mBACS enhances pseudouridine profiling through efficient and robust chemical conversion

Pseudouridine (Ψ) is the most abundant RNA modification and regulates RNA stability, splicing, and translation. Utilizing the dual nucleophilic N ¹ and O ² of Ψ, we previously developed 2-bromoacrylamide- assisted cyclization sequencing (BACS) for quantitative, single-base Ψ detection. Here, we screened new dual electrophiles and developed methyl 2-bromoacrylate-assisted cyclization sequencing (mBACS), which achieves higher Ψ conversion efficiency and lower false-positive rates, enabling more sensitive and robust Ψ detection. The enhanced sensitivity of mBACS uncovered new Ψ sites in human tRNAs. mBACS further revealed TRUB1 and PUS10 as the exclusive redundant writers of the conserved tRNA Ψ55 modification and uncovered crosstalk between Ψ55 and other tRNA modifications. It also revealed 5-fluorouracil as a pan-pseudouridine synthase inhibitor that induces widespread but site-specific pseudouridylation remodelling. Finally, mBACS supported robust Ψ profiling from as little as 10 ng of total RNA, establishing it as a sensitive, quantitative, and low-input second-generation platform.