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