Reduced sensitivity to RNA structural differences distinguishes eukaryotic Pus4 from bacterial TruB

Pseudouridine is an abundant posttranscriptional modification important to RNA structure and function. The isomerization of uridine (U) to pseudouridine (Ψ) is catalyzed by members of the pseudouridine synthase (Pus) family throughout all domains of life. All Pus enzymes modify noncoding RNAs, and a subset also pseudouridylate protein-coding messenger RNAs (mRNAs). Although the precise role of Ψ in mRNAs remains to be established, emerging evidence suggests that Ψ might contribute to the posttranscriptional control of gene expression. However, the mechanisms driving mRNA target selection by individual Pus enzymes still need to be defined. The bacterial Pus enzyme TruB has been well characterized and modifies tRNA at position U55 within T-loops. In addition to catalyzing Ψ55 in most tRNAs, eukaryotic TruB orthologs also pseudouridylate mRNAs. While it has been proposed that eukaryotic TruB orthologs modify mRNA at sites that mimic their tRNA targets in sequence and secondary structure, only a fraction of such sites are pseudouridylated in cells. Here, we demonstrate that the Saccharomyces cerevisiae TruB ortholog Pus4 binds and modifies RNAs that differ in secondary structure from its established tRNA substrates in vitro. Comparison of Pus4 and TruB activities on structurally diverse substrates reveals that while both enzymes can modify a variety of substrates, TruB does so less robustly than Pus4. We also find that the bacterial-specific PUA domain modulates TruB substrate selection. These findings are consistent with reports demonstrating that other mRNA-modifying Pus enzymes are more promiscuous in vitro than in cells. Our results suggest that Pus4 substrate selection might rely on additional factors beyond protein-RNA recognition in cells.