Rewiring the Epitranscriptome: Insights from Neurodegeneration, Viral Infections, and RNA editing

The global landscape of RNA modifications, collectively referred to as the epitranscriptome, is emerging as a key regulator of gene expression, modulating RNA stability, splicing, localization, and translation. The epitranscriptomic machinery comprises enzymes that install, recognize, or remove these modifications—known as writers, readers, and erasers—forming a complex and dynamic network that regulates RNA metabolism. Among the most abundant RNA modifications, N6-methyladenosine (m6A), adenosine-to-inosine (A-to-I) editing, and pseudouridine (Ψ) play crucial roles in fundamental physiological processes, including development, aging, immune responses, and cellular homeostasis. Dysregulation of the epitranscriptome has been increasingly associated with a wide range of pathological conditions, such as cancer, infectious diseases, neurodegeneration, cardiovascular disorders, and metabolic diseases, where RNA modifications may actively contribute to disease onset and progression. This thesis is based on the hypothesis that disruption of RNA modification homeostasis leads to a coordinated and context-dependent rewiring of the epitranscriptome, ultimately driving transcriptomic instability and altering cellular phenotypes. The main objective of this thesis is to characterize the dynamic remodeling of the transcriptome and epitranscriptome across different pathological contexts. To address this, the thesis is structured into three main sections. In the first section, I characterized the m6A methylome, A-to-I editome, and pseudouridylome in two neurodegenerative diseases, Amyotrophic Lateral Sclerosis (ALS) and Spinal Muscular Atrophy (SMA). By analyzing RNA-seq data from iPSC-derived and differentiated cell lines obtained from patients and healthy controls, I observed widespread epitranscriptomic alterations in both diseases. Although distinct sets of genes were affected, the dysregulated pathways in both diseases converged on processes related to RNA stability, splicing, and localization. In addition, potential novel biomarkers were identified for both ALS and SMA, highlighting the functional relevance of RNA modifications in these conditions. In the second section, I investigated the role of ADAR enzymes in SARS-CoV-2 infection. Endogenous deaminases such as APOBECs and ADARs have been proposed to act as antiviral or proviral factors through nucleic acid binding and deamination activities. Bioinformatic analyses of patient-derived samples revealed APOBEC-mediated and ADAR-mediated signatures in the SARS-CoV-2 transcriptome, suggesting a potential role for RNA editing in viral evolution. However, experimental evidence has remained limited and sometimes conflicting. Using ADAR-deficient cell lines infected with the native virus, I demonstrated that the A-to-I mutational component observed during infection depends on ADAR activity. Furthermore, I show that SARS-CoV-2 infection redirects ADAR-mediated RNA editing toward host transcripts involved in antiviral defense pathways. In the third section, I explored the functional impact of a novel APOBEC3A-mediated RNA editing event in the DDOST transcript. This editing introduces a premature stop codon, likely affecting transcript stability. I show that edited DDOST mRNA is associated with reduced transcript levels in large-scale transcriptomic datasets from both healthy (GTEx) and disease (TCGA) cohorts. Based on these findings, we propose that APOBEC3A-mediated editing may contribute to the modulation of N-glycosylation by regulating DDOST expression, particularly in contexts where rapid regulation of endoplasmic reticulum activity may be advantageous.