Approximately 1-2% of the global population is affected by intellectual disability (ID), representing a serious medical, social and economic problem. The clinical symptoms and aetiology of ID are highly heterogeneous, making diagnosis and treatment of the disease difficult. X-linked ID caused by UPF3B mutations is well-suited for more detailed investigation due to its clearly defined genetic cause. In this project, we will study the function of UPF3B to better understand the development of ID.
Normal brain development depends on the precise operation of molecular programs that lead to proliferation, migration, and maturation of neuronal and glial cells. Alterations in these programs and processes can impair the development of the brain and lead to intellectual disability. A varying proportion of intellectual disability cases (15% to 50%) is caused by genetic factors, such as mutations in certain genes.
Several mutations in the gene encoding UPF3B have been identified as the cause of syndromic and non-syndromic forms of X-linked intellectual disability in more than 10 families. The identified mutations include missense, frameshift and nonsense mutations presumably leading to a loss of UPF3B function. Some UPF3B patients show additional disease states, including schizophrenia, autism and attention deficit hyperactivity disorder. Thus, the UPF3B protein is crucial for normal brain development and functions by a yet unknown mechanism.
Here, we propose to dissect the mechanism of X-linked intellectual disability caused by UPF3B mutations. To gain insights into the molecular function of UPF3B, we will mutate UPF3B in cultured cells by CRISPR/Cas9 and analyse the molecular effects using different high-throughput analyses.
We have generated UPF3B K.O. HEK 293 and HeLa cells using CRISPR/Cas9 to study the cellular function of UPF3B. Preliminary analyses showed that these UPF3B K.O. cells did not differ phenotypically from their parental cells. In order to detect possible changes in gene expression, we analysed one of the HEK293 UPF3B K.O. cell lines using RNA-Seq.
To our surprise, we hardly detected any changes in gene expression in the UPF3B cells. A possible explanation for this observation is that some genes regulated by UPF3B are not expressed in HEK293 cells, or that there are very few mRNAs that are regulated only by UPF3B. We will address these two possibilities in the proposed work program by using neural-like cells for the UPF3B K.O. and by extending the analysis to additional RNA-related high-throughput assays.
Institute for Genetics
Principal Investigator - C 07
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Institute for Genetics
University of Cologne Institute for Genetics Center of Molecular Biosciences Zülpcher Str. 47a
50674 Cologne
Characterization of UPF3B knockout cells
(A) Schematic representation of genomic changes in UPF3B K.O. cells. Exons are drawn as boxes, introns as connecting lines.
(B) Western blot analysis of UPF3B protein expression in wildtype (lane 1) and K.O. (lane 2-4) cells in comparison with UPF3B knockdown (lane 6). Tubulin es shown as loading control.
UPF3B mutations
Schematic domain architecture of the UPF3B protein. Domains interacting with UPF2, eRF3a and the EJC are depicted. Positions of disease-linked missense and nonsense/frameshift mutations are shown above and below the scheme, respectively.
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Prof. Dr. med. Thomas Benzing
Chair of the CMMC
