mBACS enhances pseudouridine profiling through efficient and robust chemical conversion

Authors

Feng, F., Pisignano, G., Kong, L., Grimes, T., Brennan, P. E., Song, C.

Abstract

Pseudouridine ({Psi}) is the most abundant RNA modification and regulates RNA stability, splicing, and translation. Utilizing the dual nucleophilic N1 and O2 of {Psi}, we previously developed 2-bromoacrylamide-assisted cyclization sequencing (BACS) for quantitative, single-base {Psi} detection. Here, we screened new dual electrophiles and developed methyl 2-bromoacrylate-assisted cyclization sequencing (mBACS), which achieves higher {Psi} conversion efficiency and lower false-positive rates, enabling more sensitive and robust {Psi} detection. The enhanced sensitivity of mBACS uncovered new {Psi} sites in human tRNAs. mBACS further revealed TRUB1 and PUS10 as the exclusive redundant writers of the conserved tRNA {Psi}55 modification and uncovered crosstalk between {Psi}55 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 {Psi} profiling from as little as 10 ng of total RNA, establishing it as a sensitive, quantitative, and low-input second-generation platform.

Preprint server: bioRxiv
The authors list and abstract were imported from bioRxiv on 11 Sep 2026.

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