Pseudouridine site assignment by high-throughput in vitro RNA pseudouridylation and sequencing

Pseudouridine (Psi) is one of the most abundant modifications in cellular RNAs. High-throughput pseudouridine profiling of eukaryotic mRNAs from cells has revealed novel sites of modification across the transcriptome. Pseudouridine affects RNA structure and RNA-protein interactions with the potential to influence many steps of mRNA metabolism and thereby affect gene expression. Identifying the mechanisms by which individual pseudouridines sites are modified by pseudouridine synthases (PUS) will facilitate studies on the molecular functions of Psi. Multiple pseudouridine synthases are expressed in all organisms and might direct pseudouridylation of diverse cellular RNAs, but the RNA targets of many enzymes and their specificity determinants remain to be defined. We developed a high-throughput in vitro pseudouridylation assay followed by sequencing that allows validation of candidate sites identified in cells, assignment of sites as direct targets of PUS and interrogation of the RNA sequence and structural features that direct modification. We also implemented an analysis pipeline to assign Psi sites from these data, including an updated approach to peak-calling that accounts for noisy signal from low-abundance transcripts.