Novel frontiers in aicardi-goutières syndrome: association between a rnu7-1 variant and histone dysfunctions

Aicardi-Goutières Syndrome (AGS) is a rare and genetically determined pediatric disorder currently associated with mutations in 9 genes. Mutations in RNU7-1 (AGS9) lead to the least characterized form of the disease. This gene encodes for the U7 small nucleolar RNA (U7 snRNA), a member of the small nuclear ribonucleoprotein complex (U7 snRNP). U7 snRNP operates the cleavage of the Poly-A tail in replication-dependent histones (RDHs) pre-mRNAs. The aim of this work is to dissect the role of RNU7-1 variations in AGS pathogenesis. We both investigated canonical AGS features such as the upregulation of IFN-α, interferon-stimulated genes (ISGs) and specific outcomes of a RNU7-1 variant in primary fibroblasts obtained from an AGS9 patient and compared to a healthy matched control. Total RNA was extracted with TRIzoLTM reagent, and the gene expression was determined by Real-Time PCR. ELISA, Western Blot analysis and immunofluorescence were performed to assess protein expression. MTT assay was used to investigate cell viability. RNA immunoprecipitation (RIP) was carried out to assess the physical association between proteins and target RNAs. Transmission Electron Microscopy (TEM) was used to identify eventual alterations in subcellular compartments. Our results highlight the upregulation of ISGs and the common increase in IFN-α production. Lastly, we assessed the enrichment in Poly-A tail of RDH transcripts suggesting the lack of functionality of AGS9 U7snRNP in the cleavage of Poly-A tail. The aberrant form of U7snRNP was also investigated via RIP, and we determined the impairment of the U7 snRNA binding to ZFP100, a component of the U7snRNP complex. Lastly, TEM analysis revealed an important decrease in chromatin content and in nucleolus size in AGS9 fibroblasts. In conclusion, this work highlights novel molecular mechanisms related to AGS9 mutation which lead to the upregulation of ISGs, IFN-α overproduction, misprocessing of RDH mRNAs, and chromatin reduction.