METTL1 promotes cadmium-induced stress granules formation via enhancing translation of G3BP1 and expression of m7G- 3' tiRNA MetCAT

Abstract

Methyltransferase 1 (METTL1) is currently regarded as a key tRNA m⁷G writer. Recent studies indicate its potential role in carcinogenesis via increased m⁷G modification to stabilize tRNA and upregulate tRNA expression. Cadmium-induced stress triggers the assembly of stress granules (SGs) and production of tRNA-derived stress-induced RNAs (tiRNAs). However, whether METTL1 is involved in the formation of cadmium-induced SGs and its mechanism are still unclear. Here, we demonstrated that METTL1 promotes cadmium-induced SGs formation. Mechanistically, METTL1 not only upregulates SG's core protein Ras-GTPase-activating protein SH3 domain-binding protein 1 (G3BP1) translation through tRNAs m⁷G modification, but also enhances expression of one m⁷G-modified tiRNA, m⁷G-3' tiRNA-Met^CAT (mtiRM), which affects SGs assembly. Together, the findings concluded that the promotional effect of METTL1 on cadmium-induced SGs formation jointly through G3BP1 translation and mtiRM expression, thus providing insights into an intimate link between SGs and tumorigenesis.

Keywords: Cadmium; G3BP1; METTL1; Stress granules; TsRNAs.

Fig. 1

Overexpression of METTL1 upregulated the formation of SGs induced by cadmium. A Immunofluorescence (IF) assay was performed when T24 and SV-HUC-1 cells were exposed to CdCl₂ and NaAsO₂ (300 μM; 1 h) and stained for the SGs marker G3BP1. B T24 cell lines with CRISPR/Cas9-based knockdown METTL1 and SV-HUC-1 cell lines with LentiORF pLEX-MCS-based overexpression METTL1. Cells transfected with empty vector (T24-V2 and SV-HUC-1-pLEX) were used as control. C Compared to SV-HUC-1, IF assay showed higher expression of METTL1 and G3BP1 in METTL1-overexpressed SV-HUC-1 cells when exposed to CdCl₂ and NaAsO₂ (300 μM; 1 h). D The percentage of cells positive for SGs was quantified (= Number of cells with SGs/Total number of cells * 100%). Error bars indicate SD. The data are representative of three independent experiments

Fig. 2

Restoring METTL1 expression alleviates the inefficiency of SG formation. A-B Immunofluorescence assays of T24-V2, T24-KD-METTL1, T24-KD-METTL1/V2, T24-KD-METTL1/OE-METTL1 cells after exposure to CdCl₂ and NaAsO₂ (300 μM; 1 h) using antibodies against METTL1 and G3BP1. C Statistical analysis of the proportion of SGs-positive T24 cells. The results were obtained from three separate experiments

Fig. 3

METTL1 affects the translation of G3BP1 rather than transcription. A The Venn analysis of differentially expressed genes (DEGs) of the Ribo-seq results of T24-KD-METTL1 cells compared to T24 V2 cells(a) and SGs core proteins under arsenate stress(b). B The protein–protein interaction network of DEGs. C-F Western blot analyzed the G3BP1 level of T24-V2, T24-KD-METTL1(C), SV-HUC-1-pLEX and SV-HUC-1-OE-METTL1 cells(E), respectively. Bar-chart reflecting expression levels of G3BP1 after knockdown(D)/overexpression(F) of METTL1 in figure B. G qRT-PCR confirmed that the knockdown or overexpression of METTL1 had no significant effect on G3BP1 mRNA. H–K Polysome profile of T24-KD-METTL1/T24 V2(H) and SV-HUC-1 OE METTL1/SV-HUC-1 pLEX(J). Bar chart depicted G3BP1 mRNA level between T24-KD-METTL1/T24 V2(I) and SV-HUC-1 OE METTL1/SV-HUC-1 pLEX(K) L From 5' to 3', diagram showing the G3BP1 mRNA codons bound to m⁷G-tRNA and corresponding site-mutation plasmid construction strategies. M qRT-PCR illustrated the transfection efficiency of G3BP1 wild-type (WT) and mutant (Mut) plasmids in 293T V2 and 293T-KD-METTL1 cells. N Western blotting demonstrated the rescue effect of G3BP1 from G3BP1-Mut plasmid transfected 293T cells, detected by anti-Flag antibody. O Bar-chart reflecting expression levels of G3BP1-Flag in figure N. The results were obtained from three separate experiments

Fig. 4

Screening and validation of SG core tRFs & tiRNAs. A-C The unsupervised hierarchical heatmap of correlation coefficient (A), clustering heatmap (B) and PCA analysis (C) between SC-HUC-1 pLEX and SV-HUC-1 OE-METTL1 cells. D The volcano plot quantified the number of tRNAs with differential expression that was either up- or down-regulated between SC-HUC-1 pLEX and SV-HUC-1 OE-METTL1 cells. E Bar chart shows the intersection of the number of tsRNAs with the same trend of differential expression as m⁷G-tsRNAs (from Arraystar Human m⁷G small-RNA-modified microarrays) and tsRNAs from SG core tsRNA sequencing. F-G Venn diagram for SG core tsRNAs, m⁷G tsRNAs, and tsRNAs with m⁷G modification motifs in BC. a: Upregulated differential tsRNAs in SV-HUC-1 cells overexpressing METTL1. b: Downregulated differential m⁷G tsRNAs in T24-KD-METTL1 cells. c: tsRNAs with m⁷G modification motifs. d: Downregulated differential tsRNAs in SV-HUC-1 cells overexpressing METTL1. e: Upregulated differential m⁷G tsRNAs in T24-KD-METTL1 cells. H qRT-PCR quantified the expression level of tiRM in METTL1-overexpressed SV-HUC-1 and control SV-HUC-1 cells with a representative bar chart. I Northern blotting also validated the expression of tiRM in METTL1-overexpressed SV-HUC-1 cells. J Sanger sequence of 3' tiRNA Met^CAT, excluded amino acid arm CCA at the 3' end. K Agarose gel electrophoresis develop of 3' tiRNA Met^CAT from qRT-PCR product

Fig. 5

Effect of m⁷G modification on 3' tiRNA Met^CAT in SGs formation. A After transient transfection of tiRM inhibitor into SV-HUC-1 OE-METTL1 cells, the expression levels of tiRM were measured in transfected cells compared to cells transfected with negative control (NC). B, C METTL1-overexpressed SV-HUC-1 cells transfected tiRM inhibitor and NC were exposed to CdCl₂ (300 μM, 1 h). G3BP1 is the marker of SGs. D, E tiRM mimics and mtiRM were transfected into T24-KD-METTL1 cells, and the efficiency of overexpression was detected by qRT-PCR. F Workflow for the extraction of mtiRM (Graphing website: https://www.figdraw.com/). G The image shows the develop results of SDS-PAGE separation of tiRNAs. H mtiRM was verified by Northern Blot assay. I, J T24-KD-METTL1 transfected with tiRM mimics, mtiRM and NC were exposed to CdCl₂ (300 μM, 1 h) (I), and the percentage of positive cells for SGs was quantified (J). K, L The knockdown efficiency of total tiRM was detected by qRT-PCR after transfecting tiRM inhibitor into SV-HUC-1(K) and T24(L) cells. M–O After knocking down the expression of total tiRM in SV-HUC-1 and T24 cells, the level of G3BP1 protein(M) and mRNA (N, O) was not significantly changed

Fig. 6

Summary of the regulation of stress granule assembly by METTL1 via G3BP1 and mtiRM