Mechanisms of aberrant splicing and its implication on RNA homeostasis in CAG expansion disorders

• Applicant: Professor Dr. Andreas Neueder
• Subject Area: Molecular Biology and Physiology of Neurons and Glial Cells
• Term: from 2019 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 417976764

The dogma of protein expression – 1 gene into 1 mRNA (transcription and splicing) into 1 protein (translation) – has been challenged by the discovery of a wealth of isoforms, which are differentially spliced transcripts generated from the same gene. The family of CAG trinucleotide repeat expansion disorders consists of 9 diseases including Huntington's disease (HD). In all cases, the disease causing mutation encodes for an elongated poly-glutamine stretch in the protein. The mutated polyQ domain in turn leads to the formation of aggregates. These sequester proteins and further disturb the cellular homeostasis in a toxic gain-of-function mechanism.We have recently found that in HD, the messenger RNA of HTT, the gene that is causing the disease, is incompletely spliced. This generates a very toxic fragment of HTT, which also contains the poly-glutamine tract. To study the underlying mechanism in more detail, we have now created a novel cell model of HD that mimics this splicing block. We have first indications that the rate of transcription, which is intimately linked to splicing, influences the amount of incomplete splicing. In the current proposal we will deepen this link between transcription along the HTT gene and the amount of incomplete splicing. Furthermore, we will analyse epigenetic marks, as well as other elements that influence transcription and splicing of the HTT gene and their influence on the block of HTT splicing. In the second part, we will purify particles that contain the RNA that encodes for the toxic fragment and comprehensively analyse their composition. We will also analyse the landscape of alternative splicing events and non-coding RNA expression in HD patient samples, potentially in a longitudinal manner. For this analysis, we will use samples from which also genome-wide association (GWAS) or heteroplasmy data exists, which allows powerful correlation analyses of the observed phenotypes. Finally, in the third part, we will extend our analysis beyond HD. The analysis of RNA related pathology in the other CAG repeat expansion diseases will clarify whether splicing changes in the disease causing genes are a common feature of CAG repeat expansion diseases.Current clinical approaches to treat HD and other repeat expansion disorders use e.g. RNA lowering strategies. However, these strategies are currently not targeting the generation of the toxic fragment by incomplete splicing. A deeper understanding of how exactly this fragment is produced, as well as identifying contributing mechanisms, will help in the design of future therapeutics that specifically inhibit the generation of the toxic fragment.

• DFG Programme: Research Grants