tRNA Modifying Enzymes, NSUN2 and METTL1, Determine Sensitivity to 5-Fluorouracil in HeLa Cells

<div><p>Nonessential tRNA modifications by methyltransferases are evolutionarily conserved and have been reported to stabilize mature tRNA molecules and prevent rapid tRNA decay (RTD). The tRNA modifying enzymes, NSUN2 and METTL1, are mammalian orthologs of yeast Trm4 and Trm8, which are required for protecting tRNA against RTD. A simultaneous overexpression of NSUN2 and METTL1 is widely observed among human cancers suggesting that targeting of both proteins provides a novel powerful strategy for cancer chemotherapy. Here, we show that combined knockdown of NSUN2 and METTL1 in HeLa cells drastically potentiate sensitivity of cells to 5-fluorouracil (5-FU) whereas heat stress of cells revealed no effects. Since NSUN2 and METTL1 are phosphorylated by Aurora-B and Akt, respectively, and their tRNA modifying activities are suppressed by phosphorylation, overexpression of constitutively dephosphorylated forms of both methyltransferases is able to suppress 5-FU sensitivity. Thus, NSUN2 and METTL1 are implicated in 5-FU sensitivity in HeLa cells. Interfering with methylation of tRNAs might provide a promising rationale to improve 5-FU chemotherapy of cancer.</p></div

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Degradation of tRNAs in HeLa cells treated with heat stress or 5-FU.

<p>(A) Northern blot analysis of tRNA(Val^AAC), tRNA(iMet), tRNA(eMet), and 5S rRNA. NSUN2 and METTL1 knockdown cells and control vector-transfected cells were treated with 37°C (control), 43°C (heat stress), or 5-FU (7.69 mM) for indicated time. Total RNA (20 µg) was loaded and blotted. (B) Degradation profile of tRNA(Val^AAC), tRNA(iMet), and tRNA(eMet) in NSUN2 and METTL1 knockdown cells (closed symbols) and control vector-transfected cells (open symbols). The cells were treated with 37°C (<i>circle</i>), 43°C (<i>triangle</i>), and 5-FU (<i>square</i>). 5S rRNA transcribed by RNA polymerase III was used as a control, and quantitative data were indicated.</p

Model for how loss of NSUN2- and METTL1-dependent tRNA(Val^AAC) modifications causes 5-FU-induced tRNA destabilization.

<p>(A) Synthetic interaction between <i>trm4</i> and <i>trm8</i> mutations in yeast. The temperature-sensitive phenotype of yeast <i>trm4</i>/<i>trm8</i> double mutant is due to rapid decay of hypomodified tRNA. A melted loop indicates destabilized tRNA. Although 5-FU-induced tRNA damage(s) that mediate tRNA destabilization remains to be fully determined, reduced pseudouridylation is the most likely cause of this. Only <i>trm8</i> mutant, but not <i>trm4</i> mutant, is sensitive to 5-FU. And so hypomodified tRNA caused by <i>trm4</i> and <i>trm8</i> mutations is probably hypersensitive to 5-FU (shown as a melted loop with question mark). (B) Cooperative effect between NSUN2 and METTL1 in HeLa cells. Hypomodified tRNA caused by double knockdown of NSUN2 and METTL1 is not heat-sensitive but is 5-FU-sensitive. A melted loop indicates destabilized tRNA.</p

Colony formation assay showing synergistic effects of NSUN2 and METTL1 double knock down and 5-FU-treatment.

<p>(A) Depiction of colonies formed after 5-FU treatment at various concentrations on Giemsa-stained dishes. (B) Dose-dependent cell survival in response to 5-FU in NSUN2 and METTL1 knockdown cells (<i>open triangle</i>), the parent cells (<i>closed circle</i>), and control vector-transfected cells (<i>open circle</i>) in colony formation assay. (C) Comparison of the average IC₅₀ values for 5-FU assessed by colony formation assay between NSUN2 and METTL1 knockdown cells and control vector-transfected cells. *Significant difference compared with control vector-transfected cells (<i>P</i><0.05).</p