Analysis of FUS/TLS involvement in Amyotrophic Lateral Sclerosis

FUS is a multifunctional protein involved in almost all step of RNA metabolism, from transcription, to splicing and RNA transport and translation. FUS mutations have been associated to Amyotrophic Lateral Sclerosis (ALS) onset, a lethal neurodegenerative disease that leads to specific degeneration of upper and lower motoneurons. In this research project I demonstrated that FUS is involved in microRNA (miRNAs) biogenesis, a family of small RNAs that participate in post-transcriptional regulation of gene expression by repressing mRNA translation. In particular I demonstrated that FUS is important for the biogenesis of a group of miRNAs, including those with a pivotal role in neuronal differentiation and synaptogenesis. I showed that FUS is able to participate in miRNAs biogenesis facilitating the processing of precursor molecules (pri-miRNAs). Furthermore, I demonstrated that FUS is able to activate two feed-forward regulatory loops important for the maintenance of the correct cellular level of the FUS protein. Increased amount of FUS has been described, indeed, in ALS patients, suggesting that the overdose of FUS becomes toxic for the cellular homeostasis. In particular, a strong increase of FUS protein has been described in ALS patients carrying mutations in the 3’UTR of FUS mRNA. Even though, in this case, the protein is wild type, an ALS phenotype still occurs, and this may be due to the failure of some regulatory mechanisms that control FUS levels. I showed the existence of two mechanisms able to control FUS levels: on one side FUS induces the skipping of the exon 7 of its own pre-mRNA, leading to the formation of an out-of-frame mRNA predicted to be degraded by nonsense-mediated decay; on the other side FUS is able to upregulate miR-141 and miR-200a, which in turn repress FUS synthesis. Therefore when FUS levels increase, these two feed-forward regulatory loops, acting on pre-mRNA splicing and on mRNA translation, are able to restore the physiological levels of FUS. The failure of these mechanisms might contribute to the ALS pathogenesis, where the uncontrolled increase of FUS results toxic for the cell. Notably, one mutation found in the 3’UTR of FUS in two ALS patients, is localized in the binding site for miR-141 and miR-200a, and I demonstrated that this mutation affects the ability of these miRNAs to target FUS mRNA. So, in these patients, this regulatory process probably fails in controlling FUS protein levels, and this may be one of the mechanisms leading to ALS pathogenesis.