© 2012 Nature America, Inc. All rights reserved. 13 11 of California, San Francisco, San Francisco, California, USA. San Francisco, San Francisco, California, USA. School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania, USA. USA. USA. University of Pennsylvania, Philadelphia, Pennsylvania, USA. the define to 1 way under are Efforts patients. these for therapeutic effective strategy an be might TDP-43 targeting and cases, tia demen frontotemporal many in occur inclusions TDP-43 Notably, in not by cases caused and/or FUS be could effective cases ALS some in found been also have TLS), called also (FUS, protein RNA-binding FALS cases and SALS in tions without cases ALS most of neurons cord ALS spinal the in inclusions cytoplasmic in in found been has TDP-43 implicated been recently needed. are strategies therapeutic tions account for only a small percentage of ALS and cases, additional egies to lower SOD1 levels are being pursued by a of gain toxic to disease function cause thought 1) dismutase superoxide Cu/Zn (encoding the in mutations from result ~20% which of (FALS), ial ­diganosis of years 3–5 within death and paralysis eventual and weakness sive progres neurons, motor of loss by neurode characterized disease late-onset generative a is disease, Gehrig’s Lou as known also ALS, Zamia Diaz Maria Armakola TDP-43 toxicity in ALS models disease Inhibition of RNA lariat debranching enzyme suppresses Nature Ge Nature Received 10 July; accepted 10 September; published online 28 October 2012; cytotoxicity cell preventing absence suppressor Here, of gene Amyotrophic Aaron D Gitler Department of Genetics, Stanford University School of Medicine, Stanford, California, USA. These authors jointly directed this work. Correspondence should be addressed to A.D.G. ( Department of Biochemistry & Biophysics, University of California, San Francisco, San Francisco, California, USA.

RNA-binding proteins and RNA-processing pathways have have pathways RNA-processing and proteins RNA-binding most

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encoding Department of Neurology, University of California, San Francisco, San Francisco, California, USA. Gladstone Institute of Neurological Disease, Taube-Koret Center, Hellman Family Foundation Program, J David Gladstone Institutes, San Francisco, California,

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7 an toxicity. Here, we report results from a genome-wide loss-of-function screen We have used yeast models to illuminate mechanisms unde 3 1 Department of Cellular and Molecular Pharmacology, University of California,

, , Sami J Barmada

7 cytoplasm 22 [email protected] disease

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© 2012 Nature America, Inc. All rights reserved. tif4631 rpl16b nhx1 msn2 mrpl39 fld1 Yeast strain T ydr067c siw14 set3 rpl16a pbp1 dom34 dbr1 cce1 ing the effects on TDP-43 toxicity. In screen 1, we identified 14 yeast yeast 14 identified weTDP-43 ontoxicity. 1, screen effects In the ing confirm background, strain independent an in 2 screen from genes selected and 1 screen from hits the for deletions gene generated we the gene deletions identified from the screens modify TDP-43 toxicity, enhancement of TDP-43 toxicity). To provide independentscore quantitative(a assessmentof ofsuppression effectiveness the or evidence that (six replicates in total), which allowed the calculation of an interaction selected. Screen 2 contained three biological and two technical replicatestimes, and only gene deletions that were reproduced all three times toxicwere than in cells. wild-type Screen 1 was (suppressor) repeated three independent less or (enhancer) more was TDP-43 which in strains technology (DAmP) perturbation mRNA genes by abundance decreased essential by generated of library a and genes deletion the included screen yeast strain bydeletion mating ( galactose-inducible plasmid expressing TDP-43 into each nonessential neurodegenerative screen, we used synthetic genetic the array (SGA) analysis of modifiers proteins disease discover to used been have ( laboratories different two in screens deletion yeast genome-wide unbiased two independent, performed we toxicity, TDP-43 modify that genes identify To DBR1 RESULTS proteinopathies. TDP-43 related and ALS for target therapeutic potential a be could Dbr1 of activity enzymatic debranching the that propose We mammals. to yeast from conserved is toxicity TDP-43 on Dbr1 of effect the that suggesting toxicity, TDP-43 rescue to sufficient also is neurons rat primary in and line cell neuronal human a in Dbr1 of Knockdown and RNA-binding RNAproteins. targets cellular essential other interfering with from it preventing possibly TDP-43, cytoplasmic toxic that of in species cytoplasm the accumulate lariat intronic that evidence provide also We toxicity. the inhibiting that debranching enzymatic show activity of Dbr1 We is sufficient to rescue TDP-43 splicing. pre-mRNA during formed was screen our in discovered toxicity TDP-43 of suppressors knockout s e l c i t r A  TDP-43deletionmodifiedstrainsthattoxicity ( able able 1

∆ ∆ ∆ ∆ ∆ ∆ dbr1 ∆ ∆ ∆ ∆ ∆ ∆

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2 potent 7 ( Enhancer Enhancer Enhancer Enhancer Enhancer Enhancer Effect onTDP-43toxicity Suppressor Suppressor Suppressor Suppressor Suppressor Suppressor Suppressor Suppressor Supplementary Fig. 1b Fig. Supplementary α -synuclein, huntingtin and FUS and huntingtin -synuclein,

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of

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screen screen 2 allowed us to identify a total of 2,581 potential enhancers and 8 and ( was screens both in independently identified cussion of additional yeast TDP-43 toxicity genes). modifier inhibitorsor RNA the interference (see could these represent attractive therapeutic targets for small molecule because TDP-43toxicitysuppressors of on focus to chose we results, 2,056 potential suppressors ( these cells (data not shown). Whereas the non-targeting siRNA had had siRNA non-targeting the Whereas shown). not (data cells these ( siRNA trol human against (siRNA) inter RNA fering small a with lines cell these transfected we toxicity, TDP-43 in toxic was ( TDP-43 cells these mutant Upregulating shown). not data ( and immunoblotting and immunofluorescence by confirmed ( promoter mutant doxycycline-regulated a of control ALS-linked the under (Gln331Lys) TDP-43 epitope-tagged expresses inducibly that line mam cell in neuroblastoma M17 human toxicity a stable We generated TDP-43 cells. malian rescue could function all Dbr1 almost inhibiting to broadly contribute without to cases ALS thought is TDP-43 as activity cially enzymatic Dbr1 inhibiting ( that possibility the suggests of Identification Inhibiting FUS. and TDP-43 proteins of the cificity ( FUS ALS, to linked protein RNA-binding α proteins, disease tive of the in expression TDP-43 lower of was that not because toxicity suppressed confirmed Immunoblotting (Gln331Lys; TDP-43 of form TDP-43, as of toxicity well as wild-type that of mutant an ALS-linked the on efforts further Fig. Fig. -synuclein toxicity was slightly enhanced in the the in enhanced slightly was toxicity -synuclein One of the most effective deletion suppressors of TDP-43 toxicity toxicity TDP-43 of suppressors deletion effective most the of One DBR1

1 1

suppressors. The more sensitive and quantitative analysis in in analysis quantitative and sensitive more The suppressors. a c ). However, However, ). ) could be a promising therapeutic strategy for ALS, espe ALS, for strategy therapeutic promising a be could ) did not suppress the toxicity of two other neurodegenera other two of toxicity the suppress not did

DBR Fig. 2 Fig. Fig. Fig. 2 Fig. DBR1 a

1 2 a DVANCE ONLINE PUBLICATION ONLINE DVANCE c suppresses ). Inducible expression of TDP-43 Gln331Lys was was Gln331Lys of TDP-43 expression Inducible ). d Dbr1 ). To determine whether inhibiting Dbr1 rescues rescues Dbr1 inhibiting whether To). determine genetic interaction for ALS-linked RNA-binding for ALS-linked interaction genetic SOD1 ). Neither siRNA affected TDP-43 expression in expression TDP-43 affected siRNA Neither ). DBR1 α DBR1 as a potent modifier of TDP-43 toxicity toxicity TDP-43 of modifier potent a as -synuclein and mutant huntingtin. Indeed, Indeed, huntingtin. mutant and -synuclein S. cerevisiae mutation deletion suppressed toxicity of another another of toxicity suppressed deletion Supplementary Data

gene. gene. TDP-43 Fig. 1 Fig.

,

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. Therefore, we tested whether whether tested we Therefore, . a DBR1

and and toxicity Supplementary Note Supplementary ∆ EIF4G1 RPL13A SLC9A6 None MRPL33 ­congenital lipodystrophy);seipin BSCL2 Human homolog None None ASH1 RPL13A ATXN2 PELO DBR1 None strain ( strain deletion suppressed the the suppressed deletion or a non-targeting con non-targeting a or Supplementary Fig. 2 Fig. Supplementary Fig. 1 Fig. dbr1 (Berardinelli-Seip

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© 2012 Nature America, Inc. All rights reserved. or a non-targeting (NT) siRNA. The immunoblot shows effective knockdown of Dbr1 expression by the the by expression Dbr1 of knockdown effective shows immunoblot The siRNA. (NT) a non-targeting or mean as shown are Data ANOVA. two-way measures repeated assay. * MTT the by assessed as untransduced), or vector empty an with transduced stably (either cells control to compared manner a dose-dependent in ( right. the on shown is Quantitation TDP-43. untagged endogenous, the than slower slightly migrates which ( DBR1 toxicity. cellular in resulted and Q331K TDP-43 of expression induced (dox) doxycycline of Addition isolated. were clones stable and cells, neuroblastoma M17 into transduced were construct (Q331K) TDP-43 mutant Flag-tagged a doxycycline-regulated encoding Lentiviruses cells. human in toxicity TDP-43 on Dbr1 of effect the test to used approach the of 2 Figure vitro in proteinopathies TDP-43 of features pathological key several lating recapitu TDP-43, of aggregation and mislocalization cytoplasmic TDP-43 of neurons in cortical with primary induces neurodegeneration overexpression that previously showed We neurons. primary in We next asked knockdown of whether Inhibiting ( toxicity TDP-43 reduced significantly on effect no Nature Ge Nature * siRNA. control 5 a b Flag–TDP-43 Q331K ′ LTR ) Immunoblot showing doxycyline-induced expression of Flag-tagged TDP-43 Q331K, Q331K, TDP-43 Flag-tagged of expression doxycyline-induced showing ) Immunoblot c α a -synuclein Lentiviruses M17 neuroblastomacells htt103Q TDP-4 TDP-4 Q331K Flap expression was knocked down using siRNA, and the effect on TDP-43 toxicity was assessed. assessed. was toxicity TDP-43 on effect the and siRNA, using down knocked was expression Vector FUS 2

WT 5 8 ′ TR DBR1 . To determine whether whether To. determine 3 3 F E

Exon dbr1 dbr1 dbr1 dbr1 dbr1 dbr1 n Dbr Inducibl

etics WT WT WT WT WT WT la toxicity, transfection with the the with transfection toxicity, knockdown reduces TDP-43 toxicity in a human neuronal cell line. ( line. cell neuronal a human in toxicity TDP-43 reduces knockdown g ∆ ∆ ∆ ∆ ∆ ∆ P 1 Exon pre-mRNA 1 1 (Expression off) TDP-43 Q331K e < 0.05; repeated measures two-way ANOVA. Data are shown as mean mean as shown are Data ANOVA. two-way measures repeated < 0.05; suppresses Glucose

c mRNA 1 ) Expression of TDP-43 Q331K resulted in decreased cellular viability viability cellular decreased in resulted Q331K TDP-43 of ) Expression + G418 ADVANCE ONLINE PUBLICATION ONLINE ADVANCE Splicing Exon Exon Myc 2 Flag–TDP-43 Q331K Ubi-c 2 (Expression on) Stable cellline

Lariat TDP-43 Galactose 3 Constitutiv rtTA3 ′ Dbr1 IRE Dbr1 pSLIK TDP-43Q331K e S Toxicity?

Debranched Dbr1 + + Do Neo knockdown protects neurons neurons protects knockdown toxicity DBR1 introns + Do Fig. 2 Fig. x WR prevents TDP-43 toxicity b siRNA x E 3 TDP-43 DBR1- TDP-43 WT ′ toxicity SINLTR WT

d in ).

TDP-43 Q331K primary Degradation dbr1 WT

P specific siRNA siRNA specific dbr1 < 0.01, ** < 0.01,

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1 5 brane signifies cell death cell signifies brane mem cell of the or disruption blebbing cell of mApple fluorescence, marker, loss the as survival a as sensitive mApple serves experiment, ( d 8 for intervals 24-h imaged regular at were cohort each from neurons individual of hundreds microscopy, mated against directed siRNA (iii) and or to (TDP-43–EGFP) EGFP TDP-43 (EGFP) cent fused protein fluores green enhanced (ii) mApple, (i) expressing constructs three with them and neurons transfected cortical rat primary and cultured to survival contribute neuronal ables that vari significantly characterizes and identifies that technique a analysis, tudinal from TDP-43 cytotoxicity, we automated used and microscopy longi levels high at accumulate lariats intronic in cells, wild-type in degraded rapidly are introns Whereas splicing. following lariats debranching in Dbr1 of function the showing in affected not ( on). (TDP-43 galactose or off) (TDP-43 glucose onto spotted cells yeast of dilution serial Fivefold mutant). Q331K ALS-linked or (WT) (wild-type toxicity that showing assays spotting 1 Figure those used with traditional markers of particular cell death pathways death cell of markers particular traditional with used those sequent experiments were scaled to achieve the power necessary to necessary power the achieve to scaled were experiments sub sequent of sizes population the experiment, pilot the in noted we that size effect ofthe basis the On nM. 30 was dose effective most that the of amount optimal experiments. these from tional hazards analysis, are used topropor describe the Cox data that and are generated analysis survival Kaplan-Meier including studies, for human epidemiological developed tools statistical Powerful trial. of is to the time, analysis analogous that in clinical used a prospective in a population can be followed for longitudinally an period extended neuron each Because sensitive. more them rendering thereby assay, single a in death cell of forms all detecting of advantage the have but We initially performed a small pilot experiment to determine the the determine to experiment pilot small a performed Weinitially P 2 a < 0.001; < 0.001; ) Schematic ) Schematic

DBR1 Acti Flag–TDP-43

± TDP-43 Endogenous Flag–TDP-43

s.e.m. n

b dbr1 deletion suppresses TDP-43 toxicity in yeast. ( yeast. in toxicity TDP-43 suppresses deletion ) Immunoblotting shows that TDP-43 expression is expression TDP-43 that shows ) Immunoblotting

∆

cells compared to wild-type cells. ( cells. wild-type to compared cells Dbr1 Relative DBR1 Flag–TDP-43 levels 0. 0. 0. 0. 1. 1. d siRNA ( siRNA 0 2 4 6 8 0 2 2 9 Cell viability (%) DBR1 . These criteria are at least as specific as as specific as least at are criteria These . -specific siRNA but not the non-targeting non-targeting the not but siRNA -specific 0 10 20 40 60 80 Dbr1 Dox 0 0 0. 1 deletion ( deletion 0. ( µ Dox 5 Supplementary Fig. 3 Fig. Supplementary 1 0 g/ml) or scrambled siRNA. With auto With siRNA. scrambled or 1 ( µ 3 g/ml 2 3 . * ) dbr1

Cell viability (%) c siRNA 10 ∆ 20 40 60 80 ) suppresses TDP-43 TDP-43 ) suppresses 0 0 s e l c i t r A + NTsiRNA TDP-43 Q331 + TDP-43 Q331 TDP-43 Q331 0 0 – TDP-43 Q331 Contro DBR1 c Fig. 3 Fig. ) Schematic ) Schematic dbr1 2 Dbr1 Dox 9 . . We isolated .1 l siRN * ) and found and ) a ( µ ∆ a ) ) Yeast g/ml NT cells, cells, ). In this this In ). A 2 1 K K K ** DBR1 K ) GAPD Dbr1 ** *

H  ------­

© 2012 Nature America, Inc. All rights reserved. as the steady-state levels of lariat introns are greatly increased in in increased greatly are introns lariat of levels RNA, steady-state the intronic of as turnover the for step rate-limiting a be to seems 2 the cleaves enzyme, debranching a specialized Dbr1, until to digestion resistant 3 the digest can 5 the when 3 splicing during formed are Lariats lariats as released are which introns, remove to spliceosome the by are processed pre-mRNA transcripts primary transcription, After splicing pre-mRNA during formed are that introns 2 the cleaves that man, to yeast DBR1 Dbr separated by a billion years of evolutionary divergence. accumulation from is conserved yeast to mammalian cells, organisms that the ability of Dbr1 inhibition to suppress the toxic effects down of thatTDP-43 we observed in yeast and in neuroblastoma cells and indicate expressing human TDP-43 extend the protective effect of the presence of cytoplasmic TDP-43. These results in primary neurons siRNA( cytoplasmic TDP-43 when treated with TDP-43. In fact, we observed significantly more primary neurons with Dbr1 ( survival survival curve to display the 1.2, same ratio (hazards data alone revealed EGFP a ing similar effect on down nearly 20%, with a hazards ratio of 0.81 ( Dbr1 an approximately twofold increase in the risk of death. Knockdown of ( 2.23 proportionalwas hazardsanalysis Cox ( alone EGFP expressing neurons control in than TDP-43–EGFP expressing rons neu greater in neurons significantly was cortical primary in death of risk the expected, As survival. neuronal on effect significant a detect mean as shown are Data siRNA. scrambled control, with treated neurons to compared TDP-43 cytoplasmic and = 0.81 ratio and = 2.23 ratio hazards ***, analysis. hazards proportional Cox using determined was significance statistical and experiments, independent two from pooled were Data ( siRNA scrambled received that TDP-43–EGFP expressing neurons to compared 19% by death of risk the decreases ( TDP-43–EGFP nM 30 of Knockdown = 2.2). ratio ( alone EGFP expressing neurons of that over death of risk the ( TDP-43–EGFP of Expression time. of function a as cohort each in neurons for death of risk ( bar, 10 Scale h (arrows). 144 by died has other the (arrowheads), experiment entire the for lives neurons the of one While intervals. repeated at here depicted Twoare neurons (bottom). TDP-43–EGFP and (top) mApple encoding constructs with transfected neurons cortical rat primary of microscopy fluorescence ( neurons. rat primary in toxicity 3 Figure s e l c i t r A  dbr1 b n ′ ) Cumulative hazard plot showing the cumulative = 380 neurons counted) significantly increases splice site in a nucleophilic manner. RNA nucleases within the cell cell the manner. within RNA site in a nucleases splice nucleophilic 1 1 ∆ in neurons expressing TDP-43–EGFP reduced the risk of death by knockdown did not depend on decreased amounts of cyt encodes an RNA debranching enzyme, highly conserved from conserved highly an enzyme, RNA encodes debranching Dbr1 cells enzymatic Dbr1

Fig. 3 Fig. Dbr1 Fig. 3 Fig. had no effect on the survival of the control cohort express siRNA in neurons expressing expressing neurons in siRNA 31 n ′ = 245 neurons counted; hazards hazards counted; neurons = 245 knockdown reduces TDP-43 TDP-43 reduces knockdown , Fig. -hydroxyl-5 d 3 n 3 ). These data suggest that neurons can survive even in in even thatneuronssuggestdata survive can These ). c . P = 300 neurons counted) counted) neurons = 300 ′ ). Notably, ). improvementsurvival the in tail of the lariat introns, but the lariat structure is is structure lariat the but introns, lariat the of tail = 0.04. ( = 0.04. n

3 = 380 neurons counted). counted). neurons = 380 activity b ). The relative risk or hazard ratio calculated by calculated ratio hazard or risk relative The ). Dbr1 c ′ -phosphate bond ( bond -phosphate

) Kaplan-Meier survival curve of the same data. ( data. same the of curve survival ) Kaplan-Meier is

a required using using ) Automated ) Automated ′ –5 µ ′

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neuron 0 c 3 Dbr1 2 ). ). - - - .

Time (h) 72 h EGFP +scrambledsiRNA EGFP + siRNA 100 mRNA decay (NMD) pathway mRNA (NMD) decay within the species cell, owing to alterations in the nonsense-mediated ( strains deletion yeast other three in rescued TDP-43 Weexpressed species toxicity. RNA nonspecific any of accumulation if or lation accumu intron lariat to specific was toxicity TDP-43 of rescue the other RNA-binding proteins. To test this, we first determined whether RNAs and cellular important from away it sequestering TDP-43, for in introns lariat mulating increased of introns are levels lariat greatly of In steady-state Dbr1, the absence Lariat mammals. to yeast from conserved is Dbr1 of function ( TDP-43 toxicity to restore cells. Finally, expressing mouse Dbr1 in in yeast TDP-43 toxicity to rescue is sufficient activity ing enzymatic TDP-43 restored ( toxicity partially Dbr1 active partially and effect, no had Dbr1 fully restored TDP-43 toxicity, to whereas completely toxicity inactive TDP-43 Dbr1 restore to ability the and debranching activity partial with some and activity all lost have that some 16 Dbr1 mutants, including some that retain full debranching activity, not ( did mutants point Dbr1 to toxicity ( levels and mutant the wild-type Dbr1 proteins were at expressed equivalent activity matic enzy debranching lack that Asn85Ala) or (Asp40Ala mutants point Dbr1 independent two or Dbr1 yeast wild-type either and TDP-43 cotransformed this directly, we used ‘debranching To activity–dead’test mutantsfunction. ofDbr1 Dbr1. another We to related enzymatic potentially was or debranching activity of loss of because was rescue the whether ( cells malian d upf1 ) ) Depletion of Depletion Dbr1 reduced TDP-43 toxicity in yeast ( Dbr1 P Dbr1 ∆ < 1 × 10 97 h

Fig. Fig. 4 , , intron knockdown results in increased percentage of neurons containing containing neurons of percentage increased in results knockdown 3 150 3 upf2 siRNA . There was an exact correlation between debranching activity Supplementary Table 1) Supplementary 3 3 dbr1 . This suggested the intriguing possibility that the accu the that possibility intriguing the suggested This . a ∆ Fig. Fig. ). Whereas expressing wild-type Dbr1 restored ). TDP-43Whereas wild-type expressing

and and in in vitro accumulation −9 121 h ∆ dbr1 200 ** a * ; **, hazards ratio = 1.83 and and = 1.83 ratio hazards ; **, cells, expressing either of the debranching-inactive debranching-inactive of the either expressing cells, *** DVANCE ONLINE PUBLICATION ONLINE DVANCE 2 ) and primary neurons ( neurons ) and primary xrn1 ∆ and and yeast cells with expression plasmids for plasmids human with expression yeast cells 144 h ∆ ) that each result in accumulation of RNA of accumulation in result each that ) in in vivo c ± dbr1 Fig. 4 Fig.

s.e.m. * s.e.m. Percent survival 100 Fig. 4 Fig. 20 40 60 80

suppresses 0 34– ∆ 5 0 168 h 3 cells could act as a kind of decoy decoy of kind a as act could cells 3 b 3 . Thus, inhibiting Dbr1 debranch Dbr1 inhibiting Thus, . 8 ), indicating that the debranching debranching the that indicating ), . Immunoblotting confirmed that that confirmed . Immunoblotting b . Whereas deleting deleting . Whereas P ). ). Moreover, of a panel we tested TDP-43 +scrambledsiRNA EGFP +scrambledsiRNA TDP-43 + EGFP + < 0.0005, two-tailed two-tailed < 0.0005, dbr1 0 193

TDP-43 Fig. Fig. Dbr1 ∆ h Dbr1 P cells was also sufficient < 1 × 10 Time (h) dbr1 siRNA 3 100

siRNA ), ), but it unclear was Nature Ge Nature ∆

toxicity d cells: wild-type wild-type cells: Neurons with DBR1 −4 Fig. Fig. Scrambled cytoplasmic TDP-43 (%) 150 10 20 30 40 ; *, hazards hazards ; *, 0 t

test. siRNA 1 rescued rescued ), ), mam n * etics siRNADbr1 200 * - - - - -

© 2012 Nature America, Inc. All rights reserved. hy ooaie wt TP4 icuin. tcnqe o visual to technique A inclusions. determine TDP-43 could with we colocalized they lariats, intronic the (i) whether they localized to visualize the cytoplasm or nucleus and (ii) whether could we if in accumulate that lariats end the intronic cells living in visualize to directly, method idea a developed this we test To toxicity. TDP-43 suppress to cytoplasm cytoplasm. the is lariats ( cytoplasm in the sequestered was TDP-43 when toxicity suppressed still deletion Unexpectedly, toxic. be still would NLS an lacking mutant 43 if that, (NLS) signal localization nuclear its mutating by cytoplasm the in TDP-43 we retained case, the was this whether test the should overcome cytoplasm the toxicity suppression by cytoplasm, in TDP-43 the sequestering in model, this In toxicity. suppressing thereby aggregating from it prevent and there of TDP-43 nucleus the in accumulate might lariats cellular multiple in systems model animal and cytoplasm the in toxic is TDP-43 that shown intronic which by in toxicity TDP-43 mechanism suppress lariats the determine to sought next We Intronic accumulation. lariat intronic ( or TDP-43 toxicity, TDP-43 was toxicity not in suppressed ( RNAs linear nonspecific ( RNAs lariat (WT), species RNA excess no accumulating cells yeast of Schematic pathway. NMD cellular the in defects to owing species, RNA non-specific accumulate each which in suppressed is ( mammals. to yeast from conserved is Dbr1 of in Dbr1 mouse Expressing to toxicity TDP-43 restore to unable were activity debranching lack that N85A) and in Dbr1 yeast wild-type Expressing cells. ( levels. similar at mutants point activity–dead debranching and Dbr1 yeast (WT) wild-type HA-tagged ( accumulation. intron lariat by mediated is suppression toxicity 4 Figure Nature Ge Nature the ize Fig. 4 Fig. TDP-43 Q331K TDP-43 Q331K TDP-43 Q331K TDP-43 Q331K TDP-43 Q331K b The above genetic result suggested that intronic lariats act in the the in act lariats intronic that suggested result genetic above The xrn1 TDP-43 WT TDP-43 WT TDP-43 WT TDP-43 WT TDP-43 WT TDP-43 WT c

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. Thus, we reasoned that intronic intronic that we . reasoned Thus, toxicity or or dbr1 1 1 1 1 1 dbr1 ∆ ∆ ∆ ∆ ∆ dbr1 + + + + xrn1 ∆ ∆ ∆ cells. We and others have have We others and cells. cells. We reasoned that, that, reasoned We cells. c ∆ cells restored toxicity. Two Dbr1 mutants (D40A (D40A Two toxicity. mutants Dbr1 restored cells ) Yeast spotting assay showing that TDP-43 toxicity toxicity TDP-43 that showing assay ) Yeast spotting c TDP-43 Q331K TDP-43 Q331K TDP-43 Q331K TDP-43 Q331K TDP-43 Q331K

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(TDP-4 upf2 Galactose mulate in in mulate shown). These foci could represent the excised lariat introns that accu always formed at least one large, perfectly round focus ( ( focus lariats intronic with round colocalized always which perfectly large, one least at formed always larly shaped cytoplasmic foci ( dbr1 shape of TDP-43 inclusions were profoundly different ( in wild-type andcytoplasm the in accumulated and ments ( with our genetic results ( ( lariats intronic with colocalized TDP-43 lyzed, Notably, microscopy. every in by fluorescence lariats ( antibody TDP-43–specific a with immunocytochemistry by localization its visualized and and cells wild-type in TDP-43 untagged expressed We proteins. RNA-binding and RNAs cellular important other with interacting in lariats of cytoplasm the in accumulate lariats intronic that established Having Intronic activity. Dbr1 of absence the in accumulate that introns lariat be to likely are foci ( sequences MS2-binding which in cells wild-type in 5 Fig. ging intron an from independent another yeast gene, located in the cytoplasm ( were always and these cell, in per one or foci two bright accumulated ∆ 3 dbr1 on and and dbr1 ∆ 3 dbr1 ) 3 and and cells. In cells, TDP-43 wild-type formed multiple small, irregu are expressed expressed are ∆ Fig. Fig. 5 ∆ xrn1 upf upf dbr WT xrn1

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colocalize cells or a splicing intermediate splicing a or cells recently developed recently MS2-CP–GFP of by fluorescence microscopy. localization As the analyzed We promoter. inducible the of control under tein pro RNA-binding MS2-CP GFP–tagged 3× ( the gene of intron the MS2-binding in integrated harboring sites each cells, yeast activity of Dbr1 absence the shown by RNA blot analysis to in accumulate ( gene the of intron the in sites MS2-binding used homologous recombination to integrate 3 the tagging of Instead lariats. intronic visualize to TAG with MS2-binding sites of tion any mRNA yeast tagged that has been visualiza the enables GFP to fused MS2-CP and 3 protein region coding coat the between MS2 (MS2-CP) RNA-binding the for sites binding insert to recombination gous In contrast, in in contrast, In ( cell the throughout pattern diffuse a in expression mulate cells, in which no intronic lariats should accu In wild-type methionine). of presence the in growth (by induction without even protein fusion GFP the leaky, we detected somewhat reported Fig. Fig. 5 ′ Fig. Fig. 5 UTR of any yeast gene. Expression of of Expression gene. yeast any of UTR Fig. We transformed wild-type and and wild-type transformed We ACT1

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© 2012 Nature America, Inc. All rights reserved. test test the ability of intronic lariats to compete for binding to TDP-43 (to with TDP-43 binding a large number of RNA targets ( binding TDP-43 for competed binding to TDP-43. As expected, RNAs from wild-type and type and gel shift ( known to bind TDP-43 (ref. 46) with recombinant TDP-43 results in a mobility shift assays. Incubation of a labeled radioactively RNA probe binding to other cellular RNAs, we performed form in contained additional RNA consistentspecies, with intronic lariats. RNA. However, in ( and wild-type in structures expected, As altered their by mobility reduced ­conferred the to owing arc, separate a in migrate breakdown ­products lariat and lariat but a gels, in two-dimensional on migrate diagonal molecules RNA Linear electrophoresis. dimensional The Flag. to antibody an dbr1 and wild-type in TDP-43 Flag-tagged We complex expressed lariats. ­contains ribonucleoprotein TDP-43 the whether assessed also We Intronic RNAs. cellular important with interfering from it preventing thereby in example, (for inhibited is activity Dbr1 When proteins. RNA-binding and RNAs essential with binding, RNA via interfere, could and cytoplasm the in aggregates TDP-43 cells, wild-type In bar, 5 Scale (arrows). introns lariat tagged with colocalized and wild-type in expressed was TDP-43 in (arrowheads), foci shaped in different were inclusions cytoplasmic bar, 5 Scale cytoplasm. the in located always were these and cell, per foci bright two or one in accumulated in contrast, In cell. the throughout localized diffusely and faint was signal MS2-CP–GFP the and accumulate, not did introns the of intron the in sequence by suppressed also is 5 Figure s e l c i t r A  act as decoys), we treated RNAs isolated from wild-type and dbr1 dbr1 d a

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gene. A GFP-tagged MS2-CP protein was used to visualize accumulated intronic lariats. ( lariats. intronic accumulated visualize to used was protein MS2-CP A GFP-tagged gene. , cells, there was always at least one large, perfectly round focus per cell (arrows). Scale bar, 5 Scale (arrows). cell per focus round perfectly large, one least at always was there cells, Supplementary Fig. Supplementary c RNAs ). ). We isolated total RNA from wild- (TDP-43 on) Galactose ∆ dbr1 dbr1 cells, TDP-43 associated with with associated TDP-43 cells, dbr1

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binding cells), lariat introns accumulate in the cytoplasm and might act as decoys, sequestering TDP-43, TDP-43, sequestering decoys, as act might and cytoplasm the in accumulate introns lariat cells), e dbr1 dbr1 dbr1 TDP-43 TDP-43 TDP-43 in vitro vector WT ∆ ∆ ∆ b + + + + 4

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+ × Intron a 3) ) The toxicity of a TDP-43 mutant that is retained in the cytoplasm ( cytoplasm the in retained is that mutant a TDP-43 of toxicity ) The MS2L the the cytoplasm, sequestering essential cellular RNAs in and RNA-binding aggregates TDP-43 that is neurotoxicity of mechanism potential models animal and cellular in toxicity TDP-43 of nent for avenues intervention. new ­therapeutic open and mechanisms disease into insight more ( of TDP-43 modifiers two from uncovered toxicity yeast screens these from TDP-43cytoplasmic aggregation effects in ALS. The additional genetic toxic combat to target therapeutic novel a be could Dbr1 of activity enzymatic debranching the that suggest we Thus, proteins. RNA-binding and RNAs essential with interfering from it pre venting TDP-43, sequester to decoys as act these that propose we cytoplasm, and the in accumulate lariats intronic Dbr1, of absence the in that, evidence Weprovide toxicity. TDP-43 of modifier potent a as Dbr1 discovered we yeast, in screens genetic unbiased two Using DISCUSSION that compete for intronic lariats, to binding TDP-43. Fig. 6c ( site binding its from away TDP-43 competed cells binding reaction, only the RNase R–treated RNAs isolated from When these RNase R–treated samples were incu with RNase all RNAsR, which degrades except for branched lariats Cre recombination Homologous recombination Table Table CP DAPI GF Recent studies indicate that RNA binding is an important compo is an important RNA that binding indicate studies Recent P GF P Exon

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∆ m. ( m. cells, MS2-CP–GFP MS2-CP–GFP cells, Nature Ge Nature Merge dbr1 e Supplementary Supplementary ) Untagged ) Untagged 19 Intronic lariats ∆ , 22 , 49 , n 5 ∆ 0 dbr1 NLS) NLS) etics . One One . 4 ∆ 8 - - .

© 2012 Nature America, Inc. All rights reserved. ing patients with ALS with with ALS with patients ing progression in to mutation oligonucleotides antisense with a by (caused ALS inherited of model mouse a efficacious be might protein mutant toxic a of levels decreasing which in diseases neurodegenerative in strategies and therapeutic attractive as oligonucleotides emerging are approaches interference RNA Antisense proteinopathies. TDP-43 related inhibitors. Dbr1 molecule small ( cells living in lariats intronic visualize to not method new are our available, inhibitors Dbr1 knowledge, our to Although, analyzed. be in human cells, the effects of Dbr1 inhibition on autoimmunity should and be autoimmunity a likely would there of large pool trigger lariats to hypothesized is RNA of accumulation abnormal as Furthermore, assessed. be must this of consequences the and inhibition, Dbr1 by Dbr1 on depend activity. Expression of RNAsthat non-coding these might be dysregulated regions intronic in RNAs non-coding the is cells mammalian in approach this for consideration additional An achieved. be can index therapeutic appropriate an whether and ated is toler of inhibition what level Dbr1 to determine be important will tion (ref. and siRNA in not M17 does knockdown cells result in growth inhibi However,ous. yeast, in budding 3 Fig. ( death of risk the increased neurons rat primary in be activity toxic itself? Indeed, the highest concentration of debranching Dbr1 of inhibition could First, addressed. conformation. TDP-43 to alterations or other promote nucleation might lariat to the or binding structure, branch-point lariat the recognize preferentially also might TDP-43 away from other RNA species in the cell and suppress TDP-43 toxicity. in accumulate that (lariats) sequences or UTRs regions exonic coding than rather sites pre-mRNA intronic long to bind erentially targets for TDP-43 is emerging ( targets RNA important to cellular than rather them to bind to TDP-43 causing decoys, in species lariat intronic accumulated the that and propose We RNAs proteins. RNA-binding critical disrupting from aggregates FUS) (and TDP-43 pathogenesis. ALS to contribute all likely and exclusive mutually not are effects) dominant-negative and nucleus in the of loss function cytoplasm, in the properties toxic the protein of wild-type function the with interfering manner, dominant-negative a splicing of lation ALS dysregu widespread with to leads mouse in individuals TDP-43 of in downregulation populations neuronal affected of nuclei the from TDP-43 is depleted to disease. contributes also likely function of loss TDP-43 pathways. metabolic RNA alterations these of subtle any in to sensitive more simply were neurons motor if transcripts specific were preferentially sequestered by TDP-43 or even if to motor neuron– motor neurons more be deleterious might effect This RNAs. essential of translation and/or sta transport the splicing, bility, in defects to owing metabolism, RNA in changes strophic cata to lead could This functions. normal their from away proteins Nature Ge Nature SOD1 Fig. Fig. There are currently no TDP-43–directed therapies for ALS or or ALS for therapies TDP-43–directed no currently are There be must questions several target, therapeutic a be to Dbr1 For cytoplasmic prevent or overcome might function Dbr1 Inhibiting 4 account for only ~2–5% of all ALS cases, additional therapeutic ), suggesting that too much Dbr1 inhibition could be deleteri be could inhibition Dbr1 much too that suggesting ), ) will facilitate screening of chemical compound libraries for for libraries compound chemical of screening facilitate will ) 56 56 and M.A. and A.D.G., unpublished itdata). Nevertheless, n 4 etics . This approach offers tremendous promise for treat for promise tremendous offers approach This . dbr1 4 51 7

. Finally, TDP-43 missense variants might act in in act might variants missense TDP-43 Finally, . , 5 ADVANCE ONLINE PUBLICATION ONLINE ADVANCE 2 ∆ . These three pathogenic mechanisms (gain of (gain mechanisms pathogenic three These . Fig. 5 Fig. cells suppress TDP-43 toxicity by acting as as acting by toxicity TDP-43 suppress cells 4 7 . This might explain why the intronic RNA intronic the why explain might . This SOD1 f 47 . h ctlg f eoewd RNA genome-wide of catalog The ). , 53– DBR1 SOD1 mutations, but, as mutations in in mutations as but, mutations, 5 5 . Notably, TDP-43 seems to pref deletion is tolerated ( is tolerated deletion dbr1 significantly slowed disease disease slowed significantly ∆ cells sequester TDP-43 TDP-43 sequester cells 57– Dbr1 6 0 . Indeed, treating treating Indeed, . Supplementary Supplementary

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the the two-dimensional nucleic acid gels; S. Collins and D. Cameron for useful P experiments and analysis; C. Kurischko for advice and assistance with visualizing RNA and splicing; T. Nakaya for advice and assistance with lentivirus transduction We thank K. Lynch and S. Smith for helpful suggestions and discussions about Note: Supplementary information is available in the the in v available are references associated any and Methods M site/finkbeiner URLs. with nucleus. the in functions interfering critical potentially without cytoplasm the in TDP-43 of effects toxic the antagonize to possible be might it oligonucleotides, antisense or alternative an bystrategy: targeting Dbr1 using present small Wemolecule approaches. TDP-43–lowering pursuing in caution utes to disease via loss- or gain-of-function mechanisms However,TDP-43 contrib it whether be effective. is could unknown broadly pathogenesis ALS to contribute to appears TDP-43 Because needed. are ­strategies 2. 1. r at online available is information permissions and Reprints Published online at v The authors declare competing interests:financial details are available in the A.D.G. wrote the manuscript with contributions from all authors. A.D.G. analyzed and interpreted data. M.A., M.J.H., M.D.F., S.J.B., S.F., R.V.F. and performed the research. M.A., M.J.H., M.D.F., S.J.B., J.S., N.J.K., S.F., R.V.F. and experiments. M.A., M.J.H., M.D.F., S.J.B., J.S., E.A.S., Z.D., X.F. and A.D.G. M.A., M.J.H., M.D.F., S.J.B., J.S., N.J.K., S.F., R.V.F. and A.D.G. the designed support from National Center for Research Resources Grant RR18928. Investigator of the Gladstone Institutes. The J. David Gladstone Institutes received Scholar. N.J.K. is a Searle Scholar and a Keck Young Investigator. R.V.F. is an supported Sciences, by The Pew Charitable Trusts, and a Rita Allen Foundation Hellman Family Foundation (to S.F.). A.D.G. is a Pew Scholar in the Biomedical (to S.F.), a grant from the ALS Association (to S.F.) and the Taube-Koret Center and Consortium for Frontotemporal Research (to R.V.F.), NIH grant 2P01AG02074 Center for ALS Research at Johns Hopkins (A.D.G. and J.S.), a grant from the Award from the Ellison Medical Foundation (to J.S.), a grant from the Packard NS39074 and NS045491 (to S.F.) and NS072233 (to S.J.B.), a New Scholar in Aging NS067354 (to J.S.), GM084448, GM084279, GM081879 and GM098101 (to N.J.K.), and 1DP2OD002177 (to J.S.), NIH grants NS065317 and NS065317 (to A.D.G.), of Health (NIH) Director’s New Innovator Awards 1DP2OD004417 (to A.D.G.) for the MAT mutant Dbr1 expression plasmids. We thank C. (UniversityBoone of Toronto) We thank B. Schwer (Weill Medical Cornell forCollege) providing the yeast ( CDC48 (University of San California, Francisco) for providing temperature-sensitive for sharing the P-body and stress granule marker plasmids and J. Weibezahn for providing the yeast m-TAG plasmids, R. Parker (University of Arizona) with initial stages of this project. We are grateful to J. Gerst (Weizmann Institute) and helpful suggestions; and G. Howard for editorial assistance. A. Elden helped experiments; J. D. Hosangadi, P. advice and assistance in performing the data analysis for screen 2; B. Hodges, Ac CO AU e e e

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bodies and bodies stress granules; Q. Mitrovich and A. forPlocik advice with running r ethods r s r kn T MPE oe, D.R. Rosen, neurons motor of disease a ALS: D.W. Cleveland, & C. Velde, Vande S., Boillée, with familial amyotrophic lateral sclerosis. lateral amyotrophic familial with neighbors. nonneuronal their and s i i n o i H o t n s owl O

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© 2012 Nature America, Inc. All rights reserved. time time of death for each neuron was defined as the was last time the neuron was death seen. Cell Matlab. The in retraction. or neurite rupture membrane of by fluorescence, loss marked developed code original by channel RFP the in assembled into montages with ImagePro, and cells were prospectively counted were channels TDP-43–EGFP) (GFP; protein fluorescent mApple) and green analysis. Survival algorithms. custom-designed and Cybernetics) Media (ImagePro, software of proprietary a combination running computer host a 64-bit through trolled con were and automated fully were acquisition image and focusing filters, and emission excitation ratio. The and stage shutter fluorescence movements, xenon lamp, and a liquid light was guide to used the signal-to-noise maximize long-lasting and high-intensity adjustable, an by provided was Illumination camera. ultra-cooled 16-bit, Clara Andor an and lens objective (ELWD)20× distance working extra-long an PerfectFocus, with equipped (TE2000) scope 24–48 h after transfection, described images as were acquired system with an inverted imaging Nikon micro robotic a used we analysis. longitudinal for imaging Live-cell (Dharmacon). siRNA scrambled or Dharmacon) or TDP-43–EGFP EGFP and with against siRNA directed were transfected with plasmids (pGW1 backbone) encoding mApple and either Neurons protocols). full for URLs see (Invitrogen; 2000 Lipofectamine using d 4 for plates 96-well in medium serum-free in day pups, were rat from cultured 20–21 embryonic isolated Dawley) (Sprague neurons. cortical primary of culturing and Isolation assessed was size Colony galactose. or described as glucose on inoculation final after points time several at imaged were Plates replicate). biological per replicates technical two and biological (three replicates six generate to duplicated was replicate biological Each plates. three on sporulation after yeast haploid for described as selected were plasmid experiment or control the with gene yeast deletion/DAmP the (Mat collection DAmP knockout yeast bined BY5563 transforming (Mat by generated was strain query experimental the and (Mat BY5563 strain yeast the transforming by erated barcodes. strain deletion of sequencing DNA on analysis spore random by validation further for selected were times three all reproduced were that hits only and times, three repeated was screen described as software analysis image ImageJ by measured were expression (TDP-43 at growth galactose 30 and °C After on). colony for sizes 2 d, were photographed, plates or off) expression (TDP-43 glucose of ence pres the in grown were plasmid expression TDP-43 the harboring mutants (MAT genes tial nonessen of collection deletion haploid yeast the to mated was strain query MAT into introduced was (pAG416Gal–TDP-43) construct TDP-43 expression galactose-inducible the modifications TDP-43 toxicity. These screens were as performed described DAmP of by modifiers R.V.F.for of N.K.) and expression laboratories the in in performed 2, (screen reduced genes essential with combined knockout yeast strains nonessential of collection a or A.D.G.) of laboratory the in the collection of nonessential-only yeast knockout strains (screen 1, performed Yeast TDP-43 toxicity modifier screens. described as were (×3) pMS2-CP-GFP plasmids expression described as were plasmids expression Htt103Q (ref. TDP-43 galactose-inducible 2-micron and CEN PCR. colony by verified and strain the ing Yeast and plasmids. media strains, ONLINE doi:10.1038/ng.2434 the using generated were curves death of risk cumulative and Kaplan-Meier For screen 2, a control query strain containing the empty vector was gen was vector empty the containing strain query control a 2, screen For α ) with p415-Gal–TDP-43–GFP. Both strains were com the mated with strains ) p415-Gal–TDP-43–GFP. Both with DBR1

METHODS 6 7 28), 2-micron 2-micron 28), coding region with a KanMX4 cassette in the BY4741 or W303 or W303 BY4741 the in cassette a KanMX4 with region coding 6 . 6 , using a Singer RoToR HDA (Singer Instruments). For screen 1, α , each gene deleted by KanMX cassette; cassette; KanMX by deleted gene each , Digitized images from the red fluorescent protein (RFP; (RFP; protein fluorescent red the from images Digitized 3 6 3 5 and m-TAG plasmids pLOXHIS5MS2L, pSH47 and and pSH47 pLOXHIS5MS2L, plasmids m-TAG and . Three biological replicates were isolated by selecting selecting by isolated were replicates biological Three . α ∆ strain Y7092 to generate the query strain. This This strain. query the generate to Y7092 strain NLS-TDP-43-YFP The 4 4 We used the SGA technique to screen . dbr1 ∆ For neuronal survival analysis, analysis, survival neuronal For strain was obtained by was strain obtained replac α in vitro in 2 ). Haploid yeast containing containing yeast Haploid ). 68 2 6 , FUS , 3 , 6 . The yeast Dbr1 mutant mutant Dbr1 yeast The . 9 . Briefly, approximately approximately Briefly, . α ) with p415-Gal-GFP, p415-Gal-GFP, with ) and then transfected transfected then and Rat cortical neurons neurons cortical Rat 2 1 , , Dbr1 α Fig. 1 Fig. 26 -synuclein , 64 (SMARTpool, , 6 6 7 5 . The entire entire The . a , , with some ). Haploid Haploid ). 6 2 and and 2 7 ------

(0.1, 1 (0.1, or 2 seeded in 96-well plates. To induce TDP-43 Gln331Lys expression, doxycycline were Gln331Lys TDP-43 encoding vector the with or vector empty an either with transduced stably cells M17 assay. MTT the in proliferation cell on 43 cells. mammalian in assays toxicity TDP-43 analyses. immunofluorescence and ting immunoblot by expression inducible for analyzed and selected were clones streptomycin (complete medium) with 100 100 with medium) (complete streptomycin and 100 FBS, 100 U/ml with plemented penicillin 10% heat-inactivated sup (Invitrogen) alpha/F-12 (MEM) medium essential (minimum medium 5 growth d for in cells °C. transduced 37 by culturing at h selected were 6 lines cell for Stable supernatants lentiviral with incubated were (ATCC) cells described as produced were lentiviruses Recombinant vector. destination lentiviral Neo pSLIK- the to it transfer to (Invitrogen) reaction LR Gateway a in used was Gln331Lys TDP-43 Myc-tagged encoding clone C-terminally and Flag-tagged entry N-terminally Gateway a Gln331Lys, TDP-43 of expression tional lentiviruses. TDP-43 TDP-43. untagged detect to antibody TDP-43–specific a with istry using microscopy to fluorescence lariats detect (GFP) and immunocytochem Colocalization of an medium. mounting Vectashield in DAPI with stained and ethanol 70% with the of control the under GFP of ies to fused MS2-CP three cop intron expresses which with pMS2–CP-GFP(×3), lariats in in lariats lariats. intronic to visualize strategy tagging Genomic nm time. 302 exposure 4-s a of with wavelength excitation an at box light ultraviolet an on captured agitated gently 23–25 and at light from protected were Gels water. in prepared tion solu (Invitrogen) Gold SYBR 1× in placed then and water in briefly washed gels were acid nucleic gel. Two-dimensional of bottom the second-dimension the reached dye cyanol xylene until V 300 at electrophoresed were Samples that 10% gel, polyacrylamide. it except contained as same the first-dimension the exactly was gel second-dimension The gel. second-dimension the above gel first-dimension the from lane entire the by placing dimension second the in EDTA, separated mM V. were 300 at acids 2 them Nucleic acid, electrophoresing and 8.0) pH boric mM 90 base, Tris mM (90 TBE 1× and urea M 7 containing acrylamide:bis-acrylamide) (19:1 gel polyacrylamide 5% min) 10 for V (300 prerun a into samples loading by dimension first the in separated were acids Nucleic min. 5 for °C 85 at heated were and formamide) 100% in dye nucleic acid loading g (0.025 and cyanol xylene g 0.025 bromophenol blue 5 in resuspended were cipitation gels. acid nucleic Two-dimensional ratio. hazard the death, of risk the in change relative the measure to used was analysis hazards cohorts of neurons was by determined the log-rank test, and Cox proportional between differences survival of significance Statistical R. in package survival We transformed wild-type and and wild-type We transformed ized microscopy. fluorescence by lariats intronic tagged Visualizing DNA and sequencing. PCR by colony verified was sequences of promoter.integration Proper of a galactose-regulated control under recombinase Cre expresses which pSH47, plasmid with cells transformed we the Toexcise 311,927). to 310,967 coordinates CYH2 For 53,260). to DNA on (ACT1/YFL039C sequence chromosome VI from coordinates 54,696 ACT1 the either of the integrate to bination recom homologous using tagging, gene chromosomal PCR-based Weused MS2L , we inserted the the inserted we , genomic DNA sequence (RPL28/YGL103W on chromosome VII from on chromosome (RPL28/YGL103W DNA genomic sequence

dbr1 °C in 1× SYBR Gold solution for 45 min. Images of the gels were were gels the of Images min. 45 for solution Gold SYBR 1× in °C -tagged intronic lariats in wild-type and in wild-type lariats intronic -tagged µ g/ml) was g/ml) to added the and growth medium, were cells incubated 7 ACT1 ∆ 0 . For lentiviral transduction, human M17 neuroblastoma neuroblastoma M17 human transduction, lentiviral For . CYH2 cells, we adapted the m-TAG the protocol adapted we cells, gene or the the or gene MS2L , we inserted the the inserted we , MS2L o eeae lniia vco fr h condi the for vector lentiviral a generate To loxP -tagged -tagged intronic lariat with TDP-43 was determined cassette at nucleotide 159 of the the of 159 at nucleotide cassette ø Sphis5 dbr1 CYH2 µ ∆ l of nuclease-free water and 5 5 and water nuclease-free of l MET25 Nucleic acid pellets from immunopre from pellets acid Nucleic cells harboring an an harboring cells + gene in wild-type or or wild-type in gene MS2L ø loxP promoter. Yeast cells were fixed fixed were Yeastcells promoter. cassette at nucleotide 303 of the the of 303 at nucleotide cassette ø We assessed the effect of TDP- of effect the We assessed µ MS2L g/ml Geneticin). Individual Individual Geneticin). g/ml loxP dbr1 -flanked -flanked cassette into the intron intron the into cassette 4 2 ∆ for use with introns. introns. with use for To intronic visualize Nature Ge Nature cells as cells described MS2L dbr1 Sphis5 ACT1 -tagged -tagged ∆ We visual cells. For cells. genomic genomic cassette, cassette, µ n l of 2× 2× of l etics µ MS2L ACT1 g/ml g/ml 4 2 ------.

© 2012 Nature America, Inc. All rights reserved. while leaving lariat RNA intact. After RNase R treatment, RNA was purified purified was RNA treatment, R RNase After intact. RNA lariat leaving while structures RNA linear any degrade to R RNase with treated was or untreated or performed as described from purified was 43 TDP-43 vector. empty or siRNA control targeting with after 2 d (on day 4 TDP-43after transfection). toxicity in M17 cells transfected of by to assay expression TDP-43, induce MTT and was assessed then toxicity (0 and 1 doxycycline containing to medium 2 were d, transferred cells After (Invitrogen). Lipofectamine RNAi using siRNA control non-targeting human nM 50 with transfected was expression Gln331Lys bit polyclonal antibody, 16019-1-AP). The M17 cell line with inducible TDP-43 validated by immunoblotting with a antibody (Proteintech, Dbr1-specific rab was knockdown siRNA D-001810-01-05). non-targeting, J-008290-08-0005; ( control non-targeting siRNA were from purchased Thermo-Scientific of siRNA knockdown times. three at least repeated independently was experiment each and times, at was four replicated least condition Each significant. statistically considered to two in groups, and used means between differences evaluate was ANOVA Two-way condition. each under viability percent the calculate to used and doxycycline with treated not cells of absorbance the to with a Tecan Safire II plate reader. Absorbance measurements were normalized read was nm at 570 Absorbance crystals. formazan blue the solubilize to well ated for 4 h at 37 °C. Acidic (40 isopropanol mM HCl) to was added then each incub were cells and well, each to added was bromide) diphenyltetrazolium (3-(4,5-dimethylthiazol-2-yl)-2,5- MTT proliferation, measure To d. 3 for Nature Ge Nature dbr1 DBR1 ∆ in vitro in W303 yeast using the RNeasy Mini kit (Qiagen) and was either left left either was and (Qiagen) kit Mini RNeasy the using yeast W303 siRNA was compared to toxicity in cells transfected with non- with transfected cells in toxicity to compared was siRNA n etics RNA-binding assay. Escherichiacoli DBR1 7 1 . Briefly, competitor RNA was purified from wild-type in M17 cells. as described as Glutathione S-transferase (GST)–TDP- S-transferase Glutathione siRNA human targeting 2 0 . RNA . binding assays were P values of values were <0.05 DBR1 siRNA or or siRNA DBR1 µ DBR1 g/ml) g/ml) and ­ - ,

67. 66. 69. 68. 65. 64. 63. 62. containing 4 mM MgCl 10- a in out carried were reactions binding UTP.Standard labeled polymerase–catalyzed transcription of a DNA template in the presence of [ UG probe, using the RNeasy MinElute Cleanup kit (Qiagen). A radioactively labeled RNA 71. 70. on a 4.5% native gel(29:1), Bio-Rad). (acrylamide:bis-acrylamide heparin was added to a final concentration of 0.5 competitor RNA (0–12.9 (1 GST–TDP-43(0.5 containing reactions Binding EDTAglycerol. mM 10% Tris,0.1 and mM 7.5, KCl, 10 pH mM 75 U/ml), 40 alcohol, 25 ng of yeast tRNA, 0.8 mg of BSA, 1 mM DTT, 0.1

µ

Collins, S.R., Schuldiner, M., Krogan, N.J. & Weissman, yeast. J.S. A strategy for extracting in toxicity TDP-43 A.D. Gitler, & M.P. Hart, M., Armakola, Arrasate, M. & Finkbeiner,& M. Arrasate, factors determining for system microscope Automated S. High-throughput S. Finkbeiner, & J.A. Kaye, A., Daub, D.M., Ando, P., Sharma, Tong, A.H. & Boone, C. Synthetic genetic array analysis in and early Tong,A.H. are stress ER and ERAD Impaired S. Lindquist, & M.L. Duennwald, Cooper, A.A. Rothrock, C.R., House, A.E. & Lynch, K.W. HnRNP L represses exon splicing via a via splicing exon represses L K.W.Lynch,HnRNP & A.E. House, C.R., Rothrock, Y. Shinbo, screening in primary neurons. primary in screening (2006). R63 data. interaction epistatic quantitative large-scale analyzing and (2011). 238–245 Biol. Mol. Methods 303 toxicity. polyglutamine in events specific Parkinson in loss regulated exonic splicing silencer. splicing exonic regulated activities. fate. neuronal predict that l) were incubated for 15 min at 30 °C in the presence of various dilutions of , 808–813 (2004). 808–813 , 6 , which is known to bind TDP-43 (ref. 46), was generated by T7 T7 by generated was 46), (ref. TDP-43 bind to known is which , Cell Death Differ. Death Cell et al. et et al. et et al. Global mapping of the yeast genetic interaction network. interaction genetic yeast the of mapping Global Proper SUMO-1 conjugation is essential to DJ-1 to exert its full its exert to DJ-1 to essential is conjugation SUMO-1 Proper ′ s models. s Alpha-synuclein blocks ER-Golgi traffic and Rab1 rescues neuron

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