Promoted read-through and mutation against pseudouridine-CMC by an evolved reverse transcriptase.

ABSTRACT: Pseudouridine (Ψ) is an abundant RNA chemical modification that plays critical biological functions. Current Ψ detection methods are limited in identifying Ψs at base-resolution in U-rich sequence contexts, where Ψ occurs frequently. Here we report "Mut-Ψ-seq" that utilizes the classic N-cyclohexyl N'-(2-morpholinoethyl)carbodiimide (CMC) agent and an evolved reverse transcriptase ("RT-1306") for Ψ mapping at base-resolution. CMC selectively labels Ψs in RNA forming the CMC-Ψ adduct and we show that RT-1306 presents promoted read-through and mutation against the CMC-Ψ. We report a high-confidence list of Ψ sites in polyA-enriched RNAs from HEK-293T cells identified by orthogonal chemical treatments (CMC and bisulfite). The mutation signatures resolve the position of Ψ in UU-containing sequ

SUBMITTER: He Z 

PROVIDER: S-EPMC11724883 | biostudies-literature | 2025 Jan

REPOSITORIES: biostudies-literature