Getting close to RNF216 : proximity labeling reveals links between nuclear condensates, splicing and neurodegeneration

Background-Aims Mutations in RNF216, an E3-ubiquitin ligase, have been identified as the genetic cause of a rare recessive neurodegenerative disease. Various loss-of-function (LOF) mutations inside and outside the catalytic domain of RNF216 have been described. However, how mutations outside the catalytic domain result in LOF and how mutations in RNF216 give rise to neurodegeneration, remains unclear. We hypothesize that the heterogenous group of mutations result in a common distorted pathway resulting in the formation of toxic aggregates and hence neurodegeneration. Identifying these pathways is our main objective. Methods Like most neurodegenerative diseases, RNF216-mediated neurodegeneration is associated with the abnormal protein deposits in the brain of patients. However, the composition and formation of these aggregates is unknown and is therefore one of our primary goals to characterize. To achieve this, we will apply a multi-omics approach to unravel the function and disease pathways of RNF216. First, we performed proximity-dependent biotinylation assay to identify the interactome of RNF216. Second, RNF216 knock-down (AS0) cells were generated for proteomic and transcriptomic analysis. Last, we carried out single-cell short and long read sequencing on patient fibroblasts. The pathways identified in these experiments will initially be validated in HeLa cells and fibroblasts. To place these finding in a neurological context, we will make iPSC-derived cortical neurons out of patient fibroblasts. Results We made a specific and reliable monoclonal antibody against RNF216 as commercially available antibodies seems to be aspecific. This antibody is validated using optimized RNF216-targeting ASO’s. This antibody and ASO’s will be a valuable tool for further experiments. Gene ontology analysis of the interactome of RNF216 point towards a role in RNA splicing. Transcriptomics on patient fibroblasts and RNF216 KD cells will be used to further confirm this finding. Moreover, the interactome of RNF216 is enriched for SUMO2 substrates, which is particularly interesting given that the p62/Ub positive aggregates observed in patient brain are also positive for SUMO2/3. Conclusion The physiological and pathological mechanisms explored in this project hold the potential to greatly improve our understanding of RNF216-mediated neurodegeneration, shed light on novel key players in neurodegeneration, and open new avenues for a novel therapeutic target for this rare neurodegenerative disease.