Investigating METTL5-regulated translational changes in hepatocellular carcinoma

研究肝细胞癌中 METTL5 调节的翻译变化

• 批准号: 10220715
• 负责人: Caraline Lee Sepich
• 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: 2020-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10220715
• 关键词: Affect; Apoptotic; B Cell Proliferation; Binding; Binding Sites; Biochemical; Biogenesis; Biological Assay; Cancer Etiology; Cell Cycle; Cell Line; Cell Nucleolus; Cell Proliferation; Cells; Cellular Stress; Cessation of life; Complex; Cytosol; Data; Development; Disease; Down-Regulation; Enzymes; Evolution; Fractionation; Future; Gene Expression; Genes; Goals; Growth; Hela Cells; HepG2; Heterogeneity; High Pressure Liquid Chromatography; Image; Immunoprecipitation; Internal Ribosome Entry Site; Knock-out; Lead; Link; Liver; Luciferases; Malignant Neoplasms; Malignant neoplasm of liver; Mediating; Messenger RNA; Methylation; Methyltransferase; Modification; Monitor; Nature; Normal tissue morphology; Nuclear; Oncogenes; Oncogenic; Open Reading Frames; Pathogenesis; Pathway interactions; Patients; Phenotype; Phosphotransferases; Positioning Attribute; Primary carcinoma of the liver cells; Process; Production; Prognosis; Prognostic Factor; Protein Biosynthesis; Proteins; Proteome; RNA, Ribosomal, 18S; Recycling; Regulation; Reporter; Research; Ribosomal Proteins; Ribosomal RNA; Ribosomes; Role; Site; Stress; Structure; Supporting Cell; Testing; Transcript; Translating; Translation Initiation; Translational Regulation; Translations; Up-Regulation; Western Blotting; Xenograft procedure; base; cancer risk; cell growth; cell type; cofactor; congenital anomaly; demethylation; hepatocellular carcinoma cell line; innovation; mRNA sequencing; mouse model; new therapeutic target; novel; overexpression; response; ribosome profiling; ribosome releasing factor; stoichiometry; therapy development; transcriptome sequencing; translation factor; translational impact; treatment strategy; tumor; tumorigenesis

Project Summary — Translational regulation has emerged as a key process in the evolution of hepatocellular carcinoma (HCC)1,11. Support of rapid proliferation in cancer requires enhanced protein production as well as gene-specific translational changes that facilitate reprogrammed cellular activities. Recent research has revealed that ribosome composition, including rRNA modification stoichiometry, can affect translational function in cells and bias translation of oncogenic transcripts, altering cell state6,17–20. Although 2% of all rRNA bases are modified, only two of them are N6-methyladenosine (m6A) — 28S m6A4220 and 18S m6A1832. Our lab previously characterized the 28S m6A4220 methyltransferase (ZCCHC4)21, and we have now biochemically characterized METTL5 (M5) as the 18S m6A1832 methyltransferase, stabilized by the cofactor TRMT112. The function of this site is still unknown, but structural analysis suggests roles in translation initiation and re-initiation events29,31, as it is near the mRNA channel and at the binding site of ribosome recycling factors. Furthermore, our M5 knockout HeLa cells display a markedly hypoproliferative phenotype while overexpression of M5 has been associated with hyperproliferation, including in hepatocellular carcinoma (HCC)8,9,22. We have also shown by imaging and biochemical fractionation that M5 is localized in both the nucleolus and cytosol, suggesting that the 18S m6A site may be dynamically methylated in the cytosol, even after ribosome biogenesis, to regulate and/or fine tune translational processes. The goal of this proposal is to define the functional effects of M5 on translation and investigate the mechanism by which it supports cell proliferation and cancer development. We hypothesize that M5 dynamically methylates m6A1832 in response to oncogenic cell stress with functional consequences in ribosome composition, translational function, and ribosome recycling activities that support proliferation and tumorigenesis. To investigate this hypothesis, we will first thoroughly define the impact of M5 on ribosome composition and function as follows: quantifying M5 and m6A1832 under normal and stress conditions by LC- MS/MS and HPLC; characterizing the effect of M5 on translation through nascent protein synthesis assays, ribosome profiling, and translation reporter assays; and monitoring M5-related changes in stoichiometry of ribosomal proteins and translation-related factors. Then, we will investigate the role and mechanism of M5 in HCC proliferation and tumorigenesis by examining the effects of M5 on ribosome binding and translational activities of ribosome recycling factors, and by evaluating the role of M5 in proliferation of HCC cell lines and tumorigenesis in HCC xenograft mouse models with respect to ribosome recycling processes. Successful completion of this proposal will unveil the function of 18S m6A1832 in translation, clarify the link between M5 and HCC prognosis, potentially guide new translation-based therapy development for HCC, and contribute to our understanding of how dynamic regulation of rRNA modifications can affect the proteome and cellular state in support of cancer development.

项目摘要——翻译调控已成为肝细胞进化的关键过程 癌（HCC）1,11。支持癌症的快速增殖需要增强蛋白质的产生以及 促进重新编程的细胞活动的基因特异性翻译变化。最近的研究表明 核糖体组成，包括 rRNA 修饰化学计量，可以影响细胞中的翻译功能 致癌转录物的偏差翻译，改变细胞状态6,17-20。尽管所有 rRNA 碱基中的 2% 经过修饰，其中只有两个是 N6-甲基腺苷 (m6A) — 28S m6A4220 和 18S m6A1832。我们实验室以前 表征了 28S m6A4220 甲基转移酶 (ZCCHC4)21，现在我们已经进行了生化表征 METTL5 (M5) 作为 18S m6A1832 甲基转移酶，由辅因子 TRMT112 稳定。这个的作用 位点仍然未知，但结构分析表明在翻译起始和重新起始事件中的作用29,31，如 它靠近 mRNA 通道，位于核糖体回收因子的结合位点。此外，我们的M5淘汰赛 HeLa 细胞表现出明显的低增殖表型，而 M5 的过度表达与 过度增殖，包括肝细胞癌 (HCC)8,9,22。我们还通过成像和显示 生化分级表明 M5 位于核仁和细胞质中，表明 18S m6A 位点 即使在核糖体生物发生之后，也可以在细胞质中动态甲基化，以调节和/或微调 翻译过程。该提案的目标是定义 M5 对翻译和 研究它支持细胞增殖和癌症发展的机制。我们假设 M5 动态甲基化 m6A1832 以响应致癌细胞应激，并产生功能性后果 支持增殖和增殖的核糖体组成、翻译功能和核糖体回收活动 肿瘤发生。为了研究这个假设，我们首先要彻底定义 M5 对核糖体的影响 组成和功能如下：通过LC-在正常和应激条件下定量M5和m6A1832 MS/MS 和 HPLC；通过新生蛋白质合成分析表征 M5 对翻译的影响， 核糖体分析和翻译报告分析；并监测 M5 相关的化学计量变化 核糖体蛋白和翻译相关因子。然后，我们将研究M5在其中的作用和机制。 通过检查 M5 对核糖体结合和翻译的影响来研究 HCC 增殖和肿瘤发生 核糖体回收因子的活性，并通过评估 M5 在 HCC 细胞系增殖中的作用和 HCC 异种移植小鼠模型中的肿瘤发生与核糖体回收过程有关。成功的 该提案的完成将揭示 18S m6A1832 的翻译功能，阐明 M5 和 HCC 预后，有可能指导 HCC 新的基于转化的疗法开发，并为我们的研究做出贡献 了解 rRNA 修饰的动态调节如何影响蛋白质组和细胞状态 支持癌症的发展。