Dysregulation of alternative splicing and back-splicing processes in Multiple Sclerosis pathogenesis

Multiple sclerosis (MS) is a complex autoimmune disease of the central nervous system characterized by demyelination, chronic inflammation, neuronal loss, and axonal damage. Despite the advancement in the understanding of genetic and environmental risk factors, the molecular events underlying MS pathogenesis are still largely unknown. Among the many mechanisms with a potential role in MS, alterations of alternative splicing (AS) process have been proposed. Among factors participating in the AS regulation, the role of long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) is emerging. This project hence aimed to dissect the AS role in MS pathogenesis, with a specific focus on these classes of non-coding RNAs. As far as lncRNAs are concerned, a meta-analysis of microarray datasets led to the identification of MALAT1 (metastasis associated lung adenocarcinoma transcript 1), already known to regulate AS, as the most dysregulated lncRNA in MS patients (meta-analysis P=1.3*10-8). MALAT1 overexpression and knockdown experiments in different cell lines revealed that MALAT1 impacts both on splicing factors expression and on MS-associated AS events. RNA-seq analyses, besides confirming MALAT1 role in AS modulation (1,114 significantly modulated AS events), highlighted its effect also on back-splicing, i.e. the process involved in the biogenesis of circRNAs (49 significantly dysregulated circRNAs). As a proof of concept of the generalized dysregulation of the splicing and back-splicing processes, an in-depth investigation of the GSDMB (gasdermin B) gene was undertaken. This gene has already been associated with autoimmune disease susceptibility, and the characterization of its AS and back-splicing profiles from one side confirmed the unbalance of AS isoforms characterizing MS, from the other identified a dysregulated circRNA in MS patients, suggesting that an alteration of back-splicing profile may also occur in MS. The role of circRNAs in MS was hence further explored, by both in silico and in vivo global approaches. Using an in-house developed bioinformatics pipeline, MS genome-wide associated loci were shown to be highly enriched in circRNAs, whereas RNA-seq experiments identified 166 differentially expressed circRNAs in MS patients’ peripheral blood mononuclear cells compared to healthy controls. Finally, a correlation between the genotype of MS-associated single nucleotide polymorphisms and circRNA expression was observed. In conclusion, this work demonstrated that a dysregulation of AS and back-splicing processes occurs in MS, representing a new molecular mechanism potentially involved in the pathogenesis of the disease.