Phd in GENETICS AND MOLECULAR BIOLOGY

Thesis title: The role of m6A RNA modification in Stress Granules dynamics in the pathological context of ALS

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of upper and lower motor neurons (MNs). Genetic mutations in several RNA-binding proteins (RBPs) have been associated with ALS, such as TDP-43 and FUS, leading to the formation of pathological aggregates in affected MNs. While the exact cause remains unclear, emerging evidence suggests that abnormalities in stress granules (SGs) dynamics may contribute to the generation of pathological inclusions in ALS. SGs are cytoplasmic membrane-less assemblies of RNAs and proteins, generated in response to different stress stimuli. The molecular composition of SGs provides their liquid-like dynamics, fundamental for their rapid formation and disassembly. Impaired SGs dynamics result in the generation of permanent and solid-like condensates, leading to cellular toxicity and death. In recent years, several evidence support the idea that RNA composition is a critical determinant in regulating SGs dynamics. N6-methyladenosine (m6A) is the most abundant internal modification present on cellular mRNAs and participates in all stages of RNA metabolism. How m6A influence the RNA composition of SGs and their dynamics, including its involvement in ALS progression, is an active area of investigation. In this study, we explored the role of m6A RNA methylation in regulating SGs properties in physiological and pathological conditions. We demonstrated that m6A does not seem to play significant role in SGs physiology in normal conditions, regarding their RNA composition and physical properties, while it has a strong impact in ALS genetic contexts, particularly those where mutant FUS is mislocalized in the cytoplasm. More importantly, we showed that m6A downregulation in ALS cellular models reestablishes first the normal SGs RNA composition, restoring the localization of specific mRNAs, and second the normal SGs dynamics, such as their formation and disassembly rate. Notably, we demonstrated that the expression of mutant FUS increased the global m6A levels in our cellular systems, suggesting a possible link between m6A homeostasis and pathological aggregates. Finally, we validated our observations using STM-2457, a highly potent and selective methyltransferase inhibitor, in different cellular systems, including human spinal MNs and patient-derived fibroblasts. Colle ctively, our data propose a new interplay between m6A and altered SGs dynamics in the context of ALS, providing proof of concept that the targeting of RNA-methylating enzymes could represent a promising avenue for counteracting aggregate formation in ALS.