A joint experimental and computational approach to study the functions of the RNA-binding protein PURA and its role in the neurodevelopmental disease PURA Syndrome

• Applicants: Professor Dr. Dierk Niessing; Dr. Katharina Zarnack
• Subject Area: Molecular and Cellular Neurology and Neuropathology ; Biochemistry ; Bioinformatics and Theoretical Biology ; Cell Biology
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 541627224

This research project focuses on unraveling the functions of the RNA-binding protein PURA and the consequences of its haploinsufficiency in the neurodevelopmental disorder PURA Syndrome. This monogenetic, sporadic, and rare disorder is caused by mutations in the PURA gene, resulting in symptoms like intellectual disability, neurodevelopmental delay, hypotonia, and epilepsy. At the molecular level, the protein PURA binds both DNA and RNA, yet little is known about the molecular pathways affected when PURA function is impaired. Importantly, we recently demonstrated that PURA acts as a cytoplasmic RNA-binding protein that regulates hundreds of transcripts in human cells. Here, we propose an interdisciplinary, highly interwoven research plan to unravel the molecular mechanisms underlying PURA Syndrome. For this, we combine the expertise of the Niessing group in advanced (stem) cell biology, biochemistry, and structural biology with the Zarnack group's experience in bioinformatics, including multi-omics analyses, data mining, and machine learning. We will first investigate PURA's RNA and protein interactions in neur(on)al cells. This includes assessing transcriptome and proteome changes upon PURA depletion, developing computational models to predict PURA's RNA binding, and studying PURA's role in transporting transcripts into neurites. Additionally, the project seeks to identify the protein interaction partners of PURA in neural cells. To gain a deeper understanding, biochemical and structural characterizations of PURA-protein interactions will be conducted. The goal is to understand the joint regulation of target RNAs by PURA and its protein interactors. Experiments will be performed in 2D cultures of immortalized neural stem cells that can be differentiated into different neur(on)al cell types, allowing to obtain deep data for specific cell populations. In the second part of the project, we will focus on PURA's role in neuronal differentiation in a near-physiological setting. Here, we will employ human induced pluripotent stem cells (hiPSCs) as a powerful model that allows us to cover a wide spectrum of neurodevelopmental processes from stem cells to mature neurons. Particularly, we will use a 2D differentiation approach from hiPSCs to neural progenitor cells (NPCs) as well as a 3D model with differentiated cerebral organoids. Comparative studies with PURA KO hiPSCs will enable us to identify affected differentiation pathways in NPCs as well as differences in cellular composition in cerebral organoids. Finally, we will seek to identify robust biomarkers for abnormal gene regulation and investigate changes in gene expression profiles using patient-derived hiPSCs in 2D and 3D models. In summary, using a comprehensive and multidisciplinary effort, our project aims to shed light on the molecular functions of the RNA-binding protein PURA in a near-physiological setting and assess the disease etiology of patients with PURA Syndrome.

• DFG Programme: Research Grants