There are two main mechanisms for the formation of pseudoUridine in RNA. The one only depends on the pseudoUridine synthase, which a protein plays both a recognition and a catalytic role, that is RNA-independent pseudouridylation. And the other relies on a complex formed by a class of H/ACA box small nucleolar RNA (snRNA) and a corresponding protein, in which the RNA plays a recognition role while the protein bound to RNA acts as a catalyst, that is RNA-dependent pseudouridylation. In prokaryotes only the first mechanism exists. In eukaryotes, both mechanisms are present.
PseudoUridine synthases catalyze the isomerization of Uridine to pseudoUridine in RNA. The pseudoUridine synthases are divided into six different families, including RluA, RsuA, TruA, TruB, TruD and PUS10, which are not similar in sequence. All pseudoUridine synthases share a common core fold with a core β-sheet region and along with several conserved active site amino acid residues including an invariant Asp that is essential for activity.
In addition to this conserved region, the structures of different families of pseudoUridine synthases can differ from each other. And for these enzymes, different enzymes have the specificity of their corresponding substrates. For example, TruA family proteins catalyze the conversion of Uridine to pseudoUridine at positions 38-40 in the anticodon loop of tRNA, TruB family proteins catalyze the pseudoUridine formation at position 55 in the TΨC loop of tRNA production, and TruD family proteins isomerize Uridine at position 15 in the D-stem loop of tRNA. The RsuA family proteins mainly have a highly site-specific catalytic activity in rRNA in prokaryotes, and the RluA family also catalyzes the pseudouridylation of rRNA in prokaryotes.
Of the 13 human PUS proteins, 12 are considered stand-alone, meaning they recognize their targets without the use of accessory RNAs. With high-throughput assignment of pseudoUridine sites to specific PUS proteins, it is becoming clear that various PUS proteins rely on specific sequence and/or structural features to recognize their targets .
The catalytic subunit of the small nucleolar ribonucleoproteins (snoRNPs), DKC1, is guided by base-pairing interactions between box H/ACA snoRNAs and the target RNA, which provide the specificity for the pseudouridylation of the substrate. The conserved snoRNP complex contains four proteins, DKC1(human, the yeast homologue is CBF5), GAR1, NHP2, NOP10, and the guide RNA. The box H/ACA snRNA consists of two hairpin loops that contain an internal pseudouridylation pocket where the RNA target site is converted to pseudoUridine and two conserved sequence elements, the H and ACA motifs, that recruit the protein components. There is a conserved sequence of ANANNA between two hairpin loops, called the box H. The second hairpin loop structure is immediately followed by the three nucletides ACA sequence, which are called box ACA. In the middle of each hairpin loop structure, there is a loop formed by unpaired single-stranded RNA, which is the pseudouridylation pocket. This loop is complementarily paired with the substrate RNA to determine the site-specificity pseudouridylation.