A Yeast Model of FUS/TLS-Dependent Cytotoxicity
[["Amyotrophic lateral sclerosis ( ALS , also called Lou Gehrig's disease after one", 'of its most famous victims ) is a relentlessly progressive , fatal neurodegenerative', 'disease with a prevalence of ∼5 people out of 100 , 000 each year and an average', 'age of onset of ∼60 years .', 'Patients with ALS suffer from degeneration of motor', 'neurons in the brain and spinal cord , which leads to progressive muscular weakness .', 'ALS accounts for ∼1/300 to 1/400 of all deaths , which means that about 1 , 000 , 000', 'people now alive in the United States will develop ALS .', 'Death typically occurs', '3–5 years after disease onset , due to respiratory paralysis .', 'There is no', 'effective treatment for the disease; the only approved ALS drug ( riluzole ) extends', 'the lifespan of some ALS patients by only about 3 months .', 'While most forms of ALS are sporadic and idiopathic ( sALS ) , ∼10% of cases', 'are inherited in a Mendelian fashion and are designated familial ALS ( fALS ) .', 'As is', "the case for Parkinson's and Alzheimer's diseases , which also have", '∼10% familial forms , genetic analysis has identified several genes that', 'cause fALS .', 'The first mutations were identified in SOD1 , which', 'encodes the ubiquitously expressed copper/zinc superoxide dismutase; these variants', 'cause ∼20% of fALS worldwide .', 'More than 150 different ALS mutations ,', 'spanning virtually the entire coding sequence of the highly conserved', 'SOD1 gene , have been identified—nearly all of them', 'exhibiting autosomal dominant inheritance [1] .', 'Although inclusions containing', 'aggregated SOD1 protein have been found in the spinal motor neurons of patients with', 'SOD1-dependent fALS , they are generally not found in the sporadic disease .', 'More recently , other genes have been identified that collectively account for a', 'significant percentage of the remaining fALS cases .', 'These include the genes coding', 'for alsin ( ALS2 ) , vesicle associated membrane protein B ( VAPB ) [2] , senataxin ( SETX ) [3] , TAR-DNA-binding', 'protein ( TDP-43 ) [4] , fused in sarcoma or translocated in liposarcoma', '( FUS/TLS ) [5] , [6] , and optineurin ( OPTN ) [7] .', 'A small number of other genes', 'have been associated with increased risk for sALS , most recently ataxin-2 [8] .', 'Studies of these', 'genes have provided important information about the biochemical processes that may', 'underlie ALS .', 'Putative mechanisms of toxicity targeting motor neurons include', 'glutamate excitotoxicity , oxidative damage , proteasome inhibition , mitochondrial', 'dysfunction , ER stress , axonal transport defects , growth factor signaling', 'deficiency , and glial cell dysfunction [9] , [10] .', 'Two of the genes associated with fALS , FUS/TLS and TDP-43 , are of special interest', 'because inclusions containing these proteins have been identified in motor neurons', 'of both sporadic and familial patients [11]–[15] .', 'In addition , both of these', 'genes have been linked to rare forms of frontotemporal lobar degeneration [16] , indicating', 'that they play crucial roles in other neurons .', 'FUS/TLS and TDP-43 are both', 'predominantly nuclear RNA binding proteins , although they have also been reported to', 'bind DNA in vitro .', 'Both FUS/TLS and TDP-43 are believed to carry out important', 'functions in multiple steps of RNA processing , including transcription , splicing ,', 'transport , translation , and decay [17] .', 'The finding that both', 'are fALS genes ( each accounts for about 5% of familial ALS cases ) , and are', 'involved in sALS , raises the possibility that RNA processing or quality control', '( damage repair and decay of prematurely terminated messages ) may be central to ALS', 'pathology .', 'However , the precise connections between RNA biology and ALS remain to be', 'discovered .', 'The FUS/TLS protein , which is ubiquitously expressed in all tissues , contains an', 'N-terminal putative transcriptional activation domain ( residues 1–267 ) rich in', 'serine , tyrosine , glutamine , and glycine residues , followed by a canonical RNA', 'binding domain ( residues 285–371 ) .', 'The C-terminal region has a zinc finger', 'domain ( residues 422–453 ) interrupting a long stretch rich in arginines and', 'glycines ( residues 285–501 ) .', 'The extreme C-terminal 25 amino acids ( residues', '501–526 ) also are rich in arginines and glycines , and the majority of the', 'ALS-associated mutations are found among them .', 'Among other postulated functions ,', 'FUS/TLS is known to be a component of a large nuclear ribonucleoprotein complex that', 'functions in shuttling mRNA out of the nucleus .', 'Variants of FUS/TLS have previously been studied for their role in liposarcoma , in', 'which the N-terminal transcriptional activation domain of FUS/TLS is translocated', 'into another chromosomal locus , resulting in gene fusions and production of chimeric', 'oncoproteins ( e . g . FUS-ERG , FUS-CHOP , and FUS-CREB312 ) .', 'The fusion proteins are', 'aberrant transcription factors that contribute to the tumorigenic process by', 'altering the expression of many target genes [18] .', 'Mutations in FUS/TLS found in fALS are largely clustered at the extreme C-terminus of', 'the protein .', 'Postmortem histological analysis from patients with FUS/TLS mutations', 'indicates that the normally nuclear protein is now found more predominantly in the', 'cytosol , where it forms punctate inclusions .', 'This', 'mislocalization/inclusion-formation has been proposed to cause either a loss of', 'normal protein function in the nucleus , a gain of toxic function in the cytosol , or', 'both [5] , [17] .', 'Recently , Dormann et al . [19] reported that some of the', 'disease-causing mutations affect a non-classical PY nuclear localization signal', '( NLS ) in the extreme C-terminus of FUS/TLS and disrupt transportin-mediated nuclear', 'import of the protein .', 'As a result , FUS/TLS distribution increases in the cytosol ,', 'where the protein can be recruited into stress granules [20] .', 'These results have led to the', 'hypothesis that nuclear import defects and consequent cellular stress may be', 'necessary , and possibly sufficient , for FUS/TLS pathogenesis [19] , [20] .', 'Since FUS/TLS-immunoreactive inclusions are reported to be a common feature in both', 'sporadic and familial ALS [13] , it is likely that an understanding of FUS/TLS-associated', 'fALS could also provide valuable information about the more common sporadic form of', 'the disease .', 'Additionally , the involvement of FUS/TLS in other neurodegenerative', 'diseases , such as a subset of FTLD ( atypical FTLD-U ) [21] , neuronal intermediate', 'filament inclusion disease ( NIFID ) [22] , and polyglutamine disease [23] , suggests that several', 'neurodegenerative diseases may have similar underlying pathogenic mechanisms .', 'A', 'better understanding of the normal and aberrant functions of FUS/TLS might therefore', 'provide clues to uncovering the pathology of neurodegenerative diseases beyond ALS .', 'With this in mind , we set out to create a model for FUS/TLS-dependent cytotoxicity', 'in a genetically and biochemically tractable organism .', 'With uniquely available genetic and biochemical tools , yeast has proven to be a', 'valuable system to study the functions of human proteins involved in many diseases ,', 'including neurodegenerative disorders [24]–[29] .', 'Although yeast is a simple', 'single-cell eukaryote , many fundamental cellular processes are conserved between', 'yeast and higher eukaryotes , and a number were first discovered in S .', 'cerevisiae or its distant relative , S . pombe .', 'If', 'expression of FUS/TLS in yeast can be shown to recapitulate some of the relevant', 'features of human FUS/TLS-dependent proteotoxicity , then genetic screens can be used', 'to dissect the pathways and processes involved .', 'In this article , we report a yeast model of FUS/TLS-dependent cytotoxicity , in which', 'over-expression and mislocalization of wild-type or mutant FUS/TLS recapitulates the', 'phenotypes of toxicity and inclusion formation observed in the human disease .', 'Certain features of the model have allowed us to conclude that cytosolic', 'localization of large amounts of even the wild-type FUS protein is sufficient to', 'cause toxicity , supporting a hypothesis that had been put forward based on studies', 'in mammalian cells .', 'A genetic screen using this yeast model for suppressors of', 'toxicity identifies , among other genes , the yeast gene ECM32 , an', 'RNA helicase involved in RNA quality control , as one that rescues FUS/TLS toxicity', 'when over-expressed .', 'In addition , hUPF1 , a human homolog of', 'ECM32 , rescues FUS/TLS toxicity , as do its interacting partners', 'hUPF2 and ( to a lesser extent ) hUPF3 .', 'The', 'rescue does not involve a decrease in FUS/TLS expression or a change in its', 'localization or inclusion formation , but it does depend on intact functional domains', 'of hUPF1 .', 'Since hUPF1 plays an important function', 'in mRNA quality control , our data raise the possibility that this pathway might be', 'involved in the pathogenesis of FUS/TLS-associated ALS , and', 'possibly of the disease in general .', 'We have investigated the role of one aspect of', 'RNA quality control , nonsense-mediated decay ( NMD ) , using the yeast model but find', 'no evidence that NMD disruption is responsible for FUS/TLS toxicity or that its', 'upregulation is important for suppression .', 'Independently , Sun et al . ( 2010 , [30] ) have developed a similar yeast model for', 'FUS/TLS-associated ALS and have identified all of the same suppressor genes , plus', 'some additional ones , in a similar screen .', 'In addition , they have found genes that ,', 'when deleted , modulate FUS/TLS toxicity in yeast , and have implicated stress', 'granules ( discrete cytoplasmic phase-dense particles , observed in cells exposed to', 'heat , oxidative , hyperosmolarity , and UV stress , where non-translating mRNAs are', 'stored ) in this process .'], ['Unlike wild type FUS/TLS , which is largely found in the nucleus and somewhat', 'diffusely in the cytosol , the mutant proteins associated with fALS are', 'predominantly aggregated in the cytoplasm of neurons , where they are proposed to', 'be toxic .', 'To determine whether the budding yeast S . cerevisiae', 'may serve as a model for investigating the molecular mechanisms of FUS/TLS', 'cytotoxicity , we generated yeast strains expressing FUS/TLS .', 'The human', 'FUS/TLS gene , in both wild type and mutant ( R521G and', 'H517Q ) forms , was N-terminally fused to green fluorescent protein ( GFP ) and', 'placed under the control of the GAL1 promoter , whereby', 'expression is tightly controlled by switching the carbon source in the medium .', 'In these strains , expression of both WT and mutant FUS/TLS is highly induced by', 'shifting to galactose medium .', 'When either mutant or wild-type protein is', 'over-expressed ( induced by 2% galactose ) , the majority of the protein', 'forms punctate aggregates in the cytosol ( Figure 1A , and Figure S1 ) ,', 'recapitulating the nuclear to cytoplasmic translocation phenotype characteristic', 'of FUS/TLS-associated sporadic and familial ALS , as well as other', 'neurodegenerative diseases [5] .', 'Data from an independent yeast model of FUS/TLS', 'cytoxicity [30]', 'and expression of mutant forms of FUS/TLS in mammalian cells [20] suggest that', 'these aggregates could be localized to stress granules .', 'To test possible toxicity of aggregated FUS/TLS in the cytosol , one copy of', 'FUS/TLS ( 1XFUS; untagged ) and two copies of FUS/TLS ( 2XFUS; untagged ) were', 'integrated into the genome of yeast strain W303α .', 'Yeast strains with FUS/TLS', '( 1XFUS and 2XFUS ) were serially diluted and spotted onto plates with glucose', '( expression repressed ) and galactose ( expression induced ) .', 'As shown in Figure 1B , FUS/TLS', 'over-expression is toxic to yeast in a dose-dependent manner under these', 'proliferative growth conditions , with the 2XFUS exhibiting greater toxicity than', '1XFUS .', 'Toxicity of the over-expressed mutant proteins was comparable to that of', 'the wild-type ( Figure S1;', 'see below for an explanation ) , so the wild-type protein was used for most of the', 'remaining experiments , to avoid possible sequence-dependent peculiarities .', 'In yeast models for several other protein misfolding diseases , although the', 'morphologies of the aggregated proteins under the fluorescence microscope look', 'similar , the actual characteristics of the aggregates are sometimes quite', 'different .', 'For example , yeast toxicity and aggregation of the human', "Huntingon's disease–associated protein huntingtin harboring a", 'pathogenic polyglutamine expansion ( Htt103Q ) can be rescued by the deletion of a', 'heat shock protein HSP104 ( hsp104Δ ) or', 'yeast prion protein RNQ1 ( rnq1Δ ) .', 'However , the same', 'deletions cannot rescue the cytosolic aggregation and toxicity of TDP-43 in', 'yeast [28] .', 'Previous studies also indicate that Htt103Q forms SDS-insoluble aggregates ,', 'which cannot pass through a 0 . 2 µM cellulose acetate membrane; however ,', 'TDP43 aggregates can pass freely [28] .', 'FUS/TLS has been found to be associated with huntingtin aggregates in', "Huntington's disease patients [23] , yet the characteristics of FUS/TLS-dependent fALS", 'resemble those of TDP-43-associated ALS .', 'To test possible differences between', 'FUS/TLS aggregates and huntingtin or TDP-43 , FUS/TLS aggregates isolated from', 'over-expressing yeast were tested by a filter retardation assay .', 'As expected ,', 'Htt103Q was trapped by the membrane; however , FUS/TLS aggregates , like TDP43 ,', 'passed through the membrane freely ( Figure 2A ) .', 'Consistent with this observation , unlike their effects', 'on Htt103Q , deletion of HSP104 and RNQ1 did', 'not modify the toxicity of FUS/TLS in yeast ( Figure 2B ) , nor its aggregation or', 'localization ( Figure 2C ) .', 'In', 'addition , over-expression of HSP104 and RNQ1', 'had no effect on FUS/TLS toxicity either ( unpublished data ) .', 'These differences between FUS/TLS and Htt103Q aggregates suggest that the', 'toxicity mechanism underlying these two proteins might be different .', 'Consistent', 'with this , toxicity resulting from the over-expression of FUS/TLS and Htt103Q is', 'additive ( Figure 2D ) .', 'The full-length FUS/TLS protein has an N-terminal transcriptional activation', 'domain ( residues 1–267 ) including SYQG-rich ( residues 1–164 ) and', 'G-rich ( residues 165–267 ) subdomains , and a C-terminal RNA binding region', '( residues 285–526 ) including RNA binding ( residues 285–370 ) , Zinc', 'Finger ( residues 422–452 ) , and RGG-rich domains ( residues 371–421', 'and 453–501 ) ( Figure', '3A ) .', 'The extreme C-terminus ( residues 511–526 ) contains an', 'arginine-rich sequence and a putative nuclear localization signal [19]; every one', 'of the arginines in this region is the site of an ALS-associated mutation .', 'To', 'pinpoint which part of FUS/TLS is required for its toxicity in yeast , we', 'characterized eight fragments of the wild-type FUS/TLS protein ( tagged with GFP', 'at the N-terminus ) for their localization , aggregation , and toxicity .', 'As shown', 'in Figure 3B , after removal', 'of the C-terminal region of the protein ( constructs 1 [residues', '1–164] , 2 [residues 1–267] , and 3 [residues', '1–370] ) , the protein is no longer toxic , suggesting that the', 'C-terminal domain from residues 371 on is required for toxicity .', 'However ,', 'expression of the C-terminal domain only ( constructs 6 [residues', '165–526] , 7 [residues 268–526] , and 8 [residues', '371–526] ) is not toxic , indicating that both C- and N-terminal', 'regions of the protein are essential for toxicity .', 'To check for a possible correlation between toxicity and aggregation , all eight', 'proteins ( N-terminally GFP tagged ) were checked using fluorescence microscopy .', 'As shown in Figure 3C , all', 'the proteins , when over-expressed , show aggregation; however , only constructs 4', '( which lacks the extreme C-terminal 15 residues ) and 5 ( the full-length protein )', 'are toxic .', 'Interestingly , the aggregates formed by constructs 4 and 5 appear', 'slightly different from each other and also may differ from the aggregates', 'formed by the other constructs .', 'These data suggest that toxicity of FUS/TLS in', 'yeast involves mechanisms beyond protein aggregation , a hypothesis supported by', 'the results of the suppressor screen ( see below ) .', 'To test the possible effects of the GFP tag on toxicity , we expressed constructs', '3–5 without the N-terminal GFP fusion .', 'Consistent with non-tagged', 'constructs , construct 3 , which lacks the residues from 371 to 526 , is not toxic ,', 'and constructs 4 and 5 are still toxic .', 'Interestingly , the construct lacking the', '15 amino acids at the extreme C-terminal end ( construct 4 ) , where most of the', 'fALS mutations are clustered , is more toxic to yeast than the full-length', 'protein ( construct 5 ) .', 'This result is consistent with the recent finding that', 'patients with a nonsense mutation of FUS/TLS at position 495 have more rapidly', 'progressive neurodegeneration and earlier onset of the disease [20] , [31] and that', 'the C-terminus encodes a nuclear localization signal [19] .', 'Most mutations of FUS/TLS identified in fALS patients are clustered at the very', 'end of the C-terminus ( residues 510–526 ) , in a region enriched with', 'arginine residues [5] .', 'At least one disease-causing mutation has been', 'identified for each arginine in this region , implying that these arginine', 'residues play a critical role for the function of the protein .', 'One regulatory', 'process involving arginine residues is dimethylation , which is an important', 'signal for the nuclear/cytoplasmic translocalization of a series of RNA binding', 'proteins .', 'That more than 20 arginine residues are indeed dimethylated by the', 'enzyme PRMT1 in FUS/TLS in mammalian cells [32] , and that all the known', 'C-terminal region mutant forms of FUS/TLS do translocate to the cytosol ,', 'together suggest that arginine methylation may be involved in the shuttling of', 'FUS/TLS between nucleus and cytosol and thus may play a role in its toxicity .', 'To', 'explore the possible role of the yeast arginine methyl transferases in', 'mislocalization , localization and toxicity of FUS/TLS was studied in yeast', 'strains in which each of the two major yeast arginine methyltransferases were', 'deleted ( rmt1Δ and rmt2Δ ) .', 'As shown in', 'Figure 4 , FUS/TLS is', 'still toxic in rmt1Δ and rmt2Δ strains', '( Figure 4A ) , and the', 'protein is still aggregated in the cytosol in both cases ( Figure 4B ) .', 'To test the possible redundancy of arginine methyltransferase activity in yeast ,', 'two small molecule compounds ( AMI-1 and AMI-4 ) , previously shown to exhibit', 'broad inhibition of arginine methyl transferase activity in mammalian cells and', 'in yeast [33] ,', 'were tested on the yeast strain over-expressing FUS/TLS .', 'Consistent with the', 'results from the arginine methyl transferase deletion study , inhibition of', 'arginine methyl transferase by these two compounds does not change FUS/TLS', 'toxicity nor its localization ( unpublished data ) .', 'In addition , over-expression', 'of RMT1 and RMT2 , or of the human enzyme', 'PRMT1 , does not modify FUS/TLS toxicity ( unpublished data ) .', 'These data suggest', 'that at least the predominant yeast arginine methyl transferases are not', 'involved in the cytotoxicity of FUS/TLS in yeast .', 'We next examined the possible', 'role of nuclear localization signals in the nuclear/cytosolic distribution of', 'the human protein when expressed in yeast .', 'It was recently reported that FUS/TLS carries a non-classical PY nuclear', 'localization signal ( NLS ) in its extreme C-terminus ( approx .', 'residues', '514–526 ) and this is necessary for its nuclear import [19] .', 'The', 'disease-causing mutations clustered in this NLS affect the nuclear localization', 'of the protein .', 'The toxicity of the protein and the age of disease onset', "correlate with the protein's cytosolic mislocalization and aggregation", '[19] , [20] , [31] .', 'Since wild-type FUS was mostly aggregated in', 'punctate granules in the yeast cytosol , we posited that its NLS might not be', 'functional in this organism .', 'If so , over-expression of even the wild-type', 'protein would recapitulate the toxicity of the human mutants , as observed ,', 'through a failure in nuclear localization .', 'To test this directly , we compared', 'the ability of the FUS NLS to direct nuclear localization with a', 'well-characterized yeast NLS that uses a similar PY sequence ( from Hrp1 ) by', 'fusing them to GFP .', 'Indeed the FUS NLS was defective in nuclear localization', '( Figure 5A ) .', 'To determine if the failure in nuclear localization contributes to toxicity , we', 'tested two constructs , one in which the HRP1 NLS was simply appended to the', 'human protein ( FUS_plus ) and another in which the HRP1 NLS was used to replace', 'the FUS/TLS ( FUS_switch; Figure', '5B ) .', 'Both constructs dramatically increased nuclear localization of', 'FUS ( Figure 5B ) and both', 'reduced toxicity ( Figure', '5C ) .', 'Aggregation was also reduced when FUS was retargeted to the nucleus ,', 'but this is probably due to a generally lower level of FUS expression in these', 'constructs .', 'Toxicity was not completely ameliorated , likely because some', 'residual cytoplasmic FUS persisted even when augmented with the HRP1 NLS ( Figure 5B ) .', 'This relationship', 'between mislocalization and toxicity is consistent with data from neuronal cells', '[19] and', 'suggests that the wild-type protein is toxic in yeast because the nonfunctional', 'NLS mimics the mislocalization effect of the disease-producing mutations in', 'human cells .', 'The ability of yeast expressing human FUS/TLS to recapitulate several salient', 'features of disease prompted us to perform a genome-wide over-expression screen .', 'By identifying yeast genes that modify FUS/TLS toxicity , we hoped to identify', 'pathways or proteins that would illuminate pathogenic mechanisms .', 'We screened an', 'over-expression library containing a collection of yeast open reading frames ,', 'fully sequenced and placed under the control of a galactose-inducible promoter .', 'A total of 5 , 535 genes are covered in this library ( representing 95% of', 'the yeast genome ) .', 'We transformed each of the 5 , 535 genes into the yeast strain', 'expressing the moderately toxic one copy of FUS/TLS ( untagged FUS/TLS integrated', 'into the yeast genome at the HIS3 locus ) and selected for those', 'that suppress FUS/TLS toxicity upon over-expression .', 'All of the positive', 'over-expression plasmids were retransformed into a fresh yeast strain and', 'validated for their suppressive effects .', 'Surprisingly , after three rounds of', 'retesting , only a handful of yeast genes could suppress FUS/TLS toxicity , all of', 'which are listed in the Table', '1 .', 'FUS expression is very toxic in yeast , and the identified', 'suppressors do not completely abolish the effect of FUS on yeast growth .', 'As', 'observed for a similar screen in a yeast alpha-synuclein toxicity model [26] , several', 'transcription factors that down-regulate the GAL1 promoter activity also', 'suppress the toxicity of FUS/TLS ( Table 1 , bottom section ) .', 'These modifiers were not specific to', 'FUS/TLS; they also suppress other toxic proteins expressed under GAL1 promoter', 'control , such as TDP-43 .', 'All the FUS/TLS-specific suppressors are DNA/RNA binding proteins ( Table 1 , top section; and', 'Figure 6A ) , including', 'ECM32 , SBP1 , SKO1 , and', 'VHR1 .', 'As shown in Figure 6B , FUS/TLS protein level was not', 'altered by over-expression of these four genetic modifiers , supporting the', 'hypothesis that the rescue is not mediated by reducing the amount of', 'FUS/TLS .', 'ECM32 ( also called MTT1 ) encodes a', 'DNA-dependent ATPase/DNA helicase belonging to the Dna2p- and Nam7p-like family', 'of helicases that are involved in modulating translation termination [34] .', '( Interestingly , we also detected NAM8 , an RNA binding protein', 'that interacts genetically with NAM7 in yeast , as a suppressor', 'of FUS toxicity .', 'We include it on our list , but we are uncertain of its', 'specificity; it appears as a suppressor in many screens and may cause some', 'nonspecific downregulation of the GAL1 promoter .', 'Yeast NAM7 ,', 'which is sometimes called yeast UPF1 , is a homologue of', 'ECM32 , but neither we nor Sun et al . [30] found NAM7 as', 'a strong suppressor in our screen .', ') Over-expression of ECM32 is known to induce', 'a nonsense suppression phenotype in a wild-type yeast strain , and the ECM32 gene', 'product has been shown to interact with translation termination factors and is', 'localized to polysomes [34] .', 'ECM32 is homologous to the human gene hUPF1 , which', 'encodes a protein previously shown to function in both mRNA turnover and', 'translation termination , and which can be found in P-bodies , cytoplasmic', 'granules that are sites of mRNA sequestration and turnover , including', 'nonsense-mediated decay ( NMD ) .', 'SBP1 encodes a putative RNA binding protein that is involved in translational', 'repression and is also found in cytoplasmic P-bodies [35] .', 'SKO1 encodes a basic leucine zipper transcription factor of the ATF/CREB family ,', 'which forms a complex with Tup1p and Ssn6p that acts as a repressor of', 'transcription .', 'In response to osmotic and oxidative stress , this complex can be', 'converted into an activator that recruits SAGA and SWI/SNF [36] .', 'VHR1 is a transcriptional activator that is required for the vitamin', 'H–responsive element ( VHRE ) mediated induction of VHT1 ( Vitamin H', 'transporter ) and BIO5 ( biotin biosynthesis intermediate transporter ) in response', 'to low biotin concentrations [37] .', 'In humans , biotin deficiency leads to a variety of', 'clinical abnormalities , including neurological disorders , growth retardation ,', 'and dermal abnormalities [38] .', 'All of these genes were identified by Sun et al . in an independent screen for', 'suppressors in a similar yeast model for FUS/TLS-dependent proteotoxicity [30] .', 'Differences', 'between results of the two screens probably reflect differences in the protocol', 'of the initial pass , plus differences in stringency in retesting .', 'Of the genetic modifiers identified from our screen , ECM32 is', 'the only gene that is capable of rescuing toxicity of yeast strains integrated', 'with both one copy ( Figure', '7A ) and two copies of FUS ( Figure 7B ) .', 'The other suppressors only rescue', 'toxicity of 1XFUS ( unpublished data ) .', 'We therefore turned our attention to the', 'human homologues of this protein .', 'Based on sequence similarity ( ∼30% identity and ∼50%', 'similarity in the helicase domain ) , hUPF1 , a gene playing an', 'important role in the pathway of nonsense-mediated decay ( NMD ) , is the closest', 'human homolog of ECM32', '[39] .', 'Because of this homology , we hypothesized that the toxicity of FUS/TLS may be', 'rescued by yeast expression of hUPF1 .', 'The full-length', 'hUPF1 gene was cloned into a yeast expression vector and', 'tested on the toxicity of FUS/TLS .', 'Over-expression of hUPF1', 'rescues the toxicity of both 1XFUS and 2XFUS ( Figure 7A and B ) .', 'To check for possible', 'direct interaction between hUPF1 and FUS/TLS , we co-expressed red fluorescent', 'protein-tagged hUPF1 and GFP-tagged FUS/TLS .', 'hUPF1 is expressed mainly in the', 'cytosol in yeast but does not co-localize with FUS/TLS ( Figure 7C ) , suggesting the rescue effect by', 'hUPF1 might be indirect .', 'However , more data are needed to rule out possible', 'over-expression artifacts .', 'Next , we investigated whether the expression of hUPF2 , another', 'nonsense-mediated decay pathway gene whose protein product is known to form a', 'complex with the UPF1 protein , might also rescue FUS toxicity in yeast , and', 'found that it did , to an equal extent as that of hUPF1 ( Figure 7 ) .', 'However ,', 'over-expression of human UPF3 , another protein known to', 'interact with UPF1 , showed only moderate rescue compared to', 'hUPF1 and hUPF2 ( suppression of toxicity', 'of 1XFUS but not of 2XFUS ) .', 'We then examined the expression of the next closest', 'human homologue of ECM32 , IGHMBP2', '( ∼25% identity ) , a ribosome-associated helicase implicated in DNA', 'replication , pre-mRNA splicing , and transcription [40] .', 'Mutations in', 'IGHMBP2 cause distal spinal muscular atrophy type 1 , a', 'neuromuscular disorder [40] .', 'However , expression of hIGHMBP2 in', 'yeast did not rescue FUS/TLS toxicity ( unpublished data ) .', 'To check the possible effect of ECM32 , hUPF1 ,', 'and hUPF2 on the protein expression level or aggregation of', 'FUS/TLS , Western blots and indirect immunofluorescence using FUS/TLS antibody', 'were performed .', 'Neither protein levels ( Figure 8A ) nor localization or aggregation of', 'FUS/TLS ( Figure 8B ) was', 'modified by over-expression of ECM32 , hUPF1 ,', 'or hUPF2 .', 'hUPF1 has an N-terminal hUPF2 binding domain and a C-terminal ATPase/Helicase', 'domain .', 'To determine whether the rescue of FUS/TLS toxicity requires both', 'domains , we tested the constructs expressing only the hUPF2 binding domain', '( hUPF1-418 ) or the ATPase/Helicase domain ( hUPF419-1118 ) .', 'As shown in Figure 8C , neither domain', 'rescues the toxicity of FUS/TLS to the extent that the full-length wild type', 'protein rescues .', 'To further test whether ATPase/Helicase activity is required', 'for the rescue , we checked the full-length protein with ATPase/Helicase', 'partially inactivated by a point mutation ( R844C ) [41] and found that FUS/TLS', 'toxicity cannot be fully rescued when ATPase/Helicase activity of hUPF1 is', 'inhibited .', 'It is noteworthy that hUPF1 ( R844C ) still has 60% of the', 'activity of wild-type hUPF1 [41] , so it is quite possible that fully inactivated hUPF1', 'would not rescue at all .', 'These data suggest that both domains of hUPF1 and', 'functional ATPase/Helicase activity are required for its rescue of FUS/TLS', 'toxicity .', 'To prevent the potential accumulation of deleterious nonsense fragments of', 'polypeptides in the cytoplasm , mRNAs that retain an intron containing an', 'in-frame nonsense codon are usually degraded by the nonsense-mediated decay', '( NMD ) pathway .', 'It is long established that CYH2 and', 'MER2 pre-mRNA are among the substrates of this pathway in', 'yeast .', 'These pre-mRNAs are accumulated 2- to 5-fold when the NMD pathway is', 'deficient [42] .', 'To', 'check the potential effects of FUS expression on the NMD pathway , qRT-PCR was', 'utilized to determine CYH2 and MER2 pre-mRNA', 'levels in 1XFUS yeast , and in its suppressor strains .', 'As shown in Figure 9A ,', 'CYH2 pre-mRNA was increased about 2-fold when FUS is', 'over-expressed , and co-expression of its suppressor hUPF1 brought', 'CYH2 pre-mRNA back to the wild type level; however ,', 'co-expression of another suppressor , ECM32 , did not , suggesting', 'that these two suppressors may rescue FUS toxicity through different', 'mechanisms .', 'In contrast to CYH2 , over-expression of FUS and co-expression of', 'its suppressors did not change MER2 pre-mRNA levels ( Figure 9B ) .', 'Since', 'MER2 pre-mRNA is another substrate of the NMD pathway , this', 'result implies that accumulation of CYH2 pre-mRNA by FUS', 'over-expression is not through its direct effect on the NMD pathway but through', 'an effect on one or more additional pathways of mRNA quality control .', 'Possibly ,', 'the restoration of the level of CYH2 pre-mRNA to normal by', 'hUPF1 expression in the 1XFUS strain also does not reflect hUPF1 function in', 'NMD .'], ['Many essential cellular functions are conserved in the simple eukaryote yeast .', 'Studies from this organism have provided valuable information for our understanding', 'of many critical cellular functions , including cell cycle regulation , DNA', 'replication , RNA synthesis and processing , protein synthesis , protein trafficking ,', 'and signal transduction .', 'This simple system has also been utilized to study', 'functions of proteins involved in human diseases , including neurodegenerative', 'diseases .', 'Although a yeast model usually cannot recapit
['Introduction', 'Results', 'Discussion', 'Materials and Methods']
['FUS/TLS is a nucleic acid binding protein that , when mutated , can cause a subset', 'of familial amyotrophic lateral sclerosis ( fALS ) .', 'Although FUS/TLS is normally', 'located predominantly in the nucleus , the pathogenic mutant forms of FUS/TLS', 'traffic to , and form inclusions in , the cytoplasm of affected spinal motor', 'neurons or glia .', 'Here we report a yeast model of human FUS/TLS expression that', 'recapitulates multiple salient features of the pathology of the disease-causing', 'mutant proteins , including nuclear to cytoplasmic translocation , inclusion', 'formation , and cytotoxicity .', 'Protein domain analysis indicates that the', 'carboxyl-terminus of FUS/TLS , where most of the ALS-associated mutations are', 'clustered , is required but not sufficient for the toxicity of the protein .', 'A', 'genome-wide genetic screen using a yeast over-expression library identified five', 'yeast DNA/RNA binding proteins , encoded by the yeast genes', 'ECM32 , NAM8 , SBP1 ,', 'SKO1 , and VHR1 , that rescue the toxicity', 'of human FUS/TLS without changing its expression level , cytoplasmic', 'translocation , or inclusion formation .', 'Furthermore , hUPF1 , a', 'human homologue of ECM32 , also rescues the toxicity of FUS/TLS', 'in this model , validating the yeast model and implicating a possible', 'insufficiency in RNA processing or the RNA quality control machinery in the', 'mechanism of FUS/TLS mediated toxicity .', 'Examination of the effect of FUS/TLS', 'expression on the decay of selected mRNAs in yeast indicates that the', 'nonsense-mediated decay pathway is probably not the major determinant of either', 'toxicity or suppression .']
['Of all the thousand natural shocks that flesh is heir to , one of the most', 'devastating is amyotrophic lateral sclerosis ( ALS ) , commonly known as Lou', "Gehrig's Disease .", 'This disorder , which comes in both inherited and random', 'forms , is characterized by degeneration of spinal motor neurons , leading to', 'paralysis and death .', 'The cause of the sporadic form is unknown , but new insight', 'has come from studying the genetic variations that lead to the rarer familial', 'forms .', 'One such gene , accounting for 5%–10% of inherited', 'ALS , is FUS/TLS , which encodes a protein that normally lives in the nucleus of', 'the cell and is involved in the life-cycle of messenger RNA ( mRNA ) .', 'ALS-associated mutations in FUS/TLS cause the protein to mislocalize outside the', 'nucleus into stress granules .', 'Understanding the basis for the toxicity of', 'mislocalized FUS/TLS could lead to new approaches to the treatment of ALS .', 'We', 'have made a yeast model for FUS/TLS cellular toxicity that recapitulates the', 'mislocalization , granular accumulation , and cell death .', 'We have exploited the', 'yeast model to obtain information about what part of the protein is required for', 'proper localization and what part is essential for toxicity .', 'We have also', 'identified several human genes that , when over-expressed in yeast , are able to', 'rescue the cell from the toxicity of mislocalized FUS/TLS .', 'These genes all have', 'functions in mRNA quality control , implicating changes in this pathway in the', 'pathology of ALS .']
['medicine', 'neurodegenerative diseases', 'amyotrophic lateral sclerosis', 'microbiology', 'neuroscience', 'model organisms', 'huntington disease', 'motor neuron diseases', 'biology', 'dementia', 'genetic screens', 'neurology', 'neuromuscular diseases', 'genetics', 'neurological disorders', 'yeast and fungal models', 'saccharomyces cerevisiae', 'molecular cell biology', 'genetics of disease', 'genetics and genomics']