M6A reduction relieves FUS-associated ALS granules [RNA-Seq]

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease due to gradual motorneurons (MN) degeneration1. Among the processes associated to ALS pathogenesis, there is the formation of cytoplasmic inclusions produced by mutant protein aggregation, among which the RNA binding protein FUS2. In this work we show that such inclusions are significantly reduced in number and dissolve faster when the RNA m6A content is diminished as a consequence of the m6A writer METTL3 knock-down. These effects were obtained observed both in neuronal cell lines and in iPSC-derived human motor neurons expressing mutant FUS. Importantly, stress granules formed in mutant conditionswhen mutant FUS is expressed/ALS condition showed a distinctive transcriptome with respect to control cells; interestingly, after METTL3 downregulation, it reverted to similar to control. Finally, we show that FUS inclusions are reduced also in patient-derived fibroblasts treated with STM-2457, a well characterized inhibitor of METTL3 activity, paving the way for its possible use for counteracting aggregate formation in ALS. In order to unveil the impact of m6A on SG RNA composition in physiological and ALS-linked conditions, we sequenced RNA from input (INP) and immunoprecipitated granules (SGenr) isolated from stresses (ARS+) SK-N-BE cells carrying the doxycycline inducible overexpression of either wild type FUS (FUSWT) or mutant FUS (FUSP525L), and constitutively expressing the SG marker G3BP1 tagged with GFP. We treated FUSWT or FUSP525L cells with the combination of two shRNAs against METTL3 (shMETTL3), treated (DOXY+) or not (DOXY-) with doxycycline.