N7-methylguanosine tRNA modification enhances oncogenic mRNA translation and promotes intrahepatic cholangiocarcinoma progression. N7-methylguanosine tRNA modification enhances oncogenic mRNA translation and promotes intrahepatic cholangiocarcinoma progression

The cancer cells selectively promote translation of specific oncogenic transcripts to facilitate cancer survival and progression, while the underlying mechanisms are poorly understood. N7-methylguanosine (m7G) tRNA modification and its methyltransferase complex METTL1/WDR4 are significantly up-regulated in intrahepatic cholangiocarcinoma (ICC) and associated with poor prognosis. We developed tRNA reduction and cleavage sequencing (TRAC-Seq) to reveal the m7G tRNA methylome inICC cell line and ribosome nascent-chain complex-bound mRNAs sequencing(RNC-seq) and ribosome profiling(Ribo-seq) to study the differential translated genes and reveal the ribosome pausing. A subset of 22 tRNAs is modified at a ‘RAGGU’ motif within the variable loop. We observe increased ribosome occupancy at the corresponding codons in the Mettl1 knockdown ICC cell line implying widespread effects on tRNA function, ribosome pausing, and mRNA translation. Translation of cell cycle genes and EGFR signaling pathway genes is particularly affected. Our study uncovers the important physiological function and mechanism of METTL1-mediated m7G tRNA modification in the regulation of cancer progression. Overall design: TRAC-Seq was developed to identify the tRNA m7G methylome in the intrahepatic cholangiocarcinoma cell line. RNC-seq and Ribo-seq were used to study the differential translated genes in the Mettl1 knockdown and control cells.

癌细胞可选择性促进特定致癌转录本的翻译，以助力癌症存活与进展，但其背后的分子机制尚未被充分阐明。N7-甲基鸟苷（N7-methylguanosine, m7G）tRNA修饰及其甲基转移酶复合物METTL1/WDR4在肝内胆管癌（intrahepatic cholangiocarcinoma, ICC）中显著上调，且与不良预后密切相关。本研究开发了tRNA降解与切割测序（tRNA reduction and cleavage sequencing, TRAC-Seq），以揭示ICC细胞系中的m7G tRNA甲基化组；同时借助核糖体新生肽链复合物结合mRNA测序（ribosome nascent-chain complex-bound mRNAs sequencing, RNC-seq）与核糖体谱分析（ribosome profiling, Ribo-seq），探究差异翻译基因并解析核糖体暂停现象。
研究发现，有22种tRNA的可变环区域内的“RAGGU”基序发生了该类修饰。在METTL1敲低的ICC细胞系中，我们观察到对应密码子处的核糖体占据量升高，提示该修饰对tRNA功能、核糖体暂停及mRNA翻译存在广泛调控作用。其中，细胞周期基因与EGFR信号通路基因的翻译受影响最为显著。本研究揭示了METTL1介导的m7G tRNA修饰在调控癌症进展过程中的重要生理功能与分子机制。
整体实验设计：本研究通过TRAC-Seq鉴定肝内胆管癌细胞系中的tRNA m7G甲基化组；利用RNC-seq与Ribo-seq分析METTL1敲低组与对照组细胞中的差异翻译基因。