Detection of pseudouridine synthase activity with a fluorescence-based yeast reporter system

Pseudouridine is an isomer of uridine and a post-transcriptional RNA modification in all domains of life. Its formation is catalyzed by pseudouridine synthases functioning as standalone proteins or protein-RNA complexes called H/ACA small nucleolar ribonucleoproteins (H/ACA snoRNPs). Whereas pseudouridylation was long known to assist in stabilizing noncoding RNAs, it was discovered also in mRNAs in 2014. mRNA pseudouridylation varies in response to stress, and artificial pseudouridylation of stop codons facilitates nonsense suppression. However, the functional significance of mRNA pseudouridylation and the regulation of the pseudouridine synthases responsible are only partially understood. To study mRNA pseudouridylation by specific pseudouridine synthases, I developed a fluorescence-based reporter system in S. cerevisiae and used it to assay for standalone Pus7, snR5 and snR81 H/ACA snoRNP activity. This novel system detects snR5 H/ACA snoRNP activity and can be used to further investigate the role of pseudouridine synthases in gene expression.