mBACS enhances pseudouridine profiling through efficient and robust chemical conversion
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…
Pseudouridine ({Psi}) is a widely occurring RNA modification that influences RNA stability, splicing, and translation. Researchers previously created 2-bromoacrylamide-assisted cyclization sequencing (BACS) to quantify single-base {Psi} detection accurately. In this study, scientists discovered methyl 2-bromoacrylate-assisted cyclization sequencing (mBACS), a new method that surpasses BACS in terms of {Psi} conversion efficiency and minimizes false-positive occurrences.
This advancement enabled mBACS to detect more sensitive and robust {Psi} sites within human tRNAs. Moreover, mBACS identified TRUB1 and PUS10 as the sole redundant writers of the conserved tRNA {Psi}55 modification, unveiling a connection between {Psi}55 and other tRNA modifications. Additionally, the study revealed 5-fluorouracil as a substance capable of inhibiting pseudouridine synthesis across various spots, causing a selective remodel of pseudouridylation across the genome.
Lastly, mBACS confirmed the feasibility of high-quality {Psi} profiling even from minute samples of RNA, measuring as little as 10 ng, thereby establishing mBACS as a highly sensitive, quantitative, and low-input second-generation platform for pseudouridine analysis.
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