GSE110247
Pseudouridines have context-dependent mutation and stop rates in high-throughput sequencing
The abundant RNA modification pseudouridine (?) has been mapped transcriptome-wide by chemically modifying pseudouridines with carbodiimide and detecting the resulting reverse transcription stops in high-throughput sequencing. However, these methods have limited sensitivity and specificity, in part due to the use of reverse transcription stops. We sought to use mutations rather than just stops in sequencing data to identify pseudouridine sites. Here, we identify reverse transcription conditions that allow read-through of carbodiimide-modified pseudouridine (CMC-?), and we show that pseudouridines in carbodiimide-treated human ribosomal RNA have context-dependent mutation and stop rates in high-throughput sequencing libraries prepared under these conditions. Furthermore, accounting for the context-dependence of mutation and stop rates can enhance the detection of pseudouridine sites. Similar approaches could contribute to the sequencing-based detection of many RNA modifications. Overall design: HEK293T rRNA was treated with CMC or mock-treated, then reverse transcribed under 3 different conditions (HIV RT; SIII, Mn; SIII, Mg) for a total of 6 samples.