New insights in Aicardi-Goutières Syndrome: association between a RNU7-1 variant and novel pathologic mechanisms

Aicardi-Goutières syndrome (AGS) is a rare genetic pediatric disorder currently associated with mu-tations in 9 genes (TREX1, RNASEH2A, RNASEH2B, RNASEH2C, SAMHD1, ADAR, IFIH1, LSM11 and RNU7-1), all of which encode for proteins or transcripts involved in nucleic acid metabolism and signaling. In this thesis work, a pathologic variant of the RNU7-1 gene (AGS9) was analyzed. This gene encodes for the U7 small nucleolar RNA (U7 snRNA), an essential compo-nent of the U7 snRNP complex. The function of U7 snRNP is to operate the endonucleolytic cleavage of the poly-A tail in replication-dependent histones (RDHs) pre-mRNAs during their maturation process. RDHs genes encode the four core histones and the linker histone family. In AGS, mutations in RNU7-1 have been linked to defects in U7 complex synthesis, resulting in the production of aberrant RDHs isoforms that possess a poly-A tail. In this condition, chromatin is recognized as a foreign genome and acti-vates the innate immune cGAS-STING cascade, leading to increased type I IFN production and induction of transcription of interferon-stimulated genes (ISGs). As RNU7-1 (AGS9) is the least characterized AGS-related gene, the aim of this pro-ject is to dissect its role in AGS pathogenesis. To this end, this work both investigated canonical AGS features such as the potential upregulation of IFNα and ISGs, and spe-cific outcomes of a RNU7-1 pathologic variant in primary dermal fibroblasts obtained from an AGS9 patient and compared to a healthy age- and sex-matched control. Results of this thesis work pointed out an altered RNA morphology of U7 snRNA that leads to affects its subcellular localization in AGS9-derived fibroblasts. Moreover, al-tered expression of two AGS-related genes, (both the isoforms of ADAR1 and RNASEH2B) in fibroblasts carrying the pathologic variant of RNU7-1 was highlighted, confirming the strong cooperation between AGS genes in nucleic acid metabolism. The activation of the interferon signature (IS) in response to the mutation was ob-served, with upregulated ISGs and increased IFN score in fibroblasts and whole blood, reinforcing the connection between the RNU7-1 variant and interferon dysregulation. An anti-inflammatory compound, hydroxychloroquine, was then tested to determine its potential effectiveness in reducing IS and IFNα production. Results proven the de-crease of both ISGs and IFNα in AGS9 cells with also a partial restore of cell viability in AGS9 fibroblasts. As aberrant morphology of U7 snRNA could affect its functioning in U7 snRNP, RIP analysis was performed and a decrease in physical association between U7 snRNA and ZFP100 was reported. RNA-sequencing analysis then confirmed the alteration of multiple pathways related to neuron function and interferon signaling in AGS9 fibroblasts. Moreover, transcrip-tional changes in AGS9 cells were related to chromatin structural components. Spe-cifically, it was highlighted that genes involved in histone post-translational modifica-tions were strongly affected. Aberrant RDH histone processing was also confirmed through Real-Time qPCR, western blot and immunochemistry. Moreover, TEM anal-ysis reported a lack of chromatin in AGS9 nuclei, thus reinforcing RNAseq results. Since TEM analysis also revealed smaller mitochondria and reduced localization of ri-bosomes on ER membranes in AGS9 cells, Mitotracker, Mitosox and Click-iT staining assays were carried out to assess mitochondrial activity, ROS production and nascent protein production, respectively. Results demonstrated a reduced basal mitochondrial activity together with an increase production of ROS, and a reduced nascent protein production in AGS9 fibroblasts. In conclusion, these insights contribute to a more comprehensive characterization of AGS, shedding light on potential therapeutic targets and emphasizing the need for further research into the intricate molecular mechanisms underlying this debilitating syndrome.