Fused in sarcoma (FUS): An oncogene goes awry in neurodegeneration
Introduction
FUS, also known as Translocated in liposarcoma (TLS), was identified about 20 years ago as a fusion oncogene in human myxoid liposarcomas (Crozat et al., 1993, Rabbitts et al., 1993). In these cancers, an aberrant chromosomal translocation fuses the N-terminus of FUS to a transcription factor, such as CHOP, giving rise to a fusion oncogene (Crozat et al., 1993, Rabbitts et al., 1993). In the following decade, research on FUS was mainly focused on these fusion oncogenes and the mechanisms by which they cause oncogenic transformation (Kovar, 2011). About 15 years later, researchers interested in neurodegeneration suddenly took an interest in FUS, when mutations in FUS were reported to cause familial ALS and deposition of FUS in pathological protein inclusions (Kwiatkowski et al., 2009, Vance et al., 2009). Shortly afterwards, FUS inclusions were also found in the brains of a subset of FTLD patients, however in the absence of FUS mutations (Munoz et al., 2009, Neumann et al., 2009a, Neumann et al., 2009b). Insoluble protein deposits are observed in almost all neurodegenerative disorders, and the disease-characterizing proteinaceous components of these inclusions provided tremendous insights into the underlying disease mechanisms (Haass and Selkoe, 2007).
Section snippets
ALS and FTLD: related yet distinct neurodegenerative disorders
ALS and FTLD are considered to be related disorders with overlapping clinical symptoms, genetics and neuropathology. ALS, also known as Lou Gehrig's disease, is the most frequent motor neuron disease and is caused by degeneration of upper and lower motor neurons (Kiernan et al., 2011). The most prominent clinical symptom is muscle weakening and atrophy throughout the body. Due to progressive muscle wasting, ALS patients experience increasing difficulties in moving, swallowing, speaking and
FUS: a multifunctional nuclear protein
FUS is a 526 amino acid multidomain protein with an N-terminal transcriptional activation domain, multiple nucleic acid binding domains and a C-terminal nuclear localization signal (NLS) (Fig. 2a). The N-terminal SYGQ-rich domain functions as a potent transcriptional activation domain when fused to the DNA binding domain of other transcription factors (Prasad et al., 1994, Zinszner et al., 1994). This is the case in the above mentioned fusion oncogenes, e.g. FUS-CHOP in myxoid liposarcoma (
FET family proteins
FUS is a member of a protein family called the FET family (Tan and Manley, 2009). The other two FET family members, Ewing sarcoma protein (EWS) and TATA binding protein-associated factor 15 (TAF15), have the same domain structure as FUS (Fig. 2d) and are also imported into the nucleus via a PY-NLS (Lee et al., 2006, Marko et al., 2012, Zakaryan and Gehring, 2006). Like FUS, EWS and TAF15 are components of fusion oncogenes in a variety of cancers and have a well-established function in
Physiological roles of the FET family proteins
FUS and the FET family proteins are involved in multiple steps of DNA/RNA processing, including nuclear processes (e.g. transcription, pre-mRNA splicing and DNA repair) and cytosolic processes (e.g. mRNA transport for local translation). It is therefore not surprising that alterations in the FET proteins' subcellular localization and their sequestration into protein aggregates have deleterious consequences for cellular functions.
FET proteins in DNA damage response
Soon after its discovery as a fusion oncogene, FUS was found to be identical to the POMp75 protein, which has DNA homologous pairing activity and is important for the repair of DNA double strand breaks (Baechtold et al., 1999, Bertrand et al., 1999). FUS binds to denatured single-stranded DNA and promotes its annealing to complementary single stranded DNA and d-loop formation (Baechtold et al., 1999) (Fig. 3a). Moreover, EWS was shown to regulate DNA damage-induced alternative splicing events (
FET proteins in transcriptional regulation
Initial clues about the role of the FET proteins in gene transcription came from the observation that their N-terminal SYGQ-rich domain functions as a potent transcriptional activation domain in oncogenic fusion proteins (Bertolotti et al., 1999, May et al., 1993, Prasad et al., 1994, Zinszner et al., 1994). Moreover, the Drosophila homologue of FUS, named Cabeza (CAZ) or Sarcoma-associated RNA binding fly homologue (SARFH), was found to localize to areas of active transcription in polytene
FET proteins in pre-mRNA splicing and physiological splice targets
Around the same time that the FET proteins were identified as transcriptional activators in oncogenic fusion proteins, they were also recognized to be important splicing regulators. All three FET proteins were co-purified with the human spliceosome (Rappsilber et al., 2002, Zhou et al., 2002), different pre-mRNA splicing complexes (Calvio et al., 1995, Kameoka et al., 2004, Wu and Green, 1997) and the spliceosomal Sm core (Hackl and Luhrmann, 1996) (Fig. 3c). More recently, TAF15 and FUS were
Functions of FUS outside the nucleus
FUS not only regulates gene expression in the nucleus, but also exerts important functions in the cytoplasm. FUS has been shown to continuously shuttle between the nucleus and the cytoplasm (Zinszner et al., 1997) and therefore might play a role in mRNA export (Fig. 3e). It is still unknown if export is mediated by RNA or by specific nuclear export receptors. Whether EWS and TAF15 undergo similar shuttling and what roles they might play in the cytoplasm has not been investigated.
In neurons, FUS
Pathomechanisms in FTLD-FUS and ALS-FUS
A subset of FTLD and ALS cases (FTLD-FUS and ALS-FUS) are characterized by FUS-immunoreactive neuronal and glial inclusions (Mackenzie et al., 2011a, Mackenzie et al., 2011b, Neumann et al., 2009a). FUS inclusions are typically observed in the cytoplasm and less frequently in the nucleus of neuronal and glial cells. Moreover, FUS pathology is often accompanied by a reduction of nuclear FUS staining, consistent with the idea that defects in nuclear import of FUS contribute to FUS pathology (
FTLD-FUS: co-deposition of FET proteins and Transportin caused by defects in arginine methylation
In contrast to ALS-FUS, FTLD-FUS is usually not associated with mutations in FUS (Neumann et al., 2009a, Snowden et al., 2011, Urwin et al., 2010), so there is no obvious explanation for the cytoplasmic deposition of FUS in these cases. Moreover, EWS, TAF15 and Transportin are co-deposited along with FUS in neuronal cytoplasmic inclusions in FTLD-FUS patients (Table 1) and inclusion-bearing cells often show reduced nuclear staining of all three FET proteins (Brelstaff et al., 2011, Davidson et
ALS-FUS: selective deposition of FUS caused by FUS mutations
In contrast to FTLD-FUS, ALS-FUS cases show a selective deposition of FUS, i.e. EWS, TAF15 and Transportin are not co-deposited in FUS-positive inclusions (Neumann et al., 2011, Troakes et al., in press) (Table 1). This selective nuclear import defect in ALS-FUS can be explained by a point mutation in the FUS gene that produces a defective PY-NLS or a truncated FUS protein lacking the entire PY-NLS (Fig. 2a). This impairs Transportin binding and nuclear import of FUS and leads to its
Stress granules and p62 — further hits are required for pathological FUS deposition
The nuclear import defects described above presumably are an important first step in the pathological cascade, but are not sufficient for aggregation and deposition of cytosolically mislocalized FUS/FET proteins. The presence of stress granule marker proteins and components of the protein degradation machinery in cytoplasmic FUS inclusions (Table 1) suggests that a second hit, such as cellular stress and/or defects in protein degradation, may be involved in the formation of pathological protein
Intranuclear FUS inclusions in neurodegenerative diseases
Although FUS-immunoreactive inclusions in ALS/FTLD-FUS cases are mostly found in the cytoplasm, they are occasionally observed in the nucleus as well (Lashley et al., 2011, Mackenzie et al., 2011a, Mackenzie et al., 2011b, Neumann et al., 2009a). Nuclei containing these intranuclear inclusions often show reduced amounts of FUS in the nucleoplasm, suggesting that sequestration of FUS in intranuclear inclusions interferes with the protein's normal localization and function. How intranuclear FUS
Acknowledgments
We thank Eva Bentmann, Dieter Edbauer, Denise Orozco, Bettina Schmid and Marc Suarez-Calvet for critically reading the manuscript, Claudia Abou-Ajram for suggestions on figures and Manuela Neumann for comments regarding neuropathology. This work was supported by the Competence Network for Neurodegenerative Diseases (KNDD) of the Bundesministerium für Bildung und Forschung (BMBF) and the Consortium of Centers of Excellence in Neurodegenerative Brain Diseases (CoEN) to C.H. The research leading
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