The SONATA15 grant project “Molecular and structural insights into human pseudouridine synthase 3 (hPus3)” was initiated in 2021, with dr Ting-Yu Lin as the project leader and Rahul Mehta and Jakub Jeżowski joining the team as a doctoral student and a master’s student, respectively. This team of outstanding researchers works in close collaboration with prof. Sebastian Glatt, with the objective of characterizing biochemically and structurally a poorly understood but essential RNA-modifying enzyme, hPus3, which is implicated in phenotypes of intellectual disorders.
They describe their project:
Our first goal was to produce the recombinant human Pus3 protein for structural elucidation. With extensive efforts, we managed to obtain the protein in sufficient amounts to perform further biochemical and biophysical analyses, but not for extensive crystallization trials. Therefore, we’ve chosen cryoEM analysis as the way to go for obtaining atomic-resolution protein structures.
Secondly, we set up a functional assay to investigate hPus3 activity on tRNA pseudouridylation. We’ve made several structure-guided mutants based on the bacterial homolog protein crystal structures, which allows further in-depth analyses on catalytic reaction mechanisms.
Contemporaneously, we are in collaboration with clinicians who have provided us with patient-derived samples from patients carrying Pus3 mutations. Using a mutagenesis approach, we have then generated clinically relevant mutations for biochemical characterizations. Altogether, we believe that our work will have a fundamental impact in translational research.
Nowadays, based on our results, we are able to present an overall picture of hPus3, that its bacterial counterpart (TruA) perform the same reaction as the human Pus3, how it affects cell function, and what are the impacts of the clinical variants on enzyme activity, stability and tRNA binding.
Additionally, our focus resides not only on wet-lab work, but also in supporting our teammates in showcasing their effort in our group and the findings derived from it. Recently, Rahul has presented our work at an international workshop and Jakub at a national student conference.
P.S. The talents of the Glatt Lab’s people are also expressed through art!
Edited by: Dr A. Flores-Ibarra
