Human Trm10 orthologs function differently in vivo in S. cerevisiae. (A) Complementation assay testing ability of human TRMT10 homologs to complement the S. cerevisiae trm10Δ 5-FU hypersensitivity phenotype. The S. cerevisiae trm10Δ strain was transformed with LEU2 plasmids expressing various Trm10 homologs under control of a galactose-inducible promoter (PGAL-TRM10X in schematic). Selected transformants containing either ScTrm10, hTRMT10A, hTRMT10B, or empty vector control plasmid, as indicated, were plated by fivefold serial dilutions onto S-Gal-leu media containing increasing concentrations of 5-FU (0, 0.1, or 1 µg/mL). Images are of plates grown at the indicated temperatures for 2–3 d. (B) Western blot to test for expression of the indicated enzymes in S. cerevisiae trm10Δ strains. Antibodies targeted amino-terminal HA epitopes incorporated into each construct; detected bands matched expected sizes for the indicated enzymes as follows: ScTrm10 (36 kDa), hTRMT10A (41 kDa), hTRMT10B (37 kDa). Tubulin loading control is shown on the lower blot.
