EWS splicing regulation contributes to balancing Foxp1 isoforms required for neuronal differentiation.

Alternative splicing is a key regulatory process underlying the amplification of genomic information and the expansion of proteomic diversity, particularly in brain. Here, we identify the Ewing sarcoma protein (EWS) as a new player of alternative splicing regulation during neuronal differentiation. Knockdown of EWS in neuronal progenitor cells leads to premature differentiation. Transcriptome profiling of EWS-depleted cells revealed global changes in splicing regulation. Bioinformatic analyses and biochemical experiments demonstrated that EWS regulates alternative exons in a position-dependent fashion. Notably, several EWS-regulated splicing events are physiologically modulated during neuronal differentiation and EWS depletion in neuronal precursors anticipates the splicing-pattern of mature neurons. Among other targets, we found that EWS controls the alternative splicing of the forkhead family transcription factor FOXP1, a pivotal transcriptional regulator of neuronal differentiation, possibly contributing to the switch of gene expression underlying the neuronal differentiation program.

Post-transcriptional regulation of FUS and EWS protein expression by miR-141 during neural differentiation.

Brain development involves proliferation, migration and specification of neural progenitor cells, culminating in neuronal circuit formation. Mounting evidence indicates that improper regulation of RNA binding proteins (RBPs), including members of the FET (FUS, EWS, TAF15) family, results in defective cortical development and/or neurodegenerative disorders. However, in spite of their physiological relevance, the precise pattern of FET protein expression in developing neurons is largely unknown. Herein, we found that FUS, EWS and TAF15 expression is differentially regulated during brain development, both in time and in space. In particular, our study identifies a fine-tuned regulation of FUS and EWS during neuronal differentiation, whereas TAF15 appears to be more constitutively expressed. Mechanistically FUS and EWS protein expression is regulated at the post-transcriptional level during neuron differentiation and brain development. Moreover, we identified miR-141 as a key regulator of these FET proteins that modulate their expression levels in differentiating neuronal cells. Thus, our studies uncover a novel link between post-transcriptional regulation of FET proteins expression and neurogenesis.

Role of FET proteins in neurodegenerative disorders.

Neurodegenerative disorders such as Alzheimer disease (AD), frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), Parkinson disease (PD), Huntington's disease (HD), and multiple sclerosis (MS) affect different neuronal cells, and have a variable age of onset, clinical symptoms, and pathological features. Despite the great progress in understanding the etiology of these disorders, the underlying mechanisms remain largely unclear. Among the processes affected in neurodegenerative diseases, alteration in RNA metabolism is emerging as a crucial player. RNA-binding proteins (RBPs) are involved at all stages of RNA metabolism and display a broad range of functions, including modulation of mRNA transcription, splicing, editing, export, stability, translation and localization and miRNA biogenesis, thus enormously impacting regulation of gene expression. On the other hand, aberrant regulation of RBP expression or activity can contribute to disease onset and progression. Recent reports identified mutations causative of neurological disorders in the genes encoding a family of RBPs named FET (FUS/TLS, EWS and TAF15). This review summarizes recent works documenting the involvement of FET proteins in the pathology of ALS, FTLD, essential tremor (ET) and other neurodegenerative diseases. Moreover, clinical implications of recent advances in FET research are critically discussed.

Genotoxic stress inhibits Ewing sarcoma cell growth by modulating alternative pre-mRNA processing of the RNA helicase DHX9.

Alternative splicing plays a key role in the DNA damage response and in cancer. Ewing Sarcomas (ES) are aggressive tumors caused by different chromosomal translocations that yield in-frame fusion proteins driving transformation. RNA profiling reveals genes differentially regulated by UV light irradiation in two ES cell lines exhibiting different sensitivity to genotoxic stress. In particular, irradiation induces a new isoform of the RNA helicase DHX9 in the more sensitive SK-N-MC cells, which is targeted to nonsense-mediated decay (NMD), causing its downregulation. DHX9 protein forms a complex with RNA polymerase II (RNAPII) and EWS-FLI1 to enhance transcription. Silencing of DHX9 in ES cells sensitizes them to UV treatment and impairs recruitment of EWS-FLI1 to target genes, whereas DHX9 overexpression protects ES cells from genotoxic stress. Mechanistically, we found that UV light irradiation leads to enhanced phosphorylation and decreased processivity of RNAPII in SK-N-MC cells, which in turn causes inclusion of DHX9 exon 6A. A similar effect on DHX9 splicing was also elicited by treatment with the chemotherapeutic drug etoposide, indicating a more general mechanism of regulation in response to DNA damage. Our data identify a new NMD-linked splicing event in DHX9 with impact on EWS-FLI1 oncogenic activity and ES cell viability.

Oxidative stress affects FET proteins localization and alternative pre-mRNA processing in cellular models of ALS.

FUS/TLS, EWS and TAF15 are members of the FET family of DNA and RNA binding proteins, involved in multiple steps of DNA and RNA processing and implicated in the regulation of gene expression and cell-signaling. All members of the FET family contribute to human pathologies, as they are involved in sarcoma translocations and neurodegenerative diseases. Mutations in FUS/TLS, in EWSR1 and in TAF15 genescause Amyotrophic Lateral Sclerosis (ALS), a fatal human neurodegenerative disease that affects primarily motor neurons and is characterized by the progressive loss of motor neurons and degradation of the neuromuscular junctions.ALS-associated FET mutations cause FET protein relocalization into cytoplasmic aggregates, thus impairing their normal function. Protein aggregation has been suggested as a co-opting factor during the disease pathogenesis. Cytoplasmic mislocalization of FET proteins contributes to the formation of cytoplasmic aggregates that may alter RNA processing and initiate motor neuron degeneration. Interestingly, oxidative stress, which is implicated in the pathogenesis of ALS, triggers the accumulation of mutant FUS in cytoplasmic stress granules where it binds and sequester wild-type FUS.In order to evaluate the role of FET proteins in ALS and their involvement in the response to oxidative stress, we have developed cellular models of ALS expressing ALS-related FET mutants in neuroblastoma cell lines. Upon treatment with sodium arsenite, cells were analysed by immunofluorescence to monitor the localization of wild-type and mutated FET proteins. Furthermore, we have characterized signal transduction pathways and cell survival upon oxidative stress in our cellular models of ALS. Interestingly, we found that EWS mutant proteins display a different localization from FUS mutants and neither wild-type nor mutated EWS protein translocate into stress granules upon oxidative stress treatment. Collectively, our data provide a new link between the oxidative stress response and RNA metabolism in ALS disease.

Thapsigargin affects presenilin-2 but not presenilin-1 regulation in SK-N-BE cells.

Presenilin-1 (PS1) and presenilin-2 (PS2) are transmembrane proteins widely expressed in the central nervous system, which function as the catalytic subunits of γ-secretase, the enzyme that releases amyloid-ß protein (Aß) from ectodomain cleaved amyloid precursor protein (APP) by intramembrane proteolysis. Mutations in PS1, PS2, and Aß protein precursor are involved in the etiology of familial Alzheimer's disease (FAD), while the cause of the sporadic form of AD (SAD) is still not known. However, since similar neuropathological changes have been observed in both FAD and SAD, a common pathway in the etiology of the disease has been suggested. Given that age-related deranged Ca(2+) regulation has been hypothesized to play a role in SAD pathogenesis via PS gene regulation and γ-secretase activity, we studied the in vitro regulation of PS1 and PS2 in the human neuron-like SK-N-BE cell line treated with the specific endoplasmic reticulum (ER) calcium ATPase inhibitor Thapsigargin (THG), to introduce intracellular Ca(2+) perturbations and mimic the altered Ca(2+) homeostasis observed in AD. Our results showed a consistent and significant down-regulation of PS2, while PS1 appeared to be unmodulated. These events were accompanied by oxidative stress and a number of morphological alterations suggestive of the induction of apoptotic machinery. The administration of the antioxidant N-acetylcysteine (NAC) did not revert the THG-induced effects reported, while treatment with the Ca(2+)-independent ER stressor Brefeldin A did not modulate basal PS1 and PS2 expression. Collectively, these results suggest that Ca(2+) fluctuation rather than ER stress and/or oxidative imbalance seems to play an essential role in PS2 regulation and confirm that, despite their strong homology, PS1 and PS2 could play different roles in AD.