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. 2018 Mar 8;18(4):foy024. doi: 10.1093/femsyr/foy024

Yeast screen for modifiers of C9orf72 poly(glycine-arginine) dipeptide repeat toxicity

Noori Chai 1,2, Aaron D Gitler 1,
PMCID: PMC5946852  PMID: 29528392

Abstract

A hexanucleotide repeat expansion in the C9orf72 gene has been identified as the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia. The expanded hexanucleotide repeat is translated by an unconventional mechanism to produce five species of dipeptide repeat (DPR) proteins, glycine-proline (GP), glycine-alanine (GA), glycine-arginine (GR), proline-alanine (PA) and proline-arginine (PR). Of these, the arginine-rich ones, PR and GR, are highly toxic in a variety of model systems, ranging from human cells, to Drosophila, to even the budding yeast, Saccharomyces cerevisiae. We recently performed a genetic screen in yeast for modifiers of PR toxicity and identified suppressors and enhancers, many of which function in nucleocytoplasmic transport. Whether or not GR toxicity involves similar mechanisms to PR is unresolved. Therefore, we performed a genetic screen in yeast to identify modifiers of GR toxicity and compared the results of the GR screen to results from our previous PR screen. Surprisingly, there was only a small degree of overlap between the two screens, suggesting potential for distinct toxicity mechanisms between PR and GR.

Keywords: ALS, C9orf72, yeast, dipeptide repeat protein, screen, GR


A genetic screen in yeast reveals insight into Lou Gehrig's disease mechanisms.

INTRODUCTION

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are devastating human neurodegenerative disorders (Swinnen and Robberecht 2014). The most common genetic cause of ALS and FTD is mutations in the C9orf72 gene (Renton, Chiò and Traynor 2014). C9orf72 mutations can cause either disease or sometimes both (Taylor, Brown and Cleveland 2016). The disease-causing mutation is a massive GGGGCC hexanucleotide repeat expansion in the first intron of the C9orf72 gene (DeJesus-Hernandez et al.2011; Renton et al.2011). Normally, the C9orf72 gene harbors between 2 and 25 repeats and repeat expansions from hundreds to thousands are considered pathogenic (DeJesus-Hernandez et al.2011; Renton et al.2011). Since C9orf72 mutations are the common cause of ALS and FTD, there is intense interest in defining the mechanisms by which they cause disease so that insight could be harnessed to develop therapeutic strategies.

Several potential mechanisms could explain how the C9orf72 repeat expansion causes disease. First, the large GGGGCC repeat in the regulatory regions of C9orf72 interferes with gene expression, resulting in reduced levels of C9orf72 transcript and protein—the loss of function could contribute to disease (DeJesus-Hernandez et al.2011; Waite et al.2014). Second, the expanded repeat region is bidirectionally transcribed to form distinct RNA secondary structures that could be toxic by sequestering RNA-binding proteins and splicing factors (DeJesus-Hernandez et al.2011; Gendron et al.2013; Haeusler et al. 2014). Third, the sense and anti-sense repeat-containing RNAs are translated in multiple frames, despite the absence of a start codon, by an unconventional form of translation, called RAN (repeat-associated non-AUG) translation (Zu et al.2011), to produce dipeptide repeat (DPR) proteins (Ash et al.2013; Mori et al.2013; Zu et al.2013). The sense transcript produces glycine-alanine (GA), glycine-arginine (GR) and glycine-proline (GP) DPRs, while the anti-sense transcript produces proline-alanine (PA), proline-arginine (PR) and GP DPRs. These DPRs are themselves aggregation prone and accumulate in the brain of C9orf72 mutation carriers and could thus contribute to disease by a toxic gain-of-function mechanism (Ash et al.2013; Mori et al.2013; Zu et al.2013). These three proposed mechanisms are not mutually exclusive and there is compelling evidence for and against each of them (Gitler and Tsuiji 2016).

Of the five distinct DPRs produced from the C9orf72 repeat (GA, PA, GP, GR and PR), the arginine-rich ones (GR and PR) are particularly toxic. They are potently toxic to human cells and cause neurodegeneration in Drosophila melanogaster and human motor neurons derived from induced pluripotent stem cells (iPSCs) (Kwon et al.2014; Mizielinska et al.2014; Wen et al.2014). These phenotypes do not depend on the repeat itself, because synthetic DPRs or use of constructs codon optimized to produce the DPRs without the GGGGCC or GGCCCC repeat sequence still cause cell death (Kwon et al.2014; Mizielinska et al.2014). This permits interrogation of DPR-specific toxicity pathways and their contributions to disease, without confounds of potential RNA-mediated toxicity.

Just like for human cells and Drosophila, GR and PR DPRs are also toxic in yeast cells. We recently used this toxicity as the basis for a genetic screen for modifiers of DPR toxicity (Jovičić et al.2015). We focused on PR and identified several yeast genes that suppressed and enhanced toxicity. These studies illuminated genes in the nucleocytoplasmic transport pathway as potent modifiers of PR toxicity in yeast (Jovičić et al.2015). Studies in other systems, including Drosophila and iPSC-derived neurons also provided evidence that C9orf72 mutations disrupt nucleocytoplasmic transport (Zhang et al.2015; Freibaum et al.2015; Boeynaems et al.2016). Since our previous study focused on PR, in this paper, we performed additional screens to identify modifiers of GR toxicity, to define the commonalities and differences between how GR and PR elicit toxicity.

MATERIALS AND METHODS

Yeast strains, media and plasmids

Yeast cells were grown in rich media or in synthetic media lacking uracil and containing 2% glucose (SD/-Ura), raffinose (SRaf/-Ura) or galactose (SGal/-Ura). To generate yeast expressing a GR dipeptide protein containing 100 repeats (GR100), we utilized a codon-optimized GR sequence, as described previously (Jovičić et al.2015). The ATG-DPR construct was synthesized by Genscript (Piscataway, USA) and was flanked by attB sites. Constructs were further subcloned into a pDONR221 plasmid and subsequently used in Gateway LR reactions with pAG416GAL-ccdB (Alberti, Gitler and Lindquist 2007) to produce yeast expression vectors. The resulting pAG416GAL-GR100 construct was transformed into Y7092 yeast using the PEG/lithium acetate method. Spotting assays verified GR100 toxicity in yeast.

Yeast transformation and spotting assays

Yeast procedures were performed according to the standard protocols. We used the PEG/lithium acetate method to transform yeast with plasmid DNA. For spotting assays, yeast cells were grown overnight at 30°C in liquid media containing SRaf/-Ura until they reached log or mid-log phase. Cultures were then normalized for OD600, serially diluted and spotted with a Frogger (V&P Scientific, San Diego, USA) onto synthetic solid media containing glucose (SD/-Ura) or galactose (SGal/-Ura) lacking uracil and were grown at 30°C for 2–3 days.

Yeast genetic screen

We used synthetic genetic array analysis (Tong and Boone 2006) to identify nonessential yeast deletions that modify C9orf72 GR100 toxicity. We performed this screen as described in Jovičić et al. (2015), using a Singer RoToR HAD (Singer Instruments, Emeryville, USA). We mated MATα strain expressing GR100 under galactose promoter control to the yeast haploid deletion collection of nonessential genes (MATa, each gene deleted with KanMX cassette conferring resistance to G418). Following diploid selection and sporulation, we selected haploids carrying both deletion and GR100 expression cassette. Colony sizes were measured using the ht-colony-measurer software (Collins et al.2006). We performed the entire screen for three independent times. Individual hits were validated by independent transformations and spotting assays.

RESULTS

We screened a library of all 4850 nonessential yeast gene knockouts to identify deletions that could suppress GR100 toxicity (Fig. 1A and B). These types of genetic modifiers are an interesting class (gene deletions that suppress a phenotype) because they could represent potential drug targets. We identified 133 yeast deletions that suppressed GR100 toxicity (Table 1). We validated several modifiers from a variety of functional categories by individual transformations and spotting assays (Fig. 1C). Gene ontology analysis via YeastMine revealed an enrichment for cytoplasmic translation (P = 7.292e−7) and ribosomal small subunit biogenesis (P = 3.323e−4). The majority of the genes found in these categories encode ribosomal proteins and proteins involved in rRNA processing and ribosome synthesis in the nucleolus (Table 1). These ribosome-associated modifiers could act by reducing translation of the toxic GR100 protein. However, we did not identify these modifiers as suppressors of toxicity in deletion screens for other toxic proteins (PR50, FUS, and TDP-43) (Sun et al.2011; Armakola et al.2012; Jovičić et al.2015), suggesting that loss of these ribosomal proteins does not reduce expression of toxic proteins in general, but instead selectively affects GR100. Immunoblots to quantify GR100 were inconclusive (data not shown) and so the specific mechanism of action for these ribosomal hits remains to be determined.

Figure 1.

Figure 1.

A yeast deletion screen reveals genetic suppressors of GR100 toxicity. (A) GR toxicity is length-dependent and less severe than PR toxicity in yeast. Five-fold serial dilutions of yeast cells were spotted onto glucose- or galactose-containing plates. Galactose induced expression of GR or PR in yeast, while glucose repressed DPR expression. (B) Schematic of the yeast deletion screen. (C) Example spotting assays validating specific hits from the deletion screen. Expression of GR is no longer toxic in strains lacking Nup188 (nuclear pore protein), Rad50 (double stranded break repair protein), Erd1 (ER protein), Nop16 (nucleolar protein), Gis2 (translational activator of specific mRNAs), Stm1 (ribosome preservation factor), Bud21 (ribosomal biogenesis protein) or Ski2 (RNA helicase).

Table 1.

List of yeast deletion strains that suppress GR100 toxicity.

GR100 suppressors Systematic name PR50 suppressor FUS suppressor TDP-43 suppressor Function Human ortholog(s)
Ribosomal small subunit biogenesis (16/133, P = 3.323e–4)
rps0aΔ YGR214W ribosomal 40S subunit protein; rRNA maturation RPSA
rps1bΔ YML063W ribosomal 40S subunit protein RPS3A
rps6aΔ YPL090C rps6bΔ ribosomal 40S subunit protein RPS6
rps8aΔ YBL072C yes ribosomal 40S subunit protein RPS8
rps11aΔ YDR025W ribosomal 40S subunit protein RPS11
rps11bΔ YBR048W ribosomal 40S subunit protein RPS11
rps16bΔ YDL083C ribosomal 40S subunit protein RPS16
rps18aΔ YDR450W ribosomal 40S subunit protein RPS18
rps24aΔ YER074W ribosomal 40S subunit protein RPS24
sac3Δ YDR159W ribosome biogenesis; mRNA export SAC3D1/MCM3AP
nsr1Δ YGR159C yes yes pre-rRNA processing; ribosome biogenesis
ltv1Δ YKL143W yes Ribosomal small subunit export LTV1
hcr1Δ YLR192C pre-rRNA processing; translation initiation EIF3J
tsr2Δ YLR435W yes potential role in pre-rRNA processing TSR2
bud21Δ YOR078W part of the ribosomal small subunit processosome
bud22Δ YMR014W rRNA maturation; ribosome biogenesis SRFBP1
Additional ribosomal proteins and ribosome-associated processes (23/133)
rpl12aΔ YEL054C ribosomal 60S subunit protein RPL12
rpl19bΔ YBL027W yes ribosomal 60S subunit protein RPL19
rpl21bΔ YPL079W ribosomal 60S subunit protein RPL21
rpl34aΔ YER056C-A ribosomal 60S subunit protein RPL34
rpl37aΔ YLR185W ribosomal 60S subunit protein; pre-rRNA processing RPL37
rpl38Δ YLR325C ribosomal 60S subunit protein RPL38
rps29aΔ YLR388W ribosomal 40S subunit protein RPS29
rpp1bΔ YDL130W component of the ribosomal stalk RPLP1
rpp2bΔ YDR382W yes component of the ribosomal stalk RPLP2
cgr1Δ YGL029W pre-rRNA processing; nucleolar integrity CCDC86
hpm1Δ YIL110W methyltransferase; modification of ribosomal protein METTL18
jjj1Δ YNL227C ribosome biogenesis
kap120Δ YPL125W karyopherin; nuclear import of ribosomal maturation factor Rpf1p IPO11
kns1Δ YLL019C serine/threonine kinase; ribosome and tRNA biogenesis; rRNA transcription CLK1-4
nop12Δ YOL041C pre-rRNA processing; ribosome biogenesis HNRNPD/DL/A0/AB
nop16Δ YER002W yes ribosome biogenesis NOP16
rrp8Δ YDR083W methyltransferase; modification of ribosomal protein; pre-rRNA processing RRP8
stm1Δ YLR150W translation and ribosome preservation during nutrient stress; binds G-quadruplexes SERBP1, HABP4
tif4631Δ YGR162W ribosome biogenesis; translation initiation EIF4G
syh1Δ YPL105C unknown function, but associates with nuclear pore and ribosomes GIGYF1/2
tma19Δ YKL056C associates with ribosomes TPT1, 1P8
ygl088wΔ YGL088W yes unknown function, but partially overlaps with a snoRNA
yor309cΔ YOR309C yes dubious open reading frame (ORF), but partially overlaps with NOP58
RNA-related processes (15/133)
caf120Δ YNL278W part of a transcriptional regulatory complex; mRNA initiation, elongation, degradation PAK2
cgi121Δ YML036W yes part of a tRNA modification complex TPRKB
ebs1Δ YDR206W nonsense mediated decay; translation inhibition SMG5/6/7
gim3Δ YNL153C part of a prefoldin complex; transcriptional elongation PFDN4
gis2Δ YNL255C yes activation of translation of IRES-containing mRNAs
lrp1Δ YHR081W RNA processing, degradation, export C1D
nup188Δ YML103C part of nuclear pore complex, nucleocytoplasmic transport NUP188
nup84Δ YDL116W yes part of nuclear pore complex, nucleocytoplasmic transport NUP107
she4Δ YOR035C regulation of myosin function; asymmetric mRNA localization STIP1
ski2Δ YLR398C RNA helicase; RNA degradation
ski8Δ YGL213C yes RNA helicase; RNA degradation
sky1Δ YMR216C serine/arginine kinase; regulation of proteins involved in mRNA metabolism SRPK1/2/3
stp1Δ YDR463W yes transcription factor; potential role in tRNA processing
tex1Δ YNL253W mRNA export THOC3
tif1Δ YKR059W translation initiation; RNA helicase EIF4A2
Mitochondrial and NADPH-related metabolic pathways (12/133)
aco2Δ YJL200C mitochondrial aconitase isozyme
flx1Δ YIL134W mitochondrial flavin adenine dinucleotide transporter SLC25A32
idh2Δ YOR136W mitochondrial NAD(+)-dependent isocitrate dehydrogenase IDH3A
oxa1Δ YER154W mitochondrial inner membrane insertase OXA1L
rcf2Δ YNR018W cytochrome c oxidase subunit
zwf1Δ YNL241C glucose-6-phosphate dehydrogenase H6PD, G6PD
gor1Δ YNL274C mitochondrial glyoxylate reductase GRHPR
gpd2Δ YOL059W NAD-dependent glycerol 3-phosphate dehydrogenase GPD1, 1L
gph1Δ YPR160W glycogen phosphorylase; mobilization of glycogen PYGL/B/M
stb5Δ YHR178W transcription factor; oxidative stress, stress response
nnr2Δ YKL151C yes NADHX dehydratase CARKD
ald6Δ YPL061W aldehyde dehydrogenase ALDH1A1/A2/A3, ALDH2
Nucleotide biosynthetic pathway (7/133, P = 3.488e–5)
ade1Δ YAR015W purine nucleotide biosynthesis PAICS
ade2Δ YOR128C purine nucleotide biosynthesis
ade4Δ YMR300C purine nucleotide biosynthesis PPAT
ade5, 7Δ YGL234W purine nucleotide biosynthesis
ade6Δ S000003293 purine nucleotide biosynthesis PFAS
ade8Δ YDR408C purine nucleotide biosynthesis
bas1Δ YKR099W purine nucleotide biosynthesis; transcription factor
Amino acid and other molecular biosynthetic pathways (10/133)
alt1Δ YLR089C alanine transaminase; alanine amino acid synthesis and catabolism CCBL1/2, GPT1/2
aro1Δ YDR127W synthesis of chorismate, an amino acid precursor
cho2Δ YGR157W methyltransferase; phosphatidylcholine biosynthesis
dph6Δ YLR143W diphthamide biosynthesis DPH6
elo3Δ YLR372W fatty acid and sphingolipid biosynthesis
ilv1Δ YER086W threonine deaminase; isoleucine biosynthesis
ino1Δ YJL153C inositol, inositol-containing phospholipid biosynthesis ISYNA1
ipk1Δ YDR315C yes synthesis of phytate IPPK
met2Δ YNL277W methionine biosynthesis
met22Δ YOL064C methionine biosynthesis
ER-related processes (4/133)
erd1Δ YDR414C lumenal ER protein retention
get1Δ YGL020C insertion of proteins into the ER membrane WRB
lhs1Δ YKL073W chaperone of the ER lumen; protein translocation and folding
sse1Δ YPL106C Yes HSP90 chaperone complex; binds unfolded proteins HSPA4/A4L/H1
GTPase-related proteins (7/133)
aim44Δ YPL158C cytokinesis; regulates Rho1p
tus1Δ YLR425W GEF for Rho1p activity
lte1Δ YAL024C similar to GDP/GTP exchange factors RASGEF1A-C
msb3Δ YNL293W Rab GTPase activation; endocytosis TBC1D, SGSM3
gtr1Δ YML121W yes part of TORC1-stimulating GTPase complex RRAGA/B
tco89Δ YPL180W TORC1 subunit
tor1Δ YJR066W TORC1 subunit MTOR
DNA repair (7/133)
asf1Δ YJL115W nucleosome assembly; recovery after double-stranded DNA break repair ASF1A/B
rad50Δ YNL250W yes processing double-stranded DNA breaks RAD50
rad51Δ YER095W double-stranded DNA break repair RAD51
rad52Δ YML032C double-stranded DNA break repair RAD52
vps75Δ YNL246W histone chaperone; double-stranded DNA break repair SET/SIP, TSPYs, FAM197Y1
mms22Δ YLR320W E3 ubiquitin ligase complex involved in replication repair
slx5Δ YDL013W SUMO-targed ubiquitin ligase complex; DNA repair
Serine/threonine and serine modifiers (8/133)
fus3Δ YBL016W mitogen-activated serine/threonine protein kinase MAPK1,3,4,5,6 or NLK
ptk2Δ YJR059W serine/threonine protein kinase; regulation of ion transport TSSKs
yck3Δ YER123W vacuolar membrane serine/threonine kinase; vacuole fusion
pph21Δ YDL134C catalytic subunit of protein phosphatase 2a (serine/threonine phosphatase); mitosis
ppm1Δ YDR435C Yes methyltransferase; methylates the C terminus of Pph21p LCMT1
rts1Δ YOR014W regulatory subunit of protein phosphatase 2A PPP2R5C/D
kex2Δ YNL238W calcium-dependent serine protease
prb1Δ YEL060C vacuolar serine protease
Acetyltransferases (3/133)
eaf6Δ YJR082C part of acetyltransferase complex; histone acetylation MEAF6
hpa3Δ YEL066W D-Amino acid N-acetyltransferase; histone acetylation
mak10Δ YEL053C NatC N-terminal acetyltransferase NAA35
Other (8/133)
alf1Δ YNL148C yes alpha-tubulin folding; microtubule maintenance TBCB, CLIP3/4
atx1Δ YNL259C cytosolic copper metallochaperone ATOX1
cdc50Δ YCR094W endosomal protein; involved with Golgi membrane trafficking TMEM30A/B/C
clb2Δ YPR119W yes cell cycle progression CNTD2
fcy22Δ YER060W-A purine-cytosine permease
fen2Δ YCR028C H+-pantothenate symporterH
sho1Δ YER118C transmembrane osmosensor for filamentous growth
vps64Δ YDR200C yes cytoplasm to vacuole targeting of proteins TRAF3IP3, SLMAP, CEP170/B, CCDC136
Uncharacterized proteins (13/133)
brp1Δ YGL007W protein of unknown function
fyv1Δ YDR024W dubious ORF
fyv6Δ YNL133C protein of unknown function
gds1Δ YOR355W protein of unknown function
hhy1Δ YEL059W dubious ORF
irc14Δ YOR135C dubious ORF
mtc7Δ YEL033W protein of unknown function
rtc4Δ YNL254C protein of unknown function
sdd1Δ YEL057C protein of unknown function
ydr417cΔ YDR417C yes dubious ORF
ygl165cΔ YGL165C yes dubious ORF
ynl198cΔ YNL198C yes dubious ORF
ynr005cΔ YNR005C yes dubious ORF

Though ribosomal genes were statistically enriched in the screen, additional functional clusters emerged (Table 1). One such cluster consisted of six ADE genes (P = 3.488e−5) and BAS1, all of which are involved in purine nucleotide biosynthesis (Cherry et al.2012). Similarly, DNA damage repair genes including RAD50, RAD51 and RAD52 were identified in the screen, and this specific pathway has been implicated in GR toxicity in iPSC-derived neurons (Cherry et al.2012; Lopez-Gonzalez et al.2016). We also identified numerous genes involved with various forms of RNA-interacting processes including nucleocytoplasmic transport, tRNA synthesis and the mRNA life cycle. Similar genes, or in the case of NUP107, identical genes, involved in nucleocytoplasmic transport and RNA export and degradation were also been identified in GGGGCC repeat and PR toxicity screens in Drosophila (Freibaum et al. 2015; Zhang et al.2015; Boeynaems et al.2016).

We next compared the hits from the GR100 to hits from other screens we have performed on ALS-related proteins, including PR50, FUS and TDP-43 (Table 1). Six of the hits from the GR screen were also hits in the PR screen. This number is small, in part because there were only 13 hits from the PR deletion screen (Jovičić et al.2015) and because some hits from the PR screen were identified in only two out of three rounds of the GR screen. Nevertheless, the overlapping hits are informative, pointing to a role for the shared arginine content in the way these genes interact with and modify these arginine-rich DPRs. Also, while the individual genes between the PR and GR yeast screens diverged, the classes of genetic modifiers that have emerged from this GR100 screen have been implicated in GR and PR biology in Drosophila and mammalian cell systems (Kwon et al.2014; Boeynaems et al.2016, 2017; Lee et al.2016). There was no overlap with the TDP-43 screen (Armakola et al.2012). Surprisingly, the biggest overlap of hits came from the GR100 and FUS screens, with 22 shared suppressors of toxicity (Table 1) (Sun et al.2011). This result could be due to the fact that the FUS protein contains several domains containing arginine/glycine/glycine (RGG) repetitive sequences (Boeynaems et al.2017; Ozdilek et al.2017) that may behave similarly to the repetitive GR100 sequence when overexpressed in yeast.

DISCUSSION

Here, we have used a yeast genetic screen to identify suppressors of C9orf72 GR100 toxicity, which provide clues into the potential mechanisms of GR toxicity. While recent studies have focused on the highly toxic PR species or grouped GR and PR together due to their shared arginine content, there has been little done to parse apart potential differences in GR and PR biology, even though such differences exist. From our screen, we have discovered that there is divergence in the genes that suppress GR and PR toxicity when deleted in yeast.

Several factors could contribute to this divergence. First, PR is more toxic than GR (Fig. 1A). This increased toxicity might contribute to the low number of genetic modifiers identified in the PR deletion screen (13) compared to the GR screen (133) (Jovičić et al.2015), since the threshold for suppressing PR toxicity is greater than for GR toxicity. In that case, we could be missing real commonalities between PR and GR, which may be detectable with a less-toxic PR species. And indeed, in other experimental systems, nucleolar and ribosomal proteins, which were modifiers of GR toxicity in yeast, can interact physically with PR (Lee et al.2016; Lin et al.2016; Boeynaems et al.2017). Both PR and GR have also been shown to disrupt the nucleolus and ribosome biogenesis (Kwon et al.2014). The positively charged arginines in both species most likely contribute to these interactions.

However, when we consider the biochemistry of these species, it is important to consider the glycines and prolines in addition to the arginines. Glycine, with a single hydrogen for a sidechain, is dramatically different from proline, which contains a large cyclic side chain that imparts a high degree of structural rigidity to proline-containing peptides. Understanding why the proline content appears to confer increased toxicity at shorter lengths will be an important next step in the field. Furthermore, the specific glycine content is also biologically relevant, as repetitive glycine/arginine rich (GAR) domains occur in numerous proteins and is in fact the second-most common RNA binding domain in the human genome (Ozdilek et al.2017).

The existence of GAR domains, as opposed to proline/arginine rich domains, provides an opportunity for the GR dipeptide species to have a unique impact on the cell. The GR repeats could mimic a protein's GAR domain, thereby wreaking havoc when inserted within specific GAR domain-mediated RNA/protein or protein/protein interactions within the cell. The results from our screen suggest that this is possible, given the large number of shared hits between screens for modifiers of FUS toxicity and GR100 toxicity (Table 1). It would be interesting to see whether ectopic expression of other proteins containing GAR domains would be toxic, and if so, whether they would share significant overlap in toxicity modifiers.

Furthermore, in yeast, the majority of GAR domain proteins are nucleolar proteins involved in ribosomal biogenesis (e.g. Gar1, Nsr1, Nop1, Nop3 and Ssb1) or proteins involved in mRNA handling (e.g. Scd6, Npl3, Gbp2, Nab2, Sbp1, etc.), two major groups identified in our screen for modifiers of GR toxicity (Girard et al.1992; Inoue et al.2000; McBride et al. 2009; Rajyaguru and Parker 2012). Nsr1, which contains GAR domains, was identified in both the PR and GR screens and GR-specific hits such as Rrp8 and EIF4G have been shown to directly interact with many of the GAR domain-containing proteins listed above (Bousquet-Antonelli et al.2000; Rajyaguru, She and Parker 2012), lending credence to the possibility that GR100 toxicity occurs by interfering with the activity of GAR domain-containing proteins.

Additional experiments to investigate exactly how GR100 impacts these pathways are required, but overall, this screen has given us a look into the ways through which GR DPRs produced by the C9orf72 repeat expansion might contribute to disease, and provide potential druggable targets to ameliorate DPR toxicity. The surprising lack of overlap between hits from our GR screen here and our previous PR screen (Jovičić et al. 2015) underscores the importance of considering GR and PR toxic mechanisms as distinct and in pursuing approaches to deal with them separately.

FUNDING

This work was supported by NIH grant R35NS097263 (A.D.G.), the Robert Packard Center for ALS Research at Johns Hopkins (A.D.G.), Target ALS (A.D.G.) and the Stanford Brain Rejuvenation Project of the Stanford Neurosciences Institute (A.D.G.).

Conflict of interest. None declared.

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