Investigating METTL5-regulated translational changes in hepatocellular carcinoma

翻译调控已成为肝细胞进化的关键过程 癌症(HCC)1，11.支持癌症的快速增殖需要增加蛋白质的产生以及 促进重新编程的细胞活动的基因特有的翻译变化。最近的研究表明 核糖体的组成，包括rRNA修饰化学计量学，可以影响细胞的翻译功能 和癌基因转录物的偏向翻译，改变细胞状态6，17-20。尽管所有rRNA碱基中有2%是 其中只有两个是N6-甲基腺苷(M6A)-28S m6A4220和18S m6A1832。我们以前的实验室 鉴定了28S m6A4220甲基转移酶(ZCCHC4)21，我们现在已经进行了生化鉴定 METTL5(M5)为18S m6A1832甲基转移酶，由辅因子TRMT112稳定。它的功能是 地点尚不清楚，但结构分析表明，在翻译启动和重新启动事件中扮演了角色29，31，如 它位于mRNA通道附近，位于核糖体循环因子的结合部位。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 生化分级表明，M5定位于核仁和胞浆中，提示18S M6A位点 可能在胞浆中动态甲基化，甚至在核糖体生物发生之后，以调节和/或微调 翻译过程。这项提案的目标是定义M5对翻译的功能影响和 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，在 核糖体组成、翻译功能和核糖体循环活动，支持增殖和 肿瘤发生学。为了研究这一假说，我们将首先彻底定义M5对核糖体的影响。 组成和功能如下：正常和应激条件下的M5和M6A1832用LC- MS/MS和HPLC；通过新生蛋白质合成试验表征M5对翻译的影响， 核糖体分析和翻译报告分析；以及监测M5相关的化学计量学变化 核糖体蛋白和翻译相关因子。然后，我们将研究M5在脑内的作用和机制。 通过检测M5对核糖体结合和翻译的影响研究肝癌的增殖和肿瘤发生 核糖体循环因子的活性，并通过评估M5在肝癌细胞系增殖和 与核糖体循环过程有关的肝癌异种移植小鼠模型中的肿瘤发生。成功 这项提案的完成将揭示18S m6A1832的翻译功能，澄清M5和M5之间的联系 肝癌预后，潜在地指导新的基于翻译的肝癌治疗开发，并为我们的 了解rRNA修饰的动态调节如何影响蛋白质组和细胞状态 支持癌症发展。

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.