One of the most important post-transcriptional modification of RNA molecules in eukaryotic cells is pseudouridylation catalyzed by the multifunctional Dyskerin enzyme. Interestingly, despite its abundance and importance, we still know very little about the role of this modification during cell function. Previous results suggest that the pseudouridylation of ribosomal RNA will result in faulty ribosome function, that would manifest as a classic ribosomopathy. As the pathogenesis of this group of diseases is still mostly unexplored, dissecting the function of dyskerin will be an important step towards their possible treatment.

During our collaborative project we created an allelic series in zebrafish for the dkc1 gene (the fish gene encoding dyskerin), and we started the molecular and phenotypic characterization of these mutant lines. We were able to show that the absence of dkc1 function results in very peculiar developmental abnormalities, related to impaired cell differentiation.

In the in-vitro study of molecular complexes by pressure perturbation fluorescence spectroscopy we observed an unexpected increase in stability in the case of a hypomorphic dkc1 mutant and its partners (e.g. nop10) with respect to the wild type protein. This increase in stability is shown as a magnitude decrease in the dissociation constant (Kd). An accompanying decrease in the binding surface area was also observed, shown by the decrease in the fluorescence change upon dissociation, and also in the decrease of the activation volume of the dissociation reaction. These data point to the fact, that a massive reorganization must take place at the binding interface region of dkc1, which may result in a decreased sensitivity towards regulation due to the marked increase in the stability of the complex. Additionally an unfavorable structural and consequently functional distortion of the protein upon mutation is also expected.

Computational studies are performed to identify the reaction mechanism of uridine to pseudo-uridine transformation catalyzed by the dyskerin-containing box H/ACA pseudouridine-synthase complex. Models extracted from the dyskerin active site were investigated by quantum chemical calculations. Having identified some putative mechanisms, we are currently performing more precise, mixed quantum mechanics/molecular mechanics calculations. The mutations that have been created during the project may help us to identify the most plausible mechanism, as they reveal information about the effect of mutations on the interactions among the elements of the complex and on the catalytic properties.

György Ferenczy - Gusztáv Schay - Máté Varga


Report I. 2016. september

Report II. 2017. january