活使 METTL3 磷酸化，并且这些磷酸位点缺乏 METTL3 磷酸化 METTL3 磷酸化似乎会影响小鼠胚胎干细胞中的 m6A 甲基化。因为激酶信号传导（包括 ERK 通路）经常失调。导致癌细胞生长不受控制，并在致瘤性中发挥重要作用，我的目的是确定 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研究对象。模型，因为它们经常含有 BRAF V600E 突变，该突变会持续激活 ERK 通路。结合分子生物学、生物化学和基因组学，我将实现以下目标。（一）首先研究METTL3磷酸化对m6A甲基转移酶复合物的影响具体来说，我将研究酶活性、m6A“作家”复合物关联的变化，以及 (II) 其次，我将确定 METTL3 磷酸化如何影响黑色素瘤。因为 m6A-seq 数据表明凋亡信号通路因差异甲基化。对于 METTL3 磷酸化，我将验证凋亡途径是否具有差异活性。确定哪些基因差异表达，以及哪些读者蛋白影响这些变化这项工作将更好地阐明癌症中新的转录后调控。了解癌症中激酶信号传导如何失调 RNA 甲基化对于以下方面非常有价值未来癌症疗法的发展。