Promoted Read-through and Mutation Against Pseudouridine-CMC by an Evolved Reverse Transcriptase

Pseudouridine ({Psi}) is an abundant RNA chemical modification that can play critical roles in the biological functions of RNA, and RNA-therapeutic applications. Current {Psi} detection methods are limited in identifying {Psi}s at base-resolution in U-rich sequence contexts, where {Psi} occurs frequently. The N-cyclohexyl N-(2-morpholinoethyl)carbodiimide (CMC) can selectively label {Psi} in RNA by forming the CMC-{Psi} adduct. Here we report that an evolved reverse transcriptase ("RT-1306") shows promoted read-through and mutation against the CMC-{Psi}. The mutation signature can resolve the occurrence of {Psi}s within UU-containing sequences. We developed "Mut-{Psi}-seq" utilizing CMC and RT-1306 for transcriptome-wide mapping of {Psi} at base-resolution. The mutation signatures robustly identify reported {Psi}s in human rRNAs via the ROC analysis, and elongated CMC reaction duration increases the detection sensitivity of {Psi}. We report a high-confidence list of {Psi} sites in polyA-enriched RNAs from HEK-293T cells identified by orthogonal chemical treatments (CMC and bisulfite). The mutation signatures resolve the position of {Psi} in UU-containing sequences, revealing diverse occurrence of {Psi}s in such sequences. This work provides new methods and datasets for biological research of {Psi}, and demonstrates the potential of combining the reverse transcriptase engineering and selective chemical labeling to expand the toolkit for RNA chemical modifications studies.