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

ABSTRACT: Pseudouridine (Ψ) is an abundant modification in small RNA and is catalyzed by multiple pseudouridine synthases (PUSs). However, the substrate specificity of human PUSs are not fully understood. In this study, 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 pseudouridylation of snoRNAs can be mediated not only by RNA-guided DKC1, but also stand-alone enzyme PUS7. Interestingly, we found that several PUS enzymes, which install nearby Ψ sites within tRNA anticodon stem-loop, can influence pseudouridylation level catalyzed by other PUSs. Typical examples include PUS1, RPUSD1, and PUS7, revealing a novel interplay among human PUSs during Ψ formation. For the three RluA family enzymes, we found that in addition to the canonical substrate Ψ30, RPUSD1 also catalyzes Ψ72 of tRNA-Arg isoacceptors. RPUSD2 pseudouridylates Ψ31 of mt-tRNALeu(CUN), and Ψ32 of mt-tRNAPro and mt-tRNACys, and hence is functionally similar to yeast Pus9. RPUSD3 does not modify tRNA at all, consistent with its lack of a catalytic residue. Together, using quantitative Ψ profiling, our study characterized the tRNA substrates of PUS enzymes and revealed unexpected interplay among human PUSs in tRNA pseudouridylation.

ORGANISM(S): Homo sapiens

PROVIDER: GSE299274 | GEO | 2025/11/27

REPOSITORIES: GEO

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