Figure 5.
tRNAIleUAU receives intron-dependent pseudouridylation in vivo, and the pseudouridylation prevents the tRNA from aberrant ncm5U modification. (A) Intron-dependency of Ψ34 and Ψ36 formation on tRNAIleUAU by Pus1p was investigated by MS analyses. As described in the ‘Materials and Methods’ section, tRNAIleUAU purified from the wild-type (WT), intronless (Δintron) and PUS1-deletion (pus1Δ) strains was subjected to LC-MS analysis with or without cyanoethylation. The panels in the top row represent the LC elution patterns by base peak chromatograms, and the panels in the other three rows represent the MS signals of extracted ion chromatograms (XICs) corresponding to the non-, mono- and di-cyanoethylated RNase T1 fragments (C32-G40) of tRNAIleUAU, respectively. Information about the corresponding MS peak analyzed in each row is indicated at the top of each panel on the left. Cyanoethylated derivatives were not detected in the MS samples without acrylonitrile treatment (data not shown). (B) A typical mass spectrum of the C32–G40 fragment prepared from the tRNAIleUAU intronless strain. The m/z values of the expected mass peaks are shown in gray, and those of the unknown peaks are in black. (C) XICs of divalent negative ions at m/z 1510.7 and m/z 1539.2 (middle and bottom row, respectively) of tRNAIleUAU fragments from the wild-type (WT), intronless (Δintron) and PUS1-deletion (pus1Δ) strains. (D) Collision-induced dissociation (CID) analyses of the RNase T1 fragments with molecular masses of 3023.4 Da (upper panel) and 3080.4 Da (lower panel) derived from the tRNAIleUAU in the intronless yeast. Product ions for c- and y-series are assigned in the CID spectra and fragment sequences. (E) Identification of aberrant U + 57 Da by nucleoside analysis. The top row represents a trace of UV absorbance at 254 nm, and the second row represents XIC at m/z 300.083 corresponding to a deprotonated anion of ncm5U from co-injection of tRNAIleUAUΔint and yeast total RNA. The third and fourth rows represent XICs at the same m/z from tRNAIleUAU purified from the intronless strain and yeast total RNA alone, respectively. (F) Investigation of misdecoding of an AUG codon to Ile using a c-Myc epitope. EGFP fused with the wild-type (Wt) or an AUA-to-AUG mutant form (mut) of the c-Myc tag was expressed in the wild-type (WT) or the tRNAIleUAU intronless (Δintron) cells. The mutant c-Myc contained an AUG codon instead of the AUA codon for the 5th residue of Ile in the c-Myc epitope (possible decoding patterns of the two reporters are shown at the top). The same blot was decorated with both the anti-c-Myc 9E10 mouse monoclonal antibody (anti-c-Myc) and the anti-GFP rabbit antibodies (anti-GFP). Srp1p on the same blot was also detected as a loading control (anti-Srp1p). ‘Vec’ is a negative control without expressing any EGFP fusion. Two independent clones (‘1’ and ‘2’) of each strain were subjected to western blotting.