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

FUS affects circular RNA expression in murine embryonic stem cell-derived motor neurons

The RNA-binding protein FUS participates in several RNA biosynthetic processes and has been linked to the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. Here we report that FUS controls back-splicing reactions leading to circular RNA (circRNA) production. We identified circRNAs expressed in in vitro -derived mouse motor neurons (MNs) and determined that the production of a considerable number of these circRNAs is regulated by FUS. Using RNAi and overexpression of wild-type and ALS-asso- ciated FUS mutants, we directly correlate the modulation of circRNA biogenesis with alteration of FUS nuclear levels and with putative toxic gain of function activities. We also demonstrate that FUS regulates circRNA biogenesis by binding the introns flanking the back-splicing junctions and that this control can be reproduced with artificial constructs. Most circRNAs are conserved in humans and specific ones are deregulated in human-induced pluripotent stem cell-derived MNs carrying the FUS P525L mutation associated with AL

Comparative interactomics analysis of different ALS-associated proteins identifies converging molecular pathways

Amyotrophic lateral sclerosis (ALS) is a devastating neurological disease with no effective treatment available. An increasing number of genetic causes of ALS are being identified, but how these genetic defects lead to motor neuron degeneration and to which extent they affect common cellular pathways remains incompletely understood. To address these questions, we performed an interactomic analysis to identify binding partners of wild-type (WT) and ALS-associated mutant versions of ATXN2, C9orf72, FUS, OPTN, TDP-43 and UBQLN2 in neuronal cells. This analysis identified several known but also many novel binding partners of these proteins

Comparative interactomics analysis of different ALS-associated proteins identifies converging molecular pathways

Amyotrophic lateral sclerosis (ALS) is a devastating neurological disease with no effective treatment available. An increasing number of genetic causes of ALS are being identified, but how these genetic defects lead to motor neuron degeneration and to which extent they affect common cellular pathways remains incompletely understood. To address these questions, we performed an interactomic analysis to identify binding partners of wild-type (WT) and ALS-associated mutant versions of ATXN2, C9orf72, FUS, OPTN, TDP-43 and UBQLN2 in neuronal cells. This analysis identified several known but also many novel binding partners of these proteins. Interactomes of WT and mutant ALS proteins were very similar except for OPTN and UBQLN2, in which mutations caused loss or gain of protein interactions. Several of the identified interactomes showed a high degree of overlap: shared binding partners of ATXN2, FUS and TDP-43 had roles in RNA metabolism; OPTN- and UBQLN2-interacting proteins were related to protein degradation and protein transport, and C9orf72 interactors function in mitochondria. To conf

Invited Review: Decoding the pathophysiological mechanisms that underlie RNA dysregulation in neurodegenerative disorders: a review of the current state of the art

Altered RNA metabolism is a key pathophysiological component causing several neurodegenerative diseases. Genetic mutations causing neurodegeneration occur in coding and noncoding regions of seemingly unrelated genes whose products do not always contribute to the gene expression process. Several pathogenic mechanisms may coexist within a single neuronal cell, including RNA/protein toxic gain-of-function and/or protein loss-of-function. Genetic mutations that cause neurodegenerative disorders disrupt healthy gene expression at diverse levels, from chromatin remodelling, transcription, splicing, through to axonal transport and repeat-associated non-ATG (RAN) translation. We address neurodegeneration in repeat expansion disorders [Huntington's disease, spinocerebellar ataxias, C9ORF72-related amyotrophic lateral sclerosis (ALS)] and in diseases caused by deletions or point mutations (spinal muscular atrophy, most subtypes of familial ALS). Some neurodegenerative disorders exhibit broad dysregulation of gene expression with the synthesis of hundreds to thousands of abnormal messenger RNA (mRNA) molecules. However, the number and identity of aberrant mRNAs that are translated into proteins – and how these lead to neurodegeneration – remain unknown. The field of RNA biology research faces the challenge of identifying pathophysiological events of dysregulated gene expression. In conclusion, we discuss current research limitations and future directions to improve our characterization of pathological mechanisms that trigger disease onset and progression

An ALS-associated mutation in the FUS 3'-UTR disrupts a microRNA-FUS regulatory circuitry.

While the physiologic functions of the RNA-binding protein FUS still await thorough characterization, the pathonegetic role of FUS mutations in amyotrophic lateral sclerosis (ALS) is clearly established. Here we find that a human FUS mutation that leads to increased protein expression, and was identified in two ALS patients with severe outcome, maps to the seed sequence recognized by miR-141 and miR-200a in the 3′-UTR of FUS. We demonstrate that FUS and these microRNAs are linked by a feed-forward regulatory loop where FUS upregulates miR-141/200a, which in turn impact FUS protein synthesis. We also show that Zeb1, a target of miR-141/200a and transcriptional repressor of these two microRNAs, is part of the circuitry and reinforces it. Our results reveal a possible correlation between deregulation of this regulatory circuit and ALS pathogenesis, and open interesting perspectives in the treatment of these mutations through ad hoc-modified microRNAs. © 2014 Macmillan Publishers Limited. All rights reserved

FUS stimulates microRNA biogenesis by facilitating co-transcriptional Drosha recruitment

microRNA abundance has been shown to depend on the amount of the microprocessor components or, in some cases, on specific auxiliary co-factors. In this paper, we show that the FUS/TLS (fused in sarcoma/translocated in liposarcoma) protein, associated with familial forms of Amyotrophic Lateral Sclerosis (ALS), contributes to the biogenesis of a specific subset of microRNAs. Among them, species with roles in neuronal function, differentiation and synaptogenesis were identified. We also show that FUS/TLS is recruited to chromatin at sites of their transcription and binds the corresponding pri-microRNAs. Moreover, FUS/TLS depletion leads to decreased Drosha level at the same chromatin loci. Limited FUS/TLS depletion leads to a reduced microRNA biogenesis and we suggest a possible link between FUS mutations affecting nuclear/cytoplasmic partitioning of the protein and altered neuronal microRNA biogenesis in ALS pathogenesis. The EMBO Journal (2012) 31, 4502-4510. doi:10.1038/emboj.2012.31