METTL1 mediated tRNA m7G modification promotes leukaemogenesis of AML via tRNA regulated translational control

Abstract

Background: RNA modifications have been proven to play fundamental roles in regulating cellular biology process. Recently, maladjusted N7-methylguanosine (m⁷G) modification and its modifiers METTL1/WDR4 have been confirmed an oncogene role in multiple cancers. However, the functions and molecular mechanisms of METTL1/WDR4 in acute myeloid leukemia (AML) remain to be determined.

Methods: METTL1/WDR4 expression levels were quantified using qRT-PCR, western blot analysis on AML clinical samples, and bioinformatics analysis on publicly available AML datasets. CCK-8 assays and cell count assays were performed to determine cell proliferation. Flow cytometry assays were conducted to assess cell cycle and apoptosis rates. Multiple techniques were used for mechanism studies in vitro assays, such as northern blotting, liquid chromatography-coupled mass spectrometry (LC-MS/MS), tRNA stability analysis, transcriptome sequencing, small non-coding RNA sequencing, quantitative proteomics, and protein synthesis measurements.

Results: METTL1/WDR4 are significantly elevated in AML patients and associated with poor prognosis. METTL1 knockdown resulted in reduced cell proliferation and increased apoptosis in AML cells. Mechanically, METTL1 knockdown leads to significant decrease of m⁷G modification abundance on tRNA, which further destabilizes tRNAs and facilitates the biogenesis of tsRNAs in AML cells. In addition, profiling of nascent proteins revealed that METTL1 knockdown and transfection of total tRNAs that were isolated from METTL1 knockdown AML cells decreased global translation efficiency in AML cells.

Conclusions: Taken together, our study demonstrates the important role of METTL1/WDR4 in AML leukaemogenesis, which provides a promising target candidate for AML therapy.

Keywords: AML; METTL1/WDR4; Translation control; m7G; tRNA modification.

Fig. 1

METTL1/WDR4 were upregulated and associated with poor prognosis of AML patients. A Expression Level of METTL1 and WDR4 in healthy individuals (NC) and AML patients from the GEO dataset. B Comparison of METTL1 and WDR4 expression in bone marrow (BM) and peripheral blood (PB) samples between AML patients (red box) and healthy individuals (blue box) from the GEO dataset. C Expression level of METTL1 and WDR4 in bone marrow mononuclear cells between healthy donors (NC) and AML patients from our center. D Comparison of METTL1 and WDR4 expression in healthy individuals and various subtypes of AML patients in the GEO dataset. E Expression level of METTL1 and WDR4 between healthy donors and various subtypes of AML patients from our center. F Representative bands of METTL1 and WDR4 examined by western blot assay in healthy donors and AML patients from our center. G Correlation between METTL1/WDR4 expression and overall survival of AML patients according to data from TCGA. Data were presented as mean ± SEM (Student’s t test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: not significant)

Fig. 2

METTL1 knockdown inhibits the survival and growth of human AML cells. A Validation of the knockdown effect of METTL1 by western blot in MOLM-13 (M13) and THP-1 cells. B, C Knockdown of METTL1 suppressed cell proliferation of THP-1 and M13 cells. D, E Knockdown of METTL1 induced an increased ratio of G1-phase cells and decreased the ratio of S-phase cells. F–H Knockdown of METTL1 induced an increased cell apoptosis in THP-1 and M13 cells. F, G Representative images of flow cytometry analysis. H Quantification data. I Knockdown of METTL1 induced changes of the apoptosis-related proteins in THP-1 and M13 cells. J Validation of the overexpression effect of METTL1 by western blot in HL60 cells. K Overexpression of METTL1 promoted cell proliferation of HL60 cells. L Knockdown of METTL1 suppressed proliferation of THP-1 and M13 cells while treated with cytarabine (CYT). M Knockdown of METTL1 increased cell apoptosis in THP-1 and M13 cells while treated with CYT for 48 h. N Overexpression of METTL1 increased cell growth rates of HL60 cells while treated with CYT. O, P Overexpression of METTL1 decreased cell apoptosis in HL60 cells while treated with CYT for 48 h. O Quantification data. P Representative images of flow cytometry analysis. shNC: METTL1 control, shM1: METTL1 knockdown, CON: METTL1 control, OE-M1: METTL1 overexpression. Data were presented as mean ± SEM (Student’s t test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)

Fig. 3

DNA methylation regulates METTL1 expression and the correlation of METTL1 and WDR4 in AML cells. A Comparison of METTL1 DNA methylation level in healthy individuals (NC) and AML patients from TCGA dataset. B Correlation between mRNA expression level (FPKM) and DNA methylation level of METTL1 in AML patients from TCGA dataset. C, D The mRNA (C) and protein (D) expression level of METTL1 in AML cells while treated with bobcat339 hydrochloride. E Correlation between METTL1 and WDR4 mRNA levels in AML patients from TCGA dataset. F Correlation between the METTL1 and WDR4 expression levels and overall survival of AML patients from TCGA dataset. G The protein expression level of WDR4 in METTL1 knockdown (shM1) and METTL1 control (shNC) AML cells. H The protein expression level of WDR4 in METTL1 over-expression (OE-M1) and METTL1 control (CON) HL60 cells. I The mRNA expression level of WDR4 in METTL1 knockdown (shM1) and METTL1 control (shNC) AML cells. J The mRNA expression level of WDR4 in METTL1 over-expression (OE-M1) and METTL1 control (CON) HL60 cells. K, L The protein (K) and mRNA (L) expression level of METTL1 in WDR4 over-expression (OE-WDR4) and WDR4 control (CON) HL60 cells. Data were presented as mean ± SEM (Student’s t test, *p < 0.05, **p < 0.01, ****p < 0.0001)

Fig. 4

Knockdown of METTL1 leads to a reduction in the abundance of cellular m⁷G modification on tRNA and mRNA in THP-1 cells. A Experimental procedures for detecting RNA modifications in THP-1 cells. B, C Radar map (B) and heatmap (C) showed the comparison of RNA modification levels in tRNA-enriched fragments (tRNA, ~ 80nt) in METTL1 knockdown and control THP-1 cells. D Comparison of m⁷G modification level on tRNA in METTL1 knockdown and control THP-1 cells. E, F Radar map (E) and heatmap (F) showed the comparison of RNA modification levels in mRNA in METTL1 knockdown and control THP-1 cells. G Comparison of m⁷G modification level on mRNA in METTL1 knockdown and control THP-1 cells. Data were presented as mean ± SEM (Student’s t test, ***p < 0.001, ****p < 0.0001)

Fig. 5

METTL1 knockdown inhibits cellular nascent protein synthesis in THP-1 cells. A Western blotting confirmation of METTL1 knockdown using siRNAs in THP-1 cells. B The level of OPP means fluorescence intensity (MFI) in METTL1 knockdown (siMETTL1) and METTL1 control (siNC) THP-1 cells. Left panels: representative images. Right panels: quantification data. C Transcriptome sequencing analysis of gene changes in THP-1 cells upon METTL1 knockdown. D The top 20 down-regulation proteins in METTL1 knockdown THP1 cells compared to the controls. E GO enrichment analysis of down-regulation proteins in METTL1 knockdown THP-1 cells. Data were presented as mean ± SEM (Student’s t test, ***p < 0.001)

Fig. 6

METTL1 knockdown leads to tRNA dysregulation for nascent protein synthesis inhibition. A–D Representative tRNAs level examined by northern blot in METTL1 knockdown (shM1) and METTL1 control (shNC) THP-1 cells. Left: NB images. Right: quantification data. E Experimental procedures for OPP labeling experiments in F, G. F, G The level of OPP means fluorescence intensity (MFI) in THP-1 cells transfected with tRNAs extracted from METTL1 knockdown (shM1) or METTL1 control (shNC) THP-1 cells, respectively. F Quantification data. G Representative images of flow cytometry analysis. Data were presented as mean ± SEM (Student’s t test, ***p < 0.001, ****p < 0.0001)

Fig. 7

METTL1 knockdown promotes tsRNA biogenesis. A tRNA degradation levels on PAGE gels under RNase A/T1 in THP-1 cells upon METTL1 knockdown. B High-throughput small RNA sequencing analysis of tsRNA expression in THP-1 cells upon METTL1 knockdown. C Representative bands of tsRNA levels examined by RT-PCR in THP-1 cells upon METTL1 knockdown. D, E Northern blot analysis of 3′tsRNA^LeuCAG and 3′tsRNA^LeuTAA in THP-1 cells upon METTL1 knockdown. Left: NB images. Right: quantification data. F High-throughput small RNA sequencing analysis of tsRNAs expression in THP-1 cells upon METTL1 overexpression. G Representative bands of tsRNAs levels examined by RT-PCR in THP-1 cells upon METTL1 overexpression. shNC: METTL1 control, shM1: METTL1 knockdown, CON: METTL1 control, OE-M1: METTL1 overexpression. Data were presented as mean ± SEM (Student’s t test, ****p < 0.0001)