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

•METTL1-mediated m7G tRNA modification promotes ICC cancer progression•METTL1-mediated regulates expression and mRNA translation•m7G selectively modulates translation in a codon-dependent manner•Oncogenic function of is verified multiple mouse models Cancer cells promote specific oncogenic transcripts to facilitate survival progression, but the underlying mechanisms are poorly understood. Here, we find that N7-methylguanosine (m7G) its methyltransferase complex components, METTL1 WDR4, significantly upregulated intrahepatic cholangiocarcinoma (ICC) associated with poor prognosis. We further reveal critical role METTL1/WDR4 promoting cell progression using loss- gain-of-function assays vitro vivo. Mechanistically, transcripts, including cell-cycle epidermal growth factor receptor (EGFR) pathway genes, m7G-tRNA-decoded codon-frequency-dependent mechanisms. Moreover, overexpression knockout models, demonstrate crucial Mettl1-mediated tumorigenesis Our study uncovers important physiological mechanism METTL1-mediated regulation progression. Intrahepatic second most common devastating primary liver cancer, accounting for ∼10–20% hepatic tumors (Job et al., 2020Job S. Rapoud D. Dos S.A. Gonzalez P. Desterke C. Pascal G. Elarouci N. Ayadi M. Adam R. Azoulay al.Identification four immune subtypes characterized by distinct composition functions tumor microenvironment cholangiocarcinoma.Hepatology. 2020; 72: 965-981Crossref PubMed Scopus (89) Google Scholar). Even surgical resection highly aggressive chemotherapies, 5-year patients only 5%–40% (Mavros 2014Mavros M.N. Economopoulos K.P. Alexiou V.G. Pawlik T.M. Treatment Prognosis Patients With Cholangiocarcinoma: Systematic Review Meta-analysis.JAMA Surg. 2014; 149: 565-574Crossref (418) This discouraging prognosis largely ascribed late-stage diagnosis limited therapeutic interventions. Until now, few molecular targeted agents have been approved management ICCs (Rizvi 2018Rizvi Khan Hallemeier C.L. Kelley R.K. Gores G.J. Cholangiocarcinoma - evolving concepts strategies.Nat. Rev. Clin. Oncol. 2018; 15: 95-111Crossref (711) Therefore, investigation development advancement effective strategies. Epigenetic regulations, DNA histone modifications, play human cancers (Michalak 2019Michalak E.M. Burr M.L. Bannister A.J. Dawson M.A. The roles DNA, RNA methylation ageing cancer.Nat. Mol. Cell Biol. 2019; 20: 573-589Crossref (203) Comprehensive genetic profiling studies revealed mutation misregulation epigenetic regulators (Farshidfar 2017Farshidfar F. Zheng Gingras M.C. Newton Y. Shih J. Robertson A.G. Hinoue T. Hoadley K.A. Gibb E.A. Roszik al.Cancer Genome Atlas NetworkIntegrative Genomic Analysis Identifies Distinct IDH-Mutant Molecular Profiles.Cell Rep. 2017; 18: 2780-2794Abstract Full Text PDF (275) Scholar; Nakamura 2015Nakamura H. Arai Totoki Shirota Elzawahry A. Kato Hama Hosoda Urushidate Ohashi al.Genomic spectra biliary tract Genet. 2015; 47: 1003-1010Crossref (693) targeting misregulated promising strategy (O’Rourke 2018O’Rourke C.J. Munoz-Garrido Aguayo E.L. Andersen J.B. Epigenome dysregulation cholangiocarcinoma.Biochim. Biophys. Acta Basis Dis. 1864: 1423-1434Crossref (28) Scholar, O’Rourke 2019O’Rourke Lafuente-Barquero Remodeling Cholangiocarcinoma.Trends Cancer. 5: 335-350Abstract (26) In addition recent uncovered emerging modifications gene (Chen 2019Chen X. Li Sun B.F. Yang Han Y.N. Yuan Chen R.X. Wei W.S. Liu Gao C.C. al.5-methylcytosine pathogenesis bladder through stabilizing mRNAs.Nat. 21: 978-990Crossref (217) ICC, it has reported N6-methyladenosine (m6A) could increase stability subset like TEAD2, thus accelerating (Rong 2019Rong Z.X. Z. He J.J. L.Y. Ren X.X. Mu Guan Y.D. Duan Y.M. Zhang X.P. al.Downregulation Fat Mass Obesity Associated (FTO) Promotes Progression Cholangiocarcinoma.Front. 9: 369Crossref (65) Compared mRNAs, tRNAs more extensively modified (Schimmel, 2018Schimmel complexity world: mammalian beyond protein synthesis.Nat. 19: 45-58Crossref (212) can regulate stability, translation, synthesis rates (Kirchner Ignatova, 2015Kirchner Ignatova Emerging adaptive signalling dynamics disease.Nat. 16: 98-112Crossref (326) Mutations or enzymes widely observed diseases (Okamoto 2012Okamoto Hirata Sato Koga Fujii Qi Ogawa I. Takata Shimamoto Tatsuka Frequent increased copy number high (cytosine-5-)-methyltransferase (NSUN2) cancers.DNA 2012; 31: 660-671Crossref (67) Pan, 2018Pan Modifications functional genomics transfer RNA.Cell Res. 28: 395-404Crossref (167) Nevertheless, still remain understood, especially cancers. one prevalent occurring variable loop installed METTL1-WDR4 (Alexandrov 2005Alexandrov Grayhack E.J. Phizicky Trm8p/Trm82p: evidence linking activity phenotype implicating Trm82p maintaining levels active Trm8p.RNA. 2005; 11: 821-830Crossref (77) Lin 2018Lin Q. Lelyveld V.S. Choe Szostak J.W. Gregory R.I. Mettl1/Wdr4-Mediated Methylome Is Required Normal Translation Embryonic Stem Self-Renewal Differentiation.Mol. Cell. 71: 244-255.e5Abstract (159) catalytic enzyme, while WDR4 plays major 2002Alexandrov Martzen M.R. Two proteins form required 7-methylguanosine yeast tRNA.RNA. 2002; 8: 1253-1266Crossref (174) Alexandrov Recent from our group others widespread methylomes mammals (Enroth 2019Enroth Poulsen L.D. Iversen Kirpekar Albrechtsen Vinther Detection internal mutational sequencing.Nucleic Acids e126Crossref Malbec 2019Malbec L. Y.S. Shi B.Y. Zhao Y.L. Y.G. Dynamic methylome regulatory translation.Cell 29: 927-941Crossref (86) 2019Zhang L.S. Ma Dai H.L. Luo Hu Dong Transcriptome-wide Mapping Internal N7-Methylguanosine Mammalian mRNA.Mol. 74: 1304-1316.e8Abstract (162) Accumulating showed impaired range disorders. For example, Mettl1 impairs induces abnormal differentiation embryonic stem (Lin Mutation causes forms microcephalic primordial dwarfism Galloway-Mowat syndrome (Braun 2018Braun D.A. Shril Sinha Schneider Tan W. Ashraf Hermle Jobst-Schwan Widmeier E. Majmundar al.Mutations as new cause syndrome.Am. Med. 176: 2460-2465Crossref (31) Shaheen 2015Shaheen Abdel-Salam G.M. Guy M.P. Alomar Abdel-Hamid M.S. Afifi H.H. Ismail S.I. Emam B.A. Alkuraya F.S. m(7)G46 dwarfism.Genome 210Crossref (95) These observations indicate METTL1/WDR4-mediated fate determination growth. Interestingly, correlated sensitivity chemotherapies colon cervical cells, suggesting potential link between (Liu 2019bLiu Chi Wang B. Overexpressed methyltransferase-like 1 (METTL1) chemosensitivity cisplatin regulating miR-149-3p/S100A4/p53 axis.Aging (Albany NY). 12328-12344Crossref (47) Okamoto 2014Okamoto Fujiwara Hori Okada K. Yazama Konishi Xiao Ota al.tRNA modifying enzymes, NSUN2 METTL1, determine 5-fluorouracil HeLa cells.PLoS 10: e1004639Crossref (73) However, inconclusive. present study, elevated Impaired inhibits via mechanism. data uncover insights into selective provide basis strategies aberrant To ICCs, first examined 22 database (TCGA-CHOL). found 14 19 tRNA-modifying (73.7%) compared peri-tumor tissues (Figure S1A). core components both Further analysis different types overexpressed (63.6%), 17 (77.3%) (Figures 1A 1B). Notably, top (ranked first) METTL1/WDR4-overexpressed among all TCGA 1B), METT1/WDR4 ICCs. validate METT1/WDR4-mediated determined patterns cohort consisting 83 patients. Immunohistochemistry (IHC) staining 2-fold higher 57 (68.7%) 1C 1D). Consistently, also 1E 1F). Western blot assay aberrantly paired specimens 1G–1I). correspondingly relative 1G, lower panel; Figure 1J). performed qRT-PCR confirmed S1B). Kaplan-Meier poorer status develop frequent recurrence 1K 1L). Overall, these indicated used two independent short hairpin RNAs (shRNAs) knock down HuCCT1 RBE, commonly lines 2A, 2B, S2A, S2B). Loss 2C 2D) colony-forming abilities 2E 2F) lines. Cell-cycle bromodeoxyuridine (BrdU) incorporation proportions arrested G2/M phase METTL1-knockdown 2G 2H). addition, Annexin V/propidium iodide (PI) depletion results apoptosis 2I 2J). knockdown suppresses migratory invasive 2K–2N). then vivo xenograft model. controls, mice injected METTL1-depleted slower reflected reduced size weight 2O–2Q). Similarly, resulted defected model S2C–S2K). Collectively, understand how northwestern blotting assess control cells. leads reduction 3A, 3B, S3A, S3B). mass spectrum level S3C). employed previously established site-specific cleavage sequencing (TRAC-seq) method 2019Lin Jiang Y.Z. Nucleotide resolution TRAC-Seq.Nat. Protoc. 14: 3220-3242Crossref (29) Scholar) profile global at single-nucleotide 3C). Using TRAC-seq, identified total contain “RGGUY” motif sequence located 3D 3E; Table S1). Knockdown signals 3F 3G). Furthermore, decreased majority m7G-modified had little effect on non-m7G-modified 3H 3I). northern (LysCTT LysTTT) unmodified (GlyCCC) 3J), which consistent previous reporting loss rapid decay 2006Alexandrov Chernyakov Gu Hiley S.L. Hughes T.R. Rapid result lack nonessential modifications.Mol. 2006; 87-96Abstract (333) Taken together, reduces target tRNAs, ICC. Given involved codon recognition, next ribosome processivity pausing codons 3K), essential efficient transition codons. polysome evaluate polyribosome peak 3L, S3D, S3E), upon depletion. impairment puromycin intake assay. incorporated RBE 3M, 3N, S3F, S3G), confirming rescue overexpressing wild-type inactive mutant, respectively Overexpression not rescues response 3O, 3P, S3H, S3I), translation. support identify downstream targets modification, ribosome-nascent chain-complex-bound (RNC-seq) actively translating mRNAs. RNC-seq 1,363 mRNAs ratios (TRs) 4A; S2) 983 TRs S3). Codon frequency (TR down) decoded 4B). confirm this correlation, grouped based their (top 25%) downregulated those low (bottom 4C). length demonstrated TR-down longer coding (CDS) 4D). suggest Gene Ontology translated enriched pathways, genes 4E 4F). pathways were reproduced S4A S4B). western validated CCNA2, CCND2, CDK6, CDK8, 4G 4H). EGFR phosphorylation corresponding targets, AKT mTOR, METTL1/WDR4-depleted 4G, 4H, S4C, S4D). RNC-qPCR candidate decreases 4I 4J). isolated fractionations CCNA2 EGFR, shift fractions monosome 4K 4L). altered attributed change transcription level. deficiency disrupts activities affecting corroborate conclusion defects cell-cycle-related lead small interfering (siRNA) deplete overall levels, cycle time points. CCNA2/EGFR occur 24 h after 5A–5E S5A–S5E), point, no obvious 5F S5F). 48 depletion, was 5G S5G). occurred before arrest, indicating phenotypes separately 5H, 5L, S5H, S5L) partially invasion capacities 5I–5K, 5M–5O, S5I–S5K, S5M–S5O), demonstrating inability METLL1-depleted functionally adopted luciferase reporter system, six repeats LysCTT AAG sequences inserted front firefly (F-luc) region 5P). (Lys) selected because highest S6A), LysCTT-decoded S6B). AAG-codon-inserted 5Q), enhances 5R), supporting m7G-codon-dependent rescued well 5S–5V S5P–S5S). another tRNA, LysTTT, S6C–S6J), mediate Altogether, provided substantial strong links investigate mutant EX-NEG-Lv201 plasmid system 6A). wild-type, catalytically inactive, increases elevates GlyCCC 6B, S7A, S7B). Functionally, 6C), migration 6D 6E), 6F 6G). ectopic does 6C–6G). vivo, an hydrodynamic transfection activated (myr-AKT) Yap (YapS127A) plasmids C57BL/6 (Song 2019Song Qiao Cigliano Utpatel Ribback Pilo M.G. Serra al.Combined CDK4/6 Pan-mTOR Inhibition Synergistic Against Cholangiocarcinoma.Clin. 25: 403-413Crossref (46) 2017Zhang Song Cao Xu Fan Che al.Pan-mTOR inhibitor MLN0128 against mice.J. Hepatol. 67: 1194-1203Abstract (60) co-injected AKT/YapS127A whether AKT/YapS127A/Mettl1 (A/Y+M1) developed massive burdens 4 weeks injection, lesions AKT/YapS127A/control (A/Y+C) 6H 6I). burdens, ratio body weight, A/Y+M1 A/Y+C 6J). Mettl1-overexpressing outcome than controls 6K). Hematoxylin eosin (H&E) larger sizes 6L–6N). Histological tr