N6-Methyladenosine (m⁶A) modification has been implicated in the progression of several cancers. We reveal that during epithelial-mesenchymal transition (EMT), one important step for cancer cell metastasis, m⁶A modification of mRNAs increases in cancer cells. Deletion of methyltransferase-like 3 (METTL3) down-regulates m⁶A, impairs the migration, invasion and EMT of cancer cells both in vitro and in vivo. m⁶A-sequencing and functional studies confirm that Snail, a key transcription factor of EMT, is involved in m⁶A-regulated EMT. m⁶A in Snail CDS, but not 3’UTR, triggers polysome-mediated translation of Snail mRNA in cancer cells. Loss and gain functional studies confirm that YTHDF1 mediates m⁶A-increased translation of Snail mRNA. Moreover, the upregulation of METTL3 and YTHDF1 act as adverse prognosis factors for overall survival (OS) rate of liver cancer patients. Our study highlights the critical roles of m⁶A on regulation of EMT in cancer cells and translation of Snail during this process.

N⁶-methyladenosine (m⁶A) is the most abundant epigenetic modification in eukaryotic mRNAs and is essential for multiple RNA processing events during mammalian development and disease control. Here we show that conditional knockout of the m⁶A methyltransferase Mettl3 in bone marrow mesenchymal stem cells (MSCs) induces pathological features of osteoporosis in mice. Mettl3 loss-of-function results in impaired bone formation, incompetent osteogenic differentiation potential and increased marrow adiposity. Moreover, Mettl3 overexpression in MSCs protects the mice from estrogen deficiency-induced osteoporosis. Mechanistically, we identify PTH (parathyroid hormone)/Pth1r (parathyroid hormone receptor-1) signaling axis as an important downstream pathway for m⁶A regulation in MSCs. Knockout of Mettl3 reduces the translation efficiency of MSCs lineage allocator Pth1r, and disrupts the PTH-induced osteogenic and adipogenic responses in vivo. Our results demonstrate the pathological outcomes of m⁶A mis-regulation in MSCs and unveil novel epitranscriptomic mechanism in skeletal health and diseases.

Studies on biological functions of N⁶-methyladenosine (m⁶A) modification in mRNA have sprung up in recent years. We find m⁶A can positively regulate the glycolysis of cancer cells. Specifically, m⁶A-sequencing and functional studies confirm that pyruvate dehydrogenase kinase 4 (PDK4) is involved in m⁶A regulated glycolysis and ATP generation. The m⁶A modified 5′UTR of PDK4 positively regulates its translation elongation and mRNA stability via binding with YTHDF1/eEF-2 complex and IGF2BP3, respectively. Targeted specific demethylation of PDK4 m⁶A by dm⁶ACRISPR system can significantly decrease the expression of PDK4 and glycolysis of cancer cells. Further, TATA-binding protein (TBP) can transcriptionally increase the expression of Mettl3 in cervical cancer cells via binding to its promoter. In vivo and clinical data confirm the positive roles of m⁶A/PDK4 in tumor growth and progression of cervical and liver cancer. Our study reveals that m⁶A regulates glycolysis of cancer cells through PDK4.

Mis-regulated epigenetic modifications in RNAs are associated with human cancers. The transfer RNAs (tRNAs) are the most heavily modified RNA species in cells; however, little is known about the functions of tRNA modifications in cancers. In this study, we uncovered that the expression levels of tRNA N⁷-methylguanosine (m⁷G) methyltransferase complex components methyltransferase-like 1 (METTL1) and WD repeat domain 4 (WDR4) are significantly elevated in human lung cancer samples and negatively associated with patient prognosis. Impaired m⁷G tRNA modification upon METTL1/WDR4 depletion resulted in decreased cell proliferation, colony formation, cell invasion, and impaired tumorigenic capacities of lung cancer cells in vitro and in vivo. Moreover, gain-of-function and mutagenesis experiments revealed that METTL1 promoted lung cancer growth and invasion through regulation of m⁷G tRNA modifications. Profiling of tRNA methylation and mRNA translation revealed that highly translated mRNAs have higher frequencies of m⁷G tRNA-decoded codons, and knockdown of METTL1 resulted in decreased translation of mRNAs with higher frequencies of m⁷G tRNA codons, suggesting that tRNA modifications and codon usage play an essential function in mRNA translation regulation. Our data uncovered novel insights on mRNA translation regulation through tRNA modifications and the corresponding mRNA codon compositions in lung cancer, providing a new molecular basis underlying lung cancer progression.

The tyrosine kinase inhibitor lenvatinib is a first-line drug for treating patients with advanced hepatocellular carcinoma (HCC). However, its efficacy is severely hampered by drug resistance. Insights into the molecular mechanisms underlying lenvatinib resistance could provide new strategies to improve and prolong responses. Here, we performed unbiased proteomic screening of parental and lenvatinib-resistant HCC cells and discovered that methyltransferase-like protein-1 (METTL1) and WD repeat domain 4 protein (WDR4), the two key components of the tRNA N7-methylguanosine (m7G) methyltransferase complex, were dramatically upregulated in lenvatinib-resistant cells. METTL1 knockdown overrode resistance by impairing the proliferation capacity of HCC cells and promoting apoptosis under lenvatinib treatment. In addition, overexpression of wild-type METTL1 but not its catalytic dead mutant induced lenvatinib resistance. Animal experiments including hydrodynamic injection, subcutaneous implantation, and orthotopic xenograft mouse models further demonstrated the critical function of METTL1/WDR4-mediated m7G tRNA modification in promoting lenvatinib resistance in vivo. Mechanistically, METTL1 promoted translation of EGFR pathway genes to trigger drug resistance. This work reveals the important role of METTL1-mediated m7G tRNA modification in promoting lenvatinib resistance and provides a promising prediction marker and intervention target for resistance. Significance: Upregulation of tRNA m7G methyltransferase complex components METTL1 and WDR4 promotes lenvatinib resistance in HCC and confers a sensitivity to METTL1 targeting, providing a promising strategy to override resistance.