Arginine methylation next to the PY‐NLS modulates Transportin binding and nuclear import of FUS

Fused in sarcoma (FUS) is a nuclear protein that carries a proline‐tyrosine nuclear localization signal (PY‐NLS) and is imported into the nucleus via Transportin (TRN). Defects in nuclear import of FUS have been implicated in neurodegeneration, since mutations in the PY‐NLS of FUS cause amyotrophic lateral sclerosis (ALS). Moreover, FUS is deposited in the cytosol in a subset of frontotemporal lobar degeneration (FTLD) patients. Here, we show that arginine methylation modulates nuclear import of FUS via a novel TRN‐binding epitope. Chemical or genetic inhibition of arginine methylation restores TRN‐mediated nuclear import of ALS‐associated FUS mutants. The unmethylated arginine–glycine–glycine domain preceding the PY‐NLS interacts with TRN and arginine methylation in this domain reduces TRN binding. Inclusions in ALS‐FUS patients contain methylated FUS, while inclusions in FTLD‐FUS patients are not methylated. Together with recent findings that FUS co‐aggregates with two related proteins of the FET family and TRN in FTLD‐FUS but not in ALS‐FUS, our study provides evidence that these two diseases may be initiated by distinct pathomechanisms and implicates alterations in arginine methylation in pathogenesis.     

Intranuclear Aggregation of Mutant FUS/TLS as a Molecular Pathomechanism of Amyotrophic Lateral Sclerosis

Dominant mutations in FUS/TLS cause a familial form of amyotrophic lateral sclerosis (fALS), where abnormal accumulation of mutant FUS proteins in cytoplasm has been observed as a major pathological change. Many of pathogenic mutations have been shown to deteriorate the nuclear localization signal in FUS and thereby facilitate cytoplasmic mislocalization of mutant proteins. Several other mutations, however, exhibit no effects on the nuclear localization of FUS in cultured cells, and their roles in the pathomechanism of fALS remain obscure. Here, we show that a pathogenic mutation, G156E, significantly increases the propensities for aggregation of FUS in vitro and in vivo. Spontaneous in vitro formation of amyloid-like fibrillar aggregates was observed in mutant but not wild-type FUS, and notably, those fibrils functioned as efficient seeds to trigger the aggregation of wild-type protein. In addition, the G156E mutation did not disturb the nuclear localization of FUS but facilitated the formation of intranuclear inclusions in rat hippocampal neurons with significant cytotoxicity. We thus propose that intranuclear aggregation of FUS triggered by a subset of pathogenic mutations is an alternative pathomechanism of FUS-related fALS diseases.     

Molecular determinants and genetic modifiers of aggregation and toxicity for the ALS disease protein FUS/TLS

TDP-43 and FUS are RNA-binding proteins that form cytoplasmic inclusions in some forms of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Moreover, mutations in TDP-43 and FUS are linked to ALS and FTLD. However, it is unknown whether TDP-43 and FUS aggregate and cause toxicity by similar mechanisms. Here, we exploit a yeast model and purified FUS to elucidate mechanisms of FUS aggregation and toxicity. Like TDP-43, FUS must aggregate in the cytoplasm and bind RNA to confer toxicity in yeast. These cytoplasmic FUS aggregates partition to stress granule compartments just as they do in ALS patients. Importantly, in isolation, FUS spontaneously forms pore-like oligomers and filamentous structures reminiscent of FUS inclusions in ALS patients. FUS aggregation and toxicity requires a prion-like domain, but unlike TDP-43, additional determinants within a RGG domain are critical for FUS aggregation and toxicity. In further distinction to TDP-43, ALS-linked FUS mutations do not promote aggregation. Finally, genome-wide screens uncovered stress granule assembly and RNA metabolism genes that modify FUS toxicity but not TDP-43 toxicity. Our findings suggest that TDP-43 and FUS, though similar RNA-binding proteins, aggregate and confer disease phenotypes via distinct mechanisms. These differences will likely have important therapeutic implications.     

A Proline‐Tyrosine Nuclear Localization Signal (PY‐NLS) Is Required for the Nuclear Import of Fission Yeast PAB2, but Not of Human PABPN1

Nuclear poly(A)‐binding proteins (PABPs) are evolutionarily conserved proteins that play key roles in eukaryotic gene expression. In the fission yeast Schizosaccharomyces pombe, the major nuclear PABP, Pab2, functions in the maturation of small nucleolar RNAs as well as in nuclear RNA decay. Despite knowledge about its nuclear functions, nothing is known about how Pab2 is imported into the nucleus. Here, we show that Pab2 contains a proline‐tyrosine nuclear localization signal (PY‐NLS) that is necessary and sufficient for its nuclear localization and function. Consistent with the role of karyopherin β2 (Kapβ2)‐type receptors in the import of PY‐NLS cargoes, we show that the fission yeast ortholog of human Kapβ2, Kap104, binds to recombinant Pab2 and is required for Pab2 nuclear localization. The absence of arginine methylation in a basic region N‐terminal to the PY‐core motif of Pab2 did not affect its nuclear localization. However, in the context of a sub‐optimal PY‐NLS, we found that Pab2 was more efficiently targeted to the nucleus in the absence of arginine methylation, suggesting that this modification can affect the import kinetics of a PY‐NLS cargo. Although a sequence resembling a PY‐NLS motif can be found in the human Pab2 ortholog, PABPN1, our results indicate that neither a functional PY‐NLS nor Kapβ2 activity are required to promote entry of PABPN1 into the nucleus of human cells. Our findings describe the mechanism by which Pab2 is imported into the nucleus, providing the first example of a PY‐NLS import system in fission yeast. In addition, this study suggests the existence of alternative or redundant nuclear import pathways for human PABPN1.     

Requirements for stress granule recruitment of fused in sarcoma (FUS) and TAR DNA-binding protein of 43 kDa (TDP-43)

Cytoplasmic inclusions containing TAR DNA-binding protein of 43 kDa (TDP-43) or Fused in sarcoma (FUS) are a hallmark of amyotrophic lateral sclerosis (ALS) and several subtypes of frontotemporal lobar degeneration (FTLD). FUS-positive inclusions in FTLD and ALS patients are consistently co-labeled with stress granule (SG) marker proteins. Whether TDP-43 inclusions contain SG markers is currently still debated. We determined the requirements for SG recruitment of FUS and TDP-43 and found that cytoplasmic mislocalization is a common prerequisite for SG recruitment of FUS and TDP-43. For FUS, the arginine-glycine-glycine zinc finger domain, which is the protein's main RNA binding domain, is most important for SG recruitment, whereas the glycine-rich domain and RNA recognition motif (RRM) domain have a minor contribution and the glutamine-rich domain is dispensable. For TDP-43, both the RRM1 and the C-terminal glycine-rich domain are required for SG localization. ALS-associated point mutations located in the glycine-rich domain of TDP-43 do not affect SG recruitment. Interestingly, a 25-kDa C-terminal fragment of TDP-43, which is enriched in FTLD/ALS cortical inclusions but not spinal cord inclusions, fails to be recruited into SG. Consistently, inclusions in the cortex of FTLD patients, which are enriched for C-terminal fragments, are not co-labeled with the SG marker poly(A)-binding protein 1 (PABP-1), whereas inclusions in spinal cord, which contain full-length TDP-43, are frequently positive for this marker protein.     

Impaired nuclear transport induced by juvenile ALS causing P525L mutation in NLS domain of FUS: A molecular mechanistic study

Amyotrophic lateral sclerosis (ALS) and fronto-temporal lobar degeneration (FTLD) are progressive neurological disorders affecting motor neurons. Cellular aggregates of fused in sarcoma (FUS) protein are found in cytoplasm of ALS and FTLD patients. Nuclear localisation signal (NLS) domain of FUS binds to Karyopherin β2 (Kapβ2), which drives nuclear transport of FUS from cytoplasm. Several pathogenic mutations are reported in FUS NLS, which are associated with its impaired nuclear transport and cytoplasmic mis-localisation. P525L mutation in NLS is most commonly found in cases of juvenile ALS (jALS), which affects individuals below 25 years of age. jALS progresses aggressively causing death within a year of its onset. This study elucidates the molecular mechanism behind jALS-causing P525L mutation hindering nuclear transport of FUS. We perform multiple molecular dynamics simulations in aqueous and hydrophobic solvent to understand the effect of the mutation at molecular level. Dynamics of Kapβ2-FUS complex is better captured in hydrophobic solvent compared to aqueous solvent. P525 and Y526 (PY-motif) of NLS exhibit fine-tuned stereochemical arrangement, which is essential for optimum Kapβ2 binding. P525L causes loss of several native contacts at interface leading to weaker binding, which promotes self-aggregation of FUS in cytoplasm. Native complex samples closed conformation, while mutant complex exhibits open conformation exposing hydrophilic residues of Kapβ2 to hydrophobic solvent. Mutant complex also fails to exhibit spring-like motion essential for its transport through nuclear pore complex. This study provides a mechanistic insight of binding affinity between NLS and Kapβ2 that inhibits self-aggregation of FUS preventing the disease condition.     

The FUS about arginine methylation in ALS and FTLD

EMBO J (2012) 31 22, 4258–4275 doi:10.1038/emboj.2012.261; published online September112012In a time where links between amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) neurodegeneration are becoming increasingly clear, it is important to establish the convergent and divergent mechanisms responsible for this. Accordingly, Dormann et al (2012) have identified that methylation of the Fused in sarcoma (FUS) RGG3 domain is involved in the cytoplasmic mislocalisation of ALS-FUS mutants, through a transportin-dependent mechanism. By contrast, hypomethylation in this domain may play a role in the aberrant accumulation of FUS in FTLD-FUS. This work showcases arginine methylation as a phenomenon to watch out for in neurodegenerative pathology.The nuclear RNA/DNA-binding protein, FUS, first burst onto the ALS scene in 2009 when it was discovered that mutations in this protein are causative for familial ALS (Kwiatkowski et al, 2009; Vance et al, 2009). It is now thought to be responsible for 4% of familial (and rare sporadic) ALS cases. In ALS patients with FUS mutations (ALS-FUS), the FUS protein is deposited in abnormal protein inclusions in neurons and glia and nuclei often show a reduced FUS staining (Lagier-Tourenne et al, 2010). Fascinatingly, this abnormal FUS deposition is also observed in several subtypes of FTLD, subsequently termed as FTLD-FUS. However, unlike in ALS-FUS, there are no known FUS mutations in this disease (Da Cruz and Cleveland, 2011). Dormann et al (2012) now broaden our knowledge of how ALS-causing mutations in FUS lead to its abnormal cytoplasmic deposition in neurons and glia, and for the first time suggest a mechanism through which this could also occur in FTLD-FUS, in the absence of FUS mutations.The majority of pathogenic mutations identified in ALS-FUS are located at the C-terminus of the protein within a region identified to be a proline-tyrosine nuclear localization signal (PY-NLS) (Figure 1). The PY-NLS binds to the nuclear import receptor transportin (TRN), which facilitates FUS transport into the nucleus. Pathogenic FUS mutations affect key residues of the PY-NLS or completely delete the signal sequence and thus impair nuclear import of FUS (Dormann et al, 2010). This nuclear transport defect is likely directly involved in pathogenesis, as mutations that cause a very severe nuclear import deficit (e.g., FUS-P525L), are associated with earlier disease onset and a rapid disease course (Dormann et al, 2010).Open in a separate windowFigure 1(A) Schematic diagram showing the domain structure of FUS. SYGQ-rich, serine, tyrosine, glycine, glutamine-rich domain; RRM, RNA recognition motif; ZnF, zinc finger; PY, proline-tyrosine nuclear localization signal (PY-NLS). (B) Schematic diagram summarizing the divergent mechanisms by which arginine methylation (or absence thereof) may bring about FUS mislocalization and accumulation in ALS-FUS and FTLD-FUS. In ALS-FUS caused by FUS mutations, neuronal cytoplasmic inclusions contain methylated FUS, but are negative for EWS, TAF15 and TRN. Dormann et al (2012) propose that these FUS-specific inclusions result from the combination of a genetic defect (point mutations in the PY-NLS that impair transportin binding) and post-translational modification (arginine methylation in RGG3 domain that also impairs transportin binding). By contrast in FTLD-FUS, neuronal cytoplasmic inclusions contain all three FET proteins and transportin, but are not immunoreactive with meFUS-specific antibodies. It is therefore possible that hypomethylation of the FET proteins and thus increased transportin binding may be involved in the co-deposition of these proteins in FTLD-FUS.FUS and other related PY-NLS-containing FET proteins such as Ewing sarcoma (EWS) protein and TATA-binding protein-associated factor 15 (TAF15) have been described to undergo extensive asymmetric dimethylation in their arginine-glycine-glycine (RGG) domains (Araya et al, 2005; Hung et al, 2009; Jobert et al, 2009). In recent times, it has been established that this arginine methylation can affect their nuclear localization (Araya et al, 2005; Jobert et al, 2009; Tradewell et al, 2012). In this issue, Dormann et al (2012) sought to delve further into the mechanism by which this occurs. They began their studies in a similar fashion to Tradewell and colleagues, by confirming that inhibiting global arginine methylation using the general methylation inhibitor, adenosine-2,3-dialdehyde (AdOx) could restore the lost nuclear localization of both HA-tagged cytoplasmic ALS-causing mutants and cytoplasmic EWS and TAF15 point mutants in HeLa cells. As AdOx is capable of inhibiting protein, DNA and lipid methylation, they also investigated the effect of specifically preventing protein methylation on the localization of the severe FUS-P525L ALS mutant. In these experiments, siRNA-mediated silencing of PRMT1, the protein arginine methyltransferase responsible for the majority of cellular protein arginine dimethylation, successfully restored the nuclear localization of FUS-P525L, thus confirming the importance of arginine methylation in the cytoplasmic localization of ALS-FUS mutants. After performing this groundwork, Dormann and colleagues then began an elegant set of experiments to unravel the mechanism behind their results. They first wanted to identify whether TRN was involved. Co-expression of GFP-tagged TRN inhibitor peptide with HA-tagged FUS-P525L in HeLa cells lead to a complete prevention of the normal nuclear accumulation of FUS-P525L upon AdOx treatment. Thus, identifying a critical role for TRN in the observed nuclear import of ALS-FUS mutants upon demethylation.Dormann et al (2012) next sought to determine how arginine methylation may impact the nuclear import of FUS by TRN. Their first step was to determine which arginine residues in FUS were involved. With this came the ground-breaking finding that it is actually arginine residues in the RGG3 motif N-terminal to the PY-NLS (Figure 1), rather than in the PY-NLS itself that can modulate TRN-dependent nuclear import of mutant FUS. Through NMR spectroscopy, Dormann and colleagues were then able to show that arginine residues in the FUS RGG3 motif can bind directly to TRN. These observations were taken further via studies of the interaction of recombinant FUS-RGG3 domain or synthetic FUS peptides with TRN by isothermal titration calorimetry. Here, they showed that the FUS-P525L mutant bound weakly or not at all to TRN, but in the presence of the unmethylated RGG3 domain the binding affinity of the mutant was rescued to WT-like levels. These observations were validated by the fact that the unmethylated RGG3 domain alone was able to bind TRN in the absence of a C-terminal PY-NLS with an affinity similar to that of the WT PY-NLS. Methylation of the RGG3 peptide completely prevented this binding. Through these experiments Dormann et al (2012) were able to show for the first time that residues outside of the PY-NLS can be involved in FUS nuclear import. Furthermore, they were able to establish a working model by which this occurs: they propose that in normal situations, the PY-NLS anchors the FUS C-terminus to TRN and the adjacent RGG repeats stabilize the interaction. Methylation of the RGG repeats interferes with TRN binding, but in WT FUS the affinity of the PY-NLS for TRN is sufficient to allow nuclear import to continue. By contrast, in the methylated P525L mutant, weak binding of both the methylated RGG domain and the c-terminus of the PY-NLS to TRN abrogates FUS nuclear import, thus causing its cytoplasmic accumulation.On the basis of their model, Dormann et al (2010) predicted that cytoplasmically mislocalized ALS FUS mutants would be methylated in their RGG3 domains. To test this hypothesis, they generated two monoclonal antibodies specific to the methylated RGG3 domain (meFUS antibodies). Cytosolic FUS mutants, such as FUS-P525L, were recognized by the meFUS-specific antibodies in HeLa cells, thus strongly suggesting that methylation of FUS mutants is a contributing factor to their cytoplasmic retention.Having generated the valuable tools of meFUS-specific antibodies, Dormann et al (2010) next turned their attention to the analysis of whether the intriguing shared property of cytoplasmic FUS accumulation in ALS-FUS and FTLD-FUS could be related to arginine methylation. In direct support of their cell-culture experiments, they discovered that there was a strong and consistent co-labelling of all FUS-positive cytoplasmic neuronal and glial inclusions with the meFUS antibody in post-mortem tissue from ALS-FUS patients. However, the surprise came when they began analysing post-mortem tissues from various subtypes of FTLD-FUS patients. Here, they could not see any labelling of FUS-positive neuronal and glial cytoplasmic and intranuclear inclusions with the meFUS-specific antibody. This strongly suggests that a different FUS cytoplasmic retention mechanism is in play in FTLD-FUS patients compared to ALS-FUS patients. In support of this, inclusions in ALS-FUS patients have recently been identified to contain only the FUS protein, while inclusions in FTLD-FUS show a clear co-deposition of all FET proteins (FUS, EWS, and TAF15) along with TRN itself (Neumann et al, 2012). From this, Dormann et al (2012) conclude that mislocalization of FUS in ALS is likely a phenomenon resulting directly from mutations in the nuclear localization signal that is exacerbated by arginine methylation in the RGG3 domain, whereas FUS mislocalization in FTLD-FUS may result from a more general defect in TRN-mediated nuclear import of FET proteins (Figure 1). Indeed, they speculate that in FTLD, hypomethylation of the FET proteins by a yet to be determined mechanism may lead to excessively tight binding of these proteins to TRN. This in turn may lead to impaired dissociation of FET-TRN complexes, which could over time lead to the co-deposition of FET proteins and TRN in cytoplasmic and nuclear inclusions in FTLD patients. The results of future experiments performed to test this hypothesis will doubtless be of huge significance for the FTLD field. Furthermore, this study raises many questions about the normal cellular role of FUS arginine methylation. Is it for example involved in the fine tuning of the shuttling of FUS into and out of the nucleus and if this is the case could arginine methylation in the RGG1 and RGG2 domains, one of which (RGG1) is close to the FUS nuclear export signal, also be involved in regulating FUS localization? All this remains to be seen…     

Nucleocytoplasmic shuttling of the West Nile virus RNA‐dependent RNA polymerase NS5 is critical to infection

West Nile virus (WNV) is a single‐stranded, positive sense RNA virus of the family Flaviviridae and is a significant pathogen of global medical importance. Flavivirus replication is known to be exclusively cytoplasmic, but we show here for the first time that access to the nucleus of the WNV strain Kunjin (WNV_KUN) RNA‐dependent RNA polymerase (protein NS5) is central to WNV_KUN virus production. We show that treatment of cells with the specific nuclear export inhibitor leptomycin B (LMB) results in increased NS5 nuclear accumulation in WNV_KUN‐infected cells and NS5‐transfected cells, indicative of nucleocytoplasmic shuttling under normal conditions. We used site‐directed mutagenesis to identify the nuclear localisation sequence (NLS) responsible for WNV_KUN NS5 nuclear targeting, observing that mutation of this NLS resulted in exclusively cytoplasmic accumulation of NS5 even in the presence of leptomycin B. Introduction of NS5 NLS mutations into FLSDX, an infectious clone of WNV_KUN, resulted in lethality, suggesting that the ability of NS5 to traffic into the nucleus in integral to WNV_KUN replication. This study thus shows for the first time that NLS‐dependent trafficking into the nucleus during infection of WNV_KUN NS5 is critical for viral replication. Excitingly, specific inhibitors of NS5 nuclear import reduce WNV_KUN virus production, proving the principle that inhibition of WNV_KUN NS5 nuclear import is a viable therapeutic avenue for antiviral drug development in the future.     

Characterization of an Importin α/β‐recognized nuclear localization signal in β‐dystroglycan

β‐dystroglycan (β‐DG) is a widely expressed transmembrane protein that plays important roles in connecting the extracellular matrix to the cytoskeleton, and thereby contributing to plasma membrane integrity and signal transduction. We previously observed nuclear localization of β‐DG in cultured cell lines, implying the existence of a nuclear targeting mechanism that directs it to the nucleus instead of the plasma membrane. In this study, we delineate the nuclear import pathway of β‐DG, characterizing a functional nuclear localization signal (NLS) in the β‐DG cytoplasmic domain, within amino acids 776–782. The NLS either alone or in the context of the whole β‐DG protein was able to target the heterologous GFP protein to the nucleus, with site‐directed mutagenesis indicating that amino acids R⁷⁷⁹ and K⁷⁸⁰ are critical for NLS functionality. The nuclear transport molecules Importin (Imp)α and Impβ bound with high affinity to the NLS of β‐DG and were found to be essential for NLS‐dependent nuclear import in an in vitro reconstituted nuclear transport assay; cotransfection experiments confirmed the dependence on Ran for nuclear accumulation. Intriguingly, experiments suggested that tyrosine phosphorylation of β‐DG may result in cytoplasmic retention, with Y⁸⁹² playing a key role. β‐DG thus follows a conventional Impα/β‐dependent nuclear import pathway, with important implications for its potential function in the nucleus. J. Cell. Biochem. 110: 706–717, 2010. © 2010 Wiley‐Liss, Inc.     

Molecular and behavioural abnormalities in the FUS‐tg mice mimic frontotemporal lobar degeneration: Effects of old and new anti‐inflammatory therapies

Genetic mutations in FUS, a DNA/RNA‐binding protein, are associated with inherited forms of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). A novel transgenic FUS[1‐359]‐tg mouse line recapitulates core hallmarks of human ALS in the spinal cord, including neuroinflammation and neurodegeneration, ensuing muscle atrophy and paralysis, as well as brain pathomorphological signs of FTLD. However, a question whether FUS[1‐359]‐tg mouse displays behavioural and brain pro‐inflammatory changes characteristic for the FTLD syndrome was not addressed. Here, we studied emotional, social and cognitive behaviours, brain markers of inflammation and plasticity of pre‐symptomatic FUS[1‐359]‐tg male mice, a potential FTLD model. These animals displayed aberrant behaviours and altered brain expression of inflammatory markers and related pathways that are reminiscent to the FTLD‐like syndrome. FTLD‐related behavioural and molecular Journal of Cellular and Molecular Medicine features were studied in the pre‐symptomatic FUS[1‐359]‐tg mice that received standard or new ALS treatments, which have been reported to counteract the ALS‐like syndrome in the mutants. We used anti‐ALS drug riluzole (8 mg/kg/d), or anti‐inflammatory drug, a selective blocker of cyclooxygenase‐2 (celecoxib, 30 mg/kg/d) for 3 weeks, or a single intracerebroventricular (i.c.v.) infusion of human stem cells (Neuro‐Cells, 500 000‐CD34⁺), which showed anti‐inflammatory properties. Signs of elevated anxiety, depressive‐like behaviour, cognitive deficits and abnormal social behaviour were less marked in FUS‐tg–treated animals. Applied treatments have normalized protein expression of interleukin‐1β (IL‐1β) in the prefrontal cortex and the hippocampus, and of Iba‐1 and GSK‐3β in the hippocampus. Thus, the pre‐symptomatic FUS[1‐359]‐tg mice demonstrate FTLD‐like abnormalities that are attenuated by standard and new ALS treatments, including Neuro‐Cell preparation.     

Computational identification of post‐translational modification‐based nuclear import regulations by characterizing nuclear localization signal‐import receptor interaction

The binding affinity between a nuclear localization signal (NLS) and its import receptor is closely related to corresponding nuclear import activity. PTM‐based modulation of the NLS binding affinity to the import receptor is one of the most understood mechanisms to regulate nuclear import of proteins. However, identification of such regulation mechanisms is challenging due to the difficulty of assessing the impact of PTM on corresponding nuclear import activities. In this study we proposed NIpredict, an effective algorithm to predict nuclear import activity given its NLS, in which molecular interaction energy components (MIECs) were used to characterize the NLS‐import receptor interaction, and the support vector regression machine (SVR) was used to learn the relationship between the characterized NLS‐import receptor interaction and the corresponding nuclear import activity. Our experiments showed that nuclear import activity change due to NLS change could be accurately predicted by the NIpredict algorithm. Based on NIpredict, we developed a systematic framework to identify potential PTM‐based nuclear import regulations for human and yeast nuclear proteins. Application of this approach has identified the potential nuclear import regulation mechanisms by phosphorylation of two nuclear proteins including SF1 and ORC6. Proteins 2014; 82:2783–2796. © 2014 Wiley Periodicals, Inc.     

Treatment with a Global Methyltransferase Inhibitor Induces the Intranuclear Aggregation of ALS-Linked FUS Mutant In Vitro

FUS/TLS (fused in sarcoma/translocated in liposarcoma) encodes a multifunctional DNA/RNA binding protein with non-classical carboxy (C)-terminal nuclear localization signal (NLS). A variety of ALS-linked mutations are clustered in the C-terminal NLS, resulting in the cytoplasmic mislocalization and aggregation. Since the arginine methylations are implicated in the nuclear-cytoplasmic shuttling of FUS, a methylation inhibitor could be one of therapeutic targets for FUS-linked ALS. We here examined effects of methylation inhibitors on the cytoplasmic mislocalization and aggregates of ALS-linked C-terminal FUS mutant in a cell culture system. Treatment with adenosine dialdehyde (AdOx), a representative global methyltransferase inhibitor, remarkably mitigated the cytoplasmic mislocalization and aggregation of FUS mutant, which is consistent with previous reports. However, AdOx treatment of higher concentration and longer time period evoked the intranuclear aggregation of the ectopic expressed FUS protein. The pull down assay and the morphological analysis indicated the binding between FUS and Transportin could be potentiated by AdOx treatment through modulating methylation status in RGG domains of FUS. These findings indicated the treatment with a methylation inhibitor at the appropriate levels could alleviate the cytoplasmic mislocalization but in excess this could cause the intranuclear aggregation of FUS C-terminal mutant.

     

Identification of a Karyopherin β1/β2 Proline-Tyrosine Nuclear Localization Signal in Huntingtin Protein

Among the known pathways of protein nuclear import, the karyopherin β2/transportin pathway is only the second to have a defined nuclear localization signal (NLS) consensus. Huntingtin, a 350-kDa protein, has defined roles in the nucleus, as well as a CRM1/exportin-dependent nuclear export signal; however, the NLS and exact pathway of import have remained elusive. Here, using a live cell assay and affinity chromatography, we show that huntingtin has a karyopherin β2-dependent proline-tyrosine (PY)-NLS in the amino terminus of the protein. This NLS comprises three consensus components: a basic charged sequence, a downstream conserved arginine, and a PY sequence. Unlike the classic PY-NLS, which has an unstructured intervening sequence between the consensus components, we show that a β sheet structured region separating the consensus elements is critical for huntingtin NLS function. The huntingtin PY-NLS is also capable of import through the importin/karyopherin β1 pathway but was not functional in all cell types tested. We propose that this huntingtin PY-NLS may comprise a new class of multiple import factor-dependent NLSs with an internal structural component that may regulate NLS activity.     

Hijacking of the nucleolar protein fibrillarin by TGB1 is required for cell‐to‐cell movement of Barley stripe mosaic virus

Barley stripe mosaic virus (BSMV) Triple Gene Block1 (TGB1) is a multifunctional movement protein with RNA‐binding, ATPase and helicase activities which mainly localizes to the plasmodesmata (PD) in infected cells. Here, we show that TGB1 localizes to the nucleus and the nucleolus, as well as the cytoplasm, and that TGB1 nuclear‐cytoplasmic trafficking is required for BSMV cell‐to‐cell movement. Prediction analyses and laser scanning confocal microscopy (LSCM) experiments verified that TGB1 possesses a nucleolar localization signal (NoLS) (amino acids 95–104) and a nuclear localization signal (NLS) (amino acids 227–238). NoLS mutations reduced BSMV cell‐to‐cell movement significantly, whereas NLS mutations almost completely abolished movement. Furthermore, neither the NoLS nor NLS mutant viruses could infect Nicotiana benthamiana systemically, although the NoLS mutant virus was able to establish systemic infections of barley. Protein interaction experiments demonstrated that TGB1 interacts directly with the glycine–arginine‐rich (GAR) domain of the nucleolar protein fibrillarin (Fib2). Moreover, in BSMV‐infected cells, Fib2 accumulation increased by about 60%–70% and co‐localized with TGB1 in the plasmodesmata. In addition, BSMV cell‐to‐cell movement in fib2 knockdown transgenic plants was reduced to less than one‐third of that of non‐transgenic plants. Fib2 also co‐localized with both TGB1 and BSMV RNA, which are the main components of the ribonucleoprotein (RNP) movement complex. Collectively, these results show that TGB1–Fib2 interactions play a direct role in cell‐to‐cell movement, and we propose that Fib2 is hijacked by BSMV TGB1 to form a BSMV RNP which functions in cell‐to‐cell movement.     

The FUS gene is dual‐coding with both proteins contributing to FUS‐mediated toxicity

Novel functional coding sequences (altORFs) are camouflaged within annotated ones (CDS) in a different reading frame. We show here that an altORF is nested in the FUS CDS, encoding a conserved 170 amino acid protein, altFUS. AltFUS is endogenously expressed in human tissues, notably in the motor cortex and motor neurons. Over‐expression of wild‐type FUS and/or amyotrophic lateral sclerosis‐linked FUS mutants is known to trigger toxic mechanisms in different models. These include inhibition of autophagy, loss of mitochondrial potential and accumulation of cytoplasmic aggregates. We find that altFUS, not FUS, is responsible for the inhibition of autophagy, and pivotal in mitochondrial potential loss and accumulation of cytoplasmic aggregates. Suppression of altFUS expression in a Drosophila model of FUS‐related toxicity protects against neurodegeneration. Some mutations found in ALS patients are overlooked because of their synonymous effect on the FUS protein. Yet, we show they exert a deleterious effect causing missense mutations in the overlapping altFUS protein. These findings demonstrate that FUS is a bicistronic gene and suggests that both proteins, FUS and altFUS, cooperate in toxic mechanisms.     

Recruitment into stress granules prevents irreversible aggregation of FUS protein mislocalized to the cytoplasm

Fused in sarcoma (FUS) belongs to the group of RNA-binding proteins implicated as underlying factors in amyotrophic lateral sclerosis (ALS) and certain other neurodegenerative diseases. Multiple FUS gene mutations have been linked to hereditary forms, and aggregation of FUS protein is believed to play an important role in pathogenesis of these diseases. In cultured cells, FUS variants with disease-associated amino acid substitutions or short deletions affecting nuclear localization signal (NLS) and causing cytoplasmic mislocalization can be sequestered into stress granules (SGs). We demonstrated that disruption of motifs responsible for RNA recognition and binding not only prevents SG recruitment, but also dramatically increases the protein propensity to aggregate in the cell cytoplasm with formation of juxtanuclear structures displaying typical features of aggresomes. Functional RNA-binding domains from TAR DNA-binding protein of 43 kDa (TDP-43) fused to highly aggregation-prone C-terminally truncated FUS protein restored the ability to enter SGs and prevented aggregation of the chimeric protein. Truncated FUS was also able to trap endogenous FUS molecules in the cytoplasmic aggregates. Our data indicate that RNA binding and recruitment to SGs protect cytoplasmic FUS from aggregation, and loss of this protection may trigger its pathological aggregation in vivo.     

The Effect of PRMT1-Mediated Arginine Methylation on the Subcellular Localization,Stress Granules,and Detergent-Insoluble Aggregates of FUS/TLS

Fused in sarcoma/translocated in liposarcoma (FUS/TLS) is one of causative genes for familial amyotrophic lateral sclerosis (ALS). In order to identify binding partners for FUS/TLS, we performed a yeast two-hybrid screening and found that protein arginine methyltransferase 1 (PRMT1) is one of binding partners primarily in the nucleus. In vitro and in vivo methylation assays showed that FUS/TLS could be methylated by PRMT1. The modulation of arginine methylation levels by a general methyltransferase inhibitor or conditional over-expression of PRMT1 altered slightly the nucleus-cytoplasmic ratio of FUS/TLS in cell fractionation assays. Although co-localized primarily in the nucleus in normal condition, FUS/TLS and PRMT1 were partially recruited to the cytoplasmic granules under oxidative stress, which were merged with stress granules (SGs) markers in SH-SY5Y cell. C-terminal truncated form of FUS/TLS (FUS-dC), which lacks C-terminal nuclear localization signal (NLS), formed cytoplasmic inclusions like ALS-linked FUS mutants and was partially co-localized with PRMT1. Furthermore, conditional over-expression of PRMT1 reduced the FUS-dC-mediated SGs formation and the detergent-insoluble aggregates in HEK293 cells. These findings indicate that PRMT1-mediated arginine methylation could be implicated in the nucleus-cytoplasmic shuttling of FUS/TLS and in the SGs formation and the detergent-insoluble inclusions of ALS-linked FUS/TLS mutants.