A comprehensive tRNA pseudouridine map dependent on human stand-alone pseudouridine synthases

Pseudouridine (Ψ) is one of the most abundant RNA modifications in human cells, which is introduced post-transcriptionally by pseudouridine synthases (PUS). Despite its prevalence, the biological functions of Ψ remain poorly understood, largely due to the limited knowledge linking specific PUS enzymes to their targets. To address this gap, we systematically knocked out or knocked down 9 stand-alone PUS in HCT116 cells and mapped their Ψ profiles using 2-bromoacrylamide-assisted cyclization sequencing (BACS). Through this approach, we uncovered previously unknown targets of several PUS enzymes, including RPUSD1, RPUSD2, PUS3, PUSL1, and PUS7L. Additionally, we revealed that TRUB1 and PUS10 function redundantly to catalyze the highly conserved Ψ55 modification in cytosolic tRNAs. Intriguingly, we found that RPUSD3 and TRUB2 do not exhibit noticeable enzymatic activities in human cells. By integrating these findings with earlier results for TRUB1, PUS7, and PUS1, we constructed a comprehensive map of stand-alone PUS-dependent Ψ modifications across human tRNAs. Using this map, we further demonstrated that different PUS enzymes introduce Ψ modifications at distinct stages of pre-tRNA processing.