Pseudouridine, or Ψ, the overall most abundant post-translational RNA modification, is created when a uridine base is isomerised. In eukaryotes, this can occur by either of two distinct mechanisms; it is sometimes referred to as the ‘fifth RNA nucleotide’. It is incorporated into stable non-coding RNAs such as tRNA, rRNA, and snRNA, with roles in ribosomal ligand binding and translational fidelity in tRNA, and in fine-tuning branching events and splicing events in snRNAs. Pseudouridine has one more hydrogen bond donor from an imino group and a more stable C–C bond, since a C-glycosidic linkage has replaced the N-glycosidic linkage found in its counterpart (regular uridine). As neither of these changes affect its base-pairing properties, both will have the same output when directly sequenced; therefore methods for its detection involve prior biochemical modification.