NCN SONATA 15 2020 – 2023

NCN SONATA 15 2020 – 2023

Molecular and structural insights into human pseudouridine synthase 3

Glatt-lab

What is the project about?

Since 2020, we have started the SONATA15 grant with title Molecular and structural insights into human pseudouridine synthase 3.

Our overall objective is to biochemically and structurally characterize the human pseudouridine synthase 3 (hPus3), a poorly understood but very important RNA-modifying enzyme implicated in intellectual disorders. We believe that the results of the work will provide fundamental insights into important biological processes and show a great potential for translation into clinical research.

We have Ting-Yu Lin, PhD as the project leader and two outstanding students, including the PhD student Rahul Mehta as well as a master student Jakub Jezowski, to work on this project. In addition, the team works closely with Sebastian Glatt, PhD

How will we get to the results?

The first goal in the project is to produce recombinant human PUS3 protein.

With extensive efforts, we managed to obtain sufficient amount of protein for subsequent biochemical and biophysical analyses. As limited protein is a big challenge for protein crystallization trials, a cryoEM analysis will be applied to obtain the protein structure at atomic resolution.

Second, we setup a functional assay to investigate PUS3 activity on tRNA pseudouridylation. We have made several structural-guided mutants based on the bacterial homolog protein crystal structures. This further allows in-depth analyses on catalytic reaction mechanisms.

Third, we have a collaboration who have provided us the clinical samples that were derived from patients carrying PUS3 mutations. We also applied mutagenesis approach to generate clinical-relevant mutations for biochemical characterizations. Based on these results, we could present an overall picture of whether PUS3 conducts the same reaction as its bacterial counterparts and what the impacts of the clinical variants on enzyme activity and how they affect cells.

Not only we focus on the study but also we fully support our teammates to showcase their efforts and findings. Recently, Rahul and Jakub have presented their work in an international workshop and a national student conference, respectively.

In addition, we are very grateful that a talent colleague in Glatt’s team has created a drawing to promote our project.

Team Members

Ting-Yu Lin, PhD (Project Leader)

Rahul Mehta (PhD student)

Jakub Jeżowski (Master student)

Budget

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