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Human TRDMT1 is a transfer RNA (tRNA) methyltransferase for cytosine-5 methylation and has been suggested to be involved in the regulation of numerous developmental processes. However, little is known about the molecular mechanisms or their biological significance. In this study, we investigated the effects of CRISPR-based TRDMT1 knockdown on phenotypes, mRNA m5C modifications and gene expression changes in HEK293 cells. We found that knockdown of TRDMT1 significantly inhibited cell proliferation and migration but had no effect on clonogenic potential. The inhibitory effects could be attenuated by re-expression of TRDMT1 in HEK293 cells. RNA sequencing (RNA-Seq) and RNA bisulfite sequencing (RNA-BisSeq) were performed in TRDMT1 knockdown and wild-type HEK293 cells. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses indicated that the differentially expressed genes were associated with the cell cycle, RNA transport, and RNA degradation and were enriched in cancer and Notch signaling pathways. We also found that TRDMT1 knockdown could change mRNA methylation levels. For the first time, these findings clarify the role of TRDMT1 in regulating mRNA methylation and inhibiting the proliferation and migration of HEK293 cells. These results provide new insights into a new function of TRDMT1 and elucidate the molecular mechanisms of aberrant RNA m5C during tumorigenesis.
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