The Structure-Derived Mechanism of Box H/ACA Pseudouridine Synthase Offers a Plausible Paradigm for Programmable RNA Editing

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 C-1'-N-1 bond leading to a charge separated intermediate. The base rebounds to the ribose with its C-5 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.

Automated summary

The uridine to pseudouridine transformation is an important post-transcriptional modification of RNAs, and understanding the catalysis mechanism is crucial for potential therapeutic applications. This study reveals that destabilization of the target uridine in the active site is a key factor in the catalysis of pseudouridylation. The proposed dissociation-rebound mechanism and the use of designed guide RNAs for pseudouridylation have potential implications in RNA editing and gene lesion-related diseases.