Abstract
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.
Highlights
Box H/ACA pseudouridine synthases are protein−RNA complexes that are responsible for the isomerization of uridine to pseudouridine in specific locations of a wide range of substrate RNAs
We found that the structural ensemble generated for the model of the human box H/ACA ribonucleo-protein complex (RNP) retains the topology of the Pyrococcus furiosus RNP64 with the best fit achieved in the central, catalytic region (Figure S1)
80% of the approximately 16 hydrogen bond (H-bond) formed between the substrate and dyskerin are formed between the sugar− phosphate backbone and the protein; the only nucleobase that forms base-specific association with the protein matrix is the uridine to be isomerized. These findings suggest that guide small nucleolar guide RNA (snoRNA), which will be able to align to and activate the box H/ACA pseudouridine synthases (PSUs) enzyme maintaining the basic topology seen here, can be varied rather freely in the pseudouridylation pocket region, indicating that pseudouridylation can be induced in a great variety of RNA substrates
Summary
The isomerization of uridine to pseudouridine (Ψ) is the most abundant post-transcriptional modification of RNA,[1,2] so much so that Ψ is often referred to as the “fifth nucleotide”.3. Box H/ACA PSUs, located in the nucleolus and nucleoplasmic Cajal bodies,[14−17] take part in such fundamental functions such as telomere length maintenance and ribosome biogenesis. Mutations appearing in their protein components are linked to serious illnesses like bone marrow failure, cancer, or nephrotic syndrome.[18−21]
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