Quantitative analysis of small RNA pseudouridylation reveals interplay of PUS enzymes in tRNA anticodon stem-loop

Pseudouridine (Ψ) is an abundant modification in small RNA catalyzed by multiple pseudouridine synthases (PUSs). However, the substrate specificity of human PUSs remains elusive. Here, we adopted PRAISE, a quantitative Ψ detection method, to profile pseudouridylation in small RNA, including cytosolic and mitochondrial tRNAs, snRNA, and snoRNA. We found that snoRNA pseudouridylation is mediated not only by RNA-guided DKC1, but also by the stand-alone enzyme PUS7 at a specific site. Interestingly, several PUS enzymes, including PUS1, RPUSD1, and PUS7, which install nearby Ψ sites within tRNA anticodon stem-loop, can influence pseudouridylation catalyzed by other PUSs, revealing an unrecognized interplay during Ψ formation. For the three RluA family enzymes, RPUSD1 catalyzes the canonical Ψ30 in tRNA-Ile and Ψ72 in tRNA-Arg isoacceptors. RPUSD2 pseudouridylates Ψ31 of mt-tRNALeu(CUN), Ψ32 of mt-tRNAPro and mt-tRNACys, whereas RPUSD3 lacks tRNA activity. Together, our quantitative Ψ profiling characterized PUS tRNA substrates and revealed unexpected PUS interplay. Pseudouridine (Ψ) is an abundant modification in human small RNA but the substrate specificity of its synthesis enzymes (PUSs) remains elusive. Here, the authors quantitatively profile Ψ map in small RNA, identify the substrates of PUSs and reveal unexpected PUSs interplay in tRNA pseudouridylation.