The isolated HEPN domain of SACSIN exhibits RNA-binding activity

Abstract Autosomal Recessive Spastic Ataxia of Charlevoix–Saguenay (ARSACS) is a neurodegenerative disorder caused by mutations in the SACS gene, though the molecular function of its protein product, SACSIN, remains elusive. Therapeutic strategies for ARSACS are limited, mostly due to the exceptionally large size of SACSIN (~ 520 kDa), which precludes conventional gene therapy and standard molecular delivery methods. Over 200 pathogenic variants, including missense, nonsense and frameshift mutations, have been identified. Regardless of variant type, patients uniformly exhibit cerebellar ataxia, spasticity and peripheral neuropathy. Among them, patients with homozygous frameshift or missense mutations within the SACSIN higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domain, suggesting that the alteration of the functional role of the SACSIN HEPN domain could be linked to the manifestation of ARSACS symptoms. Here, we show that the SACSIN HEPN domain can bind RNA in overexpression, corroborating the hypothesis that SACSIN functions as an RNA-binding protein. The interaction of the SACSIN HEPN domain with the RNA regulates the cellular compartmentalization of this domain as well as its propensity to form protein condensates. Conversely, an ARSACS mutated version of the SACSIN HEPN domain (F4574C) showed an impaired RNA-binding activity, a nuclear mis-localization and the predisposition to form protein condensates. Overall, our study unveils an unknown molecular function of the SACSIN HEPN domain that, if formally confirmed also for the full-length SACSIN, can pave the way for innovative therapeutic approaches for ARSACS patients. Graphical abstract Created in BioRender. Vazzana, R. (2025) https://BioRender.com/rjn557o .