Quantitative analysis of small RNA pseudouridylation reveals interplay of PUS enzymes in tRNA anticodon stem-loop
Pseudouridine (?) 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 ? 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 ? 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 ? formation. For the three RluA family enzymes, we found that in addition to the canonical substrate ?30, RPUSD1 also catalyzes ?72 of tRNA-Arg isoacceptors. RPUSD2 pseudouridylates ?31 of mt-tRNALeu(CUN), and ?32 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 ? profiling, our study characterized the tRNA substrates of PUS enzymes and revealed unexpected interplay among human PUSs in tRNA pseudouridylation. Overall design: We detected the ? sites in small RNAs of HEK293T cells to profile the quantitative landscape. We then identified the DKC1-dependent sites of snRNA and snoRNA in DKC1 knockdown cell lines. After that, we identified substrates of 6 PUS enzymes in PUS1, PUS7, TRUB1, RPUSD1, RPUSD2, and RPUSD3 cell lines.