A protein puzzle – research team in Ulm delivers important insights into the rare PURA syndrome

Ulm University

Local biochemist and structural biologist Professor Dierk Niessing is trying to find out what occurs in the cells of people who suffer from PURA syndrome, a genetic neurodevelopmental disorder. With a study conducted in cooperation with Frankfurt University and the Helmholtz Centre Munich, he has now come a significant step closer to solving the puzzle. The study showed that not only the immune response and the “trash disposal” of the cells were affected, but also the gene regulation at the RNA level. Niessing’s findings on molecular and cellular networks may be able to provide a basis for future therapy studies for this illness, which has been incurable up to now. These findings have now been published in the journal Nucleic Acids Research.

PURA syndrome is a rare neuronal disease with severe developmental disorders from birth. In addition to learning disabilities and delays in mental development, afflicted children may also suffer from seizures and reduced muscle tension as well as problems with eating and breathing. Dierk Niessing, head of the Institute of Pharmaceutical Biotechnology at Ulm University, has been conducting research on the mechanisms of this disease for years. To date, only a little over 20 patients have been genetically identified in Germany. Niessing’s lab, which combines structural and cell biology with biophysics and biochemistry, is Germany’s leading scientific institution with regard to PURA syndrome.

At the cellular level, this disease is caused by a mutation of the PURA gene, which is found in the cell nucleus, along with the other genes, as a set of building instructions for the body. In a complicated molecular translation, in which ribonucleic acid (RNA) plays an important role, these building instructions are used to produce corresponding proteins. If the characteristic sequence of the gene building blocks is changed in the case of a mutation, the building instructions for the protein also change in terms of structure and order. Since the cell either no longer produces the faulty PURA protein or else recognises and destroys it, reduced PURA protein levels are associated with the syndrome. “Although we already had a fairly clear understanding of the structural and molecular characteristics of the PURA protein,” relates Niessing, “we were still lacking an understanding of which cell functions were controlled by PURA”. The aim of the study thus became to identify cell functions and molecular networks that might be dysregulated in patients with PURA syndrome, in a kind of highly complicated puzzle. To do this, Niessing worked closely with Dr Kathi Zarnack from the Goethe University in Frankfurt.

Which genes are dysregulated in cases of PURA syndrome?

NIessing’s team conducted large-scale experiments in the laboratory. They used several so-called high-throughput methods to understand which genes were controlled by PURA and possibly dysregulated in patients with PURA syndrome. First of all, high-throughput sequencing was used to check how often all sets of building instructions in the cell were copied into so-called messenger RNAs for producing proteins. Then, mass spectrometry was used in order to identify the type and number of nearly all proteins produced in the cell. Since the translation of some messenger RNAs into proteins is regulated by PURA, an iCLIP technique was also used to check which of these many messenger RNAs are bound by PURA. For this purpose, the team irradiated cells with UV light, which causes proteins and messenger RNAS bound together to form a very stable bond. The most important results were then confirmed using various biochemical methods and fluorescent microscopy.

The enormous volume of data collected in these experiments was analysed and interpreted by the Computational RNA Biology Group at the Goethe University in Frankfurt. “In digital multi-omics analyses, our group was able to combine the results of the various experiments and molecular classes”, says the head, Dr Kathi Zarnack. “Only with such extensive computer analysis can we make sense of the data in its abundance and begin to understand the cellular tasks of the PURA protein”.

The PURA protein is involved in many tasks within the cells

In the scientific journal Nucleic Acids Research, the scientists have now been able to show that PURA proteins are mainly found in the cytoplasm of the cells, ie the part that surrounds the nucleus with the genetic material. The research results also indicate that the PURA protein plays a role in important cell mechanisms. These include the immune response of the cells, the function of the mitochondria, ie the power plant of the cells, and autophagy, or cellular recycling. In particular, the activity and networks of the “processing bodies”, which regulate RNA, appear to be strongly and diversely affected by reduced PURA levels. “Our hope is that by solving the molecular puzzle, our two labs have made the first step toward future therapeutic approaches,” says Niessing. There is, however, still a long way to go before effective therapies are available. Further studies will need to clarify which of these abnormalities actually cause patients to have severe symptoms.

Niessing also points out that there are similarities to other neurodevelopmental disorders, such as Angelman, Pitt-Hopkins or Rett syndrome. “Although their molecular causes differ from that of PURA syndrome, there could very well be similarities relating to the similar symptoms”. Furthermore, there have been increasing reports over the last few years of defects in other RNA-binding proteins, which also result in neurodevelopmental disorders. “Studies on PURA syndrome will most likely be quite relevant for these new illnesses as well”.

In addition to investigating PURA syndrome, Niessing’s and Zarnack’s teams support patient families all over the world through lectures and counselling. They are also helping families in Germany to set up a national patient association. Niessing is setting up a “biobank” with his partner lab at the Helmholtz Centre Munich as well. Here, affected families can provide biological samples for further research on the disease.

Molitor, L.*, Klostermann, M.*, Bacher, S., Merl-Pham, J., Spranger, N.,  Burczyk, S., Ketteler, C., Rusha, E., Tews, D., Pertek, A., Proske, M., Busch, A., Reschke, S., Feederle, R.,  Hauck, S., Blum, H., Drukker, M., Fischer-Posovszky, P., König, J., Zarnack, K.#, Niessing, D. # (2022). Depletion of the RNA-binding protein PURA triggers changes in posttranscriptional gene regulation and loss of P-bodies. Nucleic Acids Research.

Text: Anja Burkel
Mediacontact: Press and Public Relations, Ulm University
Translation: Kate Gaugler

Green laser light is also used for the fluorescent microscopic analysis of the PURA protein
Green laser light is also used for the fluorescent microscopic analysis of the PURA protein (Photo: AG Niessing / Uni Ulm)
Fluorescent microscope with red laser light to analyse the PURA protein in human cells (Photo: AG Niessing / Uni Ulm)
Prof Dierk Niessing at Ulm University and researcher Dr Kathi Zarnack from Frankfurt (Photos: private)