Guanosine-7 tRNA methylation steers tRNA-derived fragment biogenesis and translational control in prostate cancer

Abstract Transfer RNAs (tRNAs) are exceptionally subject to modifications, including methylation. While mRNA methylation is emerging as an important regulator of biological and pathological processes in cancer, how post-transcriptional methylation of tRNAs contributes to cancer is largely unknown. Here we show that the RNA N7-methylguanosine (m7G) methyltransferase METTL1 is highly differentially expressed in prostate cancer compared to non-tumour prostate tissues. METTL1 expression regulation is mediated by the oncogenic regulator PI3K, which is altered in most advanced prostate tumours. Knockdown of METTL1 dramatically inhibits prostate cancer cell growth and tumour progression in vivo. In contrast, overexpression of the wild type but not the catalytically inactive METTL1 potentiates cell growth. Thus, METTL1-mediated methylation is important for prostate tumorigenesis. Mechanistically we find that METTL1 depletion causes loss of m7G tRNA methylation and increases endonucleolytic cleavage of Cysteine tRNA leading to an accumulation of 5′ tRNA-derived small RNA fragments. 5′ tRNA-derived fragments steer translation control to favour synthesis of key regulators of tumour growth suppression and immune rejection. In summary, our findings uncover a critical function of m7G tRNA methylation in directing translation control in cancer cells with important implications for tumour growth and unveil METTL1 inhibition as a promising anti-cancer therapeutic strategy. induction and maintenance of naïve human pluripotency are governed by distinct signaling requirements. tRNAs from WT and METTL1 KO cells were subjected to NaBH4-Aniline treatment followed by RNA-seq to unveil methylation of guanosine-7 in tRNA with nucleotie resolution. RNA-seq libraries were also analysed to unveil tRNA stability or processing into tRNA-derived fragments in METTL1 KO cells. Samll RNA from prostate cancer cells PC3 were extrated. Cells were either control CRIPSR (WT) or METTL1KO (using CRISPR). RNA were either non chemically treated (non-treated NT) or treated wih NaBH4 and aniline. Three replicates were used per condition. A total of 12 samples were sequenced.

摘要：转运RNA（Transfer RNA, tRNAs）广泛存在包括甲基化在内的转录后修饰。尽管mRNA甲基化已被证实是癌症中生物学与病理过程的关键调控因子，但tRNA的转录后甲基化如何参与癌症进程仍尚未明确。本研究发现，与非肿瘤前列腺组织相比，RNA N7-甲基鸟苷（m7G）甲基转移酶METTL1在前列腺癌组织中呈现显著差异表达。METTL1的表达调控由致癌调控因子PI3K介导，而PI3K在多数晚期前列腺肿瘤中存在异常改变。体内实验显示，敲低METTL1可显著抑制前列腺癌细胞增殖与肿瘤进展；与之相反，过表达野生型METTL1（而非催化失活型METTL1）可增强细胞增殖能力。由此可见，METTL1介导的甲基化修饰在前列腺肿瘤发生过程中发挥关键作用。机制研究表明，METTL1缺失会导致tRNA的m7G甲基化丢失，并增强半胱氨酸tRNA的核糖核酸内切酶切割，进而造成5′端tRNA衍生小RNA片段的积累。这类5′端tRNA衍生片段可通过调控翻译过程，优先促进肿瘤生长抑制因子与免疫排斥相关关键调控因子的合成。综上，本研究揭示了m7G tRNA甲基化在调控癌细胞翻译过程中的关键功能，其对肿瘤生长具有重要影响，并证实METTL1抑制是一种极具潜力的抗癌治疗策略。初始态人多能性的诱导与维持受不同信号通路需求调控。本研究对野生型（WT）和METTL1敲除（KO）细胞的tRNA进行NaBH4-苯胺处理，随后进行RNA测序（RNA-seq），以核苷酸分辨率解析tRNA中鸟苷-7位的甲基化修饰情况；同时对RNA测序文库进行分析，以探究METTL1 KO细胞中tRNA的稳定性及其加工为tRNA衍生片段的过程。从前列腺癌细胞PC3中提取小RNA，细胞分为对照成簇规律间隔短回文重复序列（CRISPR）（WT）组与METTL1敲除（KO，通过CRISPR技术构建）组，提取的RNA分为未化学处理（未处理组，NT）与经NaBH4和苯胺处理两组。每个实验条件设置3个生物学重复，共计12个样本进行测序。