METTL1/WDR4-mediated m7G tRNA modifications and m7G codon usage promote mRNA translation and lung cancer progression

• DOI: 10.1016/j.ymthe.2021.08.005
• Publication Date: 2021-08-08T08:59:09Z
• Authors: Jieyi Ma; Hui Han; Ying Huang; Chunlong Yang; Siyi Zheng; Tiancai Cai; Jiong Bi; Xiaohui Huang; Ruiming Liu; Libin Huang; Yifeng Luo; Wen Li; Shuibin Lin
• Keywords: 0301 basic medicine; 03 medical and health sciences; Lung Neoplasms; RNA, Transfer; GTP-Binding Proteins; Protein Biosynthesis; Humans; Methyltransferases; Codon Usage; 3. Good health

Mis-regulated epigenetic modifications in RNAs are associated with human cancers. The transfer (tRNAs) the most heavily modified RNA species cells; however, little is known about functions of tRNA In this study, we uncovered that expression levels N7-methylguanosine (m7G) methyltransferase complex components methyltransferase-like 1 (METTL1) and WD repeat domain 4 (WDR4) significantly elevated lung cancer samples negatively patient prognosis. Impaired m7G modification upon METTL1/WDR4 depletion resulted decreased cell proliferation, colony formation, invasion, impaired tumorigenic capacities cells vitro vivo. Moreover, gain-of-function mutagenesis experiments revealed METTL1 promoted growth invasion through regulation modifications. Profiling methylation mRNA translation highly translated mRNAs have higher frequencies tRNA-decoded codons, knockdown suggesting codon usage play an essential function regulation. 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Interestingly, co-related (Figures 1E 1F), interplay WDR4. high survival, indicating potential 1G 1H). To further confirm these results, immunohistochemistry (IHC) tissue array. Based staining intensities, were divided two groups: low group (immunoreactivity score [IRS] < 3) (IRS ≧ 3). representative cases different groups Figure 1I. confirmed 1J 1K). Western blot analysis also verified specimens, peri-tumor 1L−1N). correspondingly 1L 1O). Overall, results expressions patients, oncogenic cancer.METTL1 inhibition suppresses migration, vitroTo investigate effect knocked down A549 H1299 lentiviruses expressing short hairpin (sh)GFP (negative control [NC]), shMETTL1-1 (shM1-1), shMETTL1-2 (shM1-2) 2A 2B). reduced but other methyltransferases METTL3 METTL5 2B; S1A), stabilizes cofactor cells. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay showed inhibited proliferation 2C 2D). depleted exhibited colony-formation 2E 2F). Cell-cycle induced increased ratio G0/G1-phase G2/M-phase 2G 2H). We carried out wound-healing assays evaluate migration metastasis delayed wound closure suppressed invasive abilities 2I−2L). growth, vitro.Figure 2Inhibition vitroShow full caption(A B) Validation (sh)METTL1 western (C D) Knockdown (E F) METTL1-depleted formed less colonies assays. (G H) (I−L) (I J) (K L) Data presented mean ± SD. Scale bar, 100 μm. (One-way ANOVA, ∗p 0.05, ∗∗p 0.01, ∗∗∗p 0.001, #p ##p shGFP.)View Large Image ViewerDownload Hi-res image Download (PPT)Knockdown vitroWDR4 tRNAs.16Lin infected shGFP, shWDR4-1, shWDR4-2 3A 3B). decreases maintaining 3B), consistent finding yeast.23Alexandrov Grayhack E.J. Trm8p/Trm82p: linking activity implicating Trm82p active Trm8p.RNA. 2005; 821-830Crossref (76) next subjected led activities 3C−3F). 3G−3J), confirming Taken together, 3WDR4 cellsShow confirmation independent shRNAs was knockdown. formation Wound-healing inhibitory migration. invasion. shGFP group.)View inhibits vivoWe performed xenograft tumor-formation Briefly, subcutaneously injected right dorsal flank nude mice, volumes measured weekly. larger subcutaneous visible tumors observed group, whereas only 3 smaller shM1-1 no shM1-2 (Figure 4A). Xenograft curve weight reduces vivo 4B 4C). IHC shMETTL1 (shM1) 4D 4E). shM1 lower Ki67 level, METTL1-knockdown malignant than those 4F). WDR4-knockdown Similarly, model, reflected size 4G−4I). shWDR4 4J 4K). WDR4-depleted proliferative 4L). our supported progression.Figure 4Knockdown vivoShow caption(A) (n = 6). (B C) Growth (B) final weights (C) without depletion. (D−F) Representative images quantitative scores (D), (E), (F) tumors. (G) (H I) (H) (I) (J−L) (J), (K), (L) SEM. bars, μm 50 (Two-way one-way Student's t test, (PPT)Overexpression promotes capacitiesIn order validate Results wild-type (M1-WT) 5A−5C). enhanced 5D 5E). catalytic dead mutant (M1-Mut) had 5A−5E), METTL1's enzyme promoting 5F−5J). 5Overexpressed upregulation Overexpressed Overexpression instead A549. (D E) (D) (E) (J) I); 200 J). negative [NC] (PPT)METTL1 expressionIn unveil how regulates northwestern analyze notable reduction 6A 6B; S1B). cleavage sequencing (TRAC-seq) profile after total extracted, small isolated oxidative demethylase AlkB demethylation then cleaved NaBH4/aniline treatment. Following purification, purified used high-throughput identify single-nucleotide resolution. TRAC-seq identified series isoacceptors containing 6C). 6D 6E). Analysis majority m7G-modified 6F 6G). Northern ValAAC ProAGG non-m7G-modifiied GlnCTG 6H). On hand, non-m7G 6I). expression.Figure 6METTL1 modification, translationShow list TRAC-seq. indicated sties. Decreased non-m7G-modified tRNAs. Relative based relative each type calculated combined all belonging same type. blotting U6 snRNA loading control. Polysome profiling (K) efficiency (TE) analyzing puromycin intake inactive rescued deficiency Scatterplot TE dividing RNC-mRNA signals input signals. (M) Frequency m7G-related codons up, down, (others) (N) frequency top 25% bottom NC group. (O) TEs up (P) ontology enrichment biological process (Q) Pathway (R S) qRT-PCR changes cell-cycle-related genes. (T) CCND3 sections. translationGiven factors translation, determined whether polysome factions 6J), th