Fig. 3
The WDR4-R170L mutation results in decreased levels of m7G on tRNA. a Predicted location of R170 residue of WDR4, based on the yeast Trm8-Trm82 crystal structure. Representation of S. cerevisiae Trm8-Trm82 holoenzyme (PDB 2VDU), Trm82 is light gray with residue K223 in red, and Trm8 is in dark gray with residue E204 in blue. Corresponding WDR4 and METTL1 residues are in brackets. b A trm82Δ trm4Δ (CEN LEU2 P TRM82 -trm82-K223L) strain has a growth defect at high temperature. Wild type and trm82Δ trm4Δ strains with (LEU2) plasmids expressing TRM82 variants as indicated were grown overnight in S -Leu medium containing dextrose, diluted to OD600 of approximately 0.5 in H2O, and serially diluted 10-fold in H2O, and then 2 μL was spotted onto indicated media, followed by incubation for 3 days as indicated. c Nucleoside analysis of tRNAPhe purified from human LCLs derived from a PD patient homozygous for the WDR4-R170L allele. tRNAPhe isolated from LCLs derived from the WDR4-R170L proband with PD, and from control LCLs, was digested to nucleosides and analyzed by HPLC as described in Materials and Methods