Ribonucleome analysis identified enzyme genes responsible for wybutosine synthesis.

Wybutosine (yW) is a hyper modified guanosine with a tricyclic base found at the 3'-position adjacent to the anticodon of eukaryotic phenylalanine tRNA. yW supports reading frame maintenance by stabilizing codon-anticodon interactions during decoding on the ribosome. To identify genes responsible for yW synthesis from uncharacterized genes of Saccharomyces cerevisiae, we employed a systematic reverse genetic approach combined with mass spectrometry ('ribonucleome analysis'). We found four new genes YPL207w, YML005w, YOL141w and YGL050w (named TYW1, TYW2, TYW3 and TYW4, respectively) to be responsible for wybutosine (yW) synthesis. Mass spectrometric analysis of each modification intermediate of yW revealed its sequential biosynthetic pathway. Multi-step enzymatic formation of yW was partially reconstituted in vitro using recombinant proteins. The results suggested that yW synthesis might proceed through sequential reactions in a complex formed by multiple protein components assembled with the precursor tRNA.