The uridine to pseudouridine transformation, one of the most abundant and essential
post-transcriptional modification of RNAs, is carried out by pseudouridine synthases
(PSUs). Aside from a few very specific targets, pseudouridylation is performed by
a ribonucleo-protein complex, box H/ACA PSU, containing four different proteins and
a guide RNA. Mutations of PSUs cause serious diseases including dyskeratosis congenita
(DC), various types of cancers, and nephrotic syndrome. Here, we combined homology
modeling, classical force-field-based molecular dynamics, and quantum mechanics/molecular
mechanics-based enhanced sampling free energy simulations to show that reactant destabilization
through the severe distortion of the target uridine in the active site of box H/ACA
PSU is a key factor in the catalysis of pseudouridylation. We propose a dissociation-rebound
mechanism where the uracil detaches from the ribose by the cleavage of the C1′−N1
bond leading to a charge separated intermediate. The base rebounds to the ribose with
its C5 carbon with a very small barrier. The subsequent tautomerization step is proposed
to be coupled to the tilting of the upper dyskerin region, comprising the thumb loop,
and product release. The proposed mechanism does not impose sequence restriction on
the substrate; it only requires a complementary guide RNA coordinated to the protein
components of the enzyme complex. We also found that the interactions of the guide
RNA with the proteins of the complex in the vicinity of the active site are overwhelmingly
formed by the sugar−phosphate backbone, indicating that designed guide RNAs could
be applied to carry out pseudouridylation of substrates with a great variety of different
sequence motifs. Therefore, the endogenous box H/ACA PSU system may be used to target
premature stop codons, for example, to induce their read through serving as a vehicle
for RNA editing and therapeutics for gene lesion-related diseases.
