© 2012 Nature America, Inc. All rights reserved. Singapore. Singapore. of California, Santa Cruz, California, USA. of Cell Biology, University of Manitoba, Winnipeg, Canada. USA. California at San Diego, La Jolla, California, USA. 1 Comprehensive maps of TDP-43–bindingnuclearcytoplasmicand sitesstepsRNAprocessingof (reviewedin ref. inRNAs of normal mouse unknown. is disease in dysfunction neuronal to contributes toxicity acquired an or function nuclear FUS/TLS of tions FUS/TLS with patients mutations of system nervous the in FUS/TLS of tion ALS portion inherited of additional an for responsible were that FUS/TLS, protein, by the ofidentification mutations in another RNA- and DNA-binding ALS inherited of instances some ALS (reviewed in ref. recognizedgenetically,asbeing clinically and pathologically linkedto nent feature of frontotemporal lobar degeneration (FTLD), insoluble,which is ubiquitinatednow formsinclusions it where patients, ALS sporadic all essentially in cells affected and DNA-binding protein TDP-43 is mislocalized in the cytoplasm of primarilynuclearRNA-the that finding the was discovery seminal A with dominant mutations identified in several genes (reviewed in ref. sive, fatal paralysis. Approximately 10% of disease instances areprogres a familial,in resultingneurons,motor premature of degenerationby ALS is an adult-onset neurodegenerative disorder that is characterized a common loss-of-function pathway as one component underlying motor neuron death from misregulationdepletion inof stemTDP-43 cell–derivedor FUS/TLS. human neurons and in TDP-43 aggregate–containing motor neuronsproteins in sporadicthat areALS, essentialsupporting for neuronal integrity. Expression levels of a subset offrom thesemouse werebrain, lowered but afteramong TDP-43these wereor FUS/TLSmRNAs that were transcribed from genes with exceptionallydistinct fromlong RNAsintrons dependent and onthat TDP-43.encode Abundance of only 45 RNAs was reduced after depletionFUS/TLS. Depletionof either of TDP-43FUS/TLS or fromFUS/TLS the adult nervous system altered the levels or splicingthrough of a >950GUGGU-binding mRNAs, mostmotif. of Wewhich identifiedare a sawtooth-like binding pattern, consistent andwith frontotemporalco-transcriptional dementia. deposition We of found that FUS/TLS binds to RNAs from >5,500 genes inFUS/TLS mouse (fusedand inhuman sarcoma/translocatedbrain, primarily in liposarcoma) and TDP-43 are integrally involved in amyotrophic lateral sclerosis (ALS) John Paul Donohue Curt Mazur Michael Baughn Lagier-TourenneClotilde TDP-43 intersect in processing long pre-mRNAs Divergent roles of ALS-linked proteins FUS/TLS and nature nature Received 12 June; accepted 6 September; published online 30 September 2012; Ludwig Ludwig Institute for Cancer Research, University of California at San Diego, La Jolla, California, USA. oh D-3 n FSTS ae en soitd ih multiple with associated been have FUS/TLS and TDP-43 Both cause TDP-43 in mutations point dominant that discovery The 4 10– Department Department of University Neurosciences, of California at San Diego, La Jolla, California, USA. 1 NEUR 2 , , disrupts its normal nuclear localization 9 8 These These authors contributed equally to this work. should Correspondence be addressed to D.W.C. ( , 9 , and in some instances of FTLD in the absence of muta of in absence , the FTLD and in instances some OSCI 5 , , Edward Wancewicz 8 , 9 and rare cases of FTLD. Cytoplasmic accumula Cytoplasmic FTLD. of cases rare and EN 4 4). , , Stephanie C Huelga C E 7

, Lily , Shiue Lily advance online publication online advance 1 2 , 5– 2 , 3 , 7 . TDP-43 pathology is also a promi 9 8 and FTLD was rapidly followed followed rapidly was FTLD and , , Magdalini Polymenidou Molecular Molecular Engineering Laboratory, A*STAR and Department of Biological Sciences, National University of Singapore, 7 , , C Frank Bennett 5 3 , Aneeza , S Aneeza Kim Stem Stem Cell Program and Institute for Genomic Medicine, University of California at San Diego, La Jolla, California, 2 , 3 1 7 3 , , Kevin M Clutario RNA RNA Center, Department of Molecular, Cell and Biology,Developmental Sinsheimer Labs, University . . Whether the loss

5 5 , , Andy Watt , , John Ravits 14). 1). 1 5 - - - - 1

, doi:10.1038/nn.323 2 , 9 light were efficiently immunoprecipitated using antibodies to three three to antibodies using immunoprecipitated efficiently were light cross-linked complexes protein–RNA FUS/TLS RNA targets of FUS/TLS in mouse and human brain RESULTS FUS/TLS. or TDP-43 either of misregulation the from neurons motor in pathway pathogenic common a identifying in level neurons post-mortem affected of patients protein with ALS, sporadic thereby the at reduced substantially were these targets of common Several targets. FUS/TLS and TDP-43 intron–containing long of downregulation the confirmed cells stem pluripotent from differentiated neurons human in TDP-43 or FUS/TLS of Depletion FUS/TLS. and TDP-43 both for sites binding multiple and introns RNAs long identified with targets exceptionally FUS/TLS-dependent and TDP-43– the between comparison Systematic TDP-43. on ent were genes and on only 112 of FUS/TLS, 610 dependent the were depend genes also of levels expression The brain. human and mouse both in FUS/TLS of targets RNA ~5,500 identified we approaches, for neuronal function derived from very long intron–containing pre-mRNAsrevealingessentialan roleTDP-43for thatsustaining in arelevelsRNAs theof important sion in the adult nervous system alters pre-mRNA splicingor diseased of >900 genes,human , , Kasey R Hutt 1 Using similar high-throughput sequencing and computational computational and sequencing high-throughput similar Using , 2 , , Shuo-Chien Ling 5 , , Sue Freier 4 , , Don W Cleveland 5 Isis Pharmaceuticals, Isis Carlsbad, Pharmaceuticals, California, USA. 2 0 Department Department of Cellular and Molecular Medicine, University of 1 6 2 CNS have been determined. Reducing TDP-43 expres d , 3 1 [email protected] 5 5 , 9 , Geoffrey G , Hicks Geoffrey , some of which are reduced in human disease , , Anthony Q Vu 1 , 2 , , Tiffany Y Liang 1 , 2 & Gene W Yeo u ) ) or G.W.Y. ( 2 , 3 6 , ,

g in in vivo [email protected] t r a 2 , 3 , 2 by ultraviolet by ultraviolet ,

6 3 MB MB Institute , 8 C I e l

u ). 1 6 . s  - - © 2012 Nature America, Inc. All rights reserved. nonredundant mapped reads represented the sense strand of the the of strand sense the represented reads mapped nonredundant Most from of the three antibodies. (88.6%) the >93 combined million ( sites ( sites for were FUS/TLS quite from distinct those obtained for TDP-43 ( neurofilament in ple, mouse the exam for including, antibodies, FUS/TLS three all by precipitated ( genome mm9 the onto high-throughput by libraries sequencing) followed immunoprecipitation, and linking mm9; in the mouse genome (University Santa of reference Cruz, California, nonredundant 37,586,201 reads that mapped to pre-mRNAs encoded precipitated with each antibody andproduced 32,427,431 12,344,760, cross- ( linking ultraviolet of absence the in or IgG nonspecific using when ( complexes mobility lower some as well as RNA, to bound molecule FUS/TLS for a single mobility expected the with complexes cipitated brain lysates ( mouse from FUS/TLS of reduction stantial Fig. Supplementary 1a ( FUS/TLS of regions distinct the in positions intronic distinct, but neighboring, in ( (right). sites FUS/TLS-binding and TDP-43– both with genes or (left) nucleotide one least at by overlapping clusters CLIP FUS/TLS and ( the 5 of exon orthologous in brain ( panel. top the in defined as regions pre-mRNAs in clusters CLIP FUS/TLS and TDP-43 of ( brain. mouse and human in clusters CLIP FUS/TLS in motif GUGGU the of ( clusters. FUS/TLS define to experiments CLIP-seq three from ( base. per coverage read the represents bar scale The motifs. GUGGU of positions the show lines red ( subunit neurofilament molecular-weight low to binding (purple) ( panel). (fourth immunoprecipitation after remained FUS/TLS no and panel), (third mobility TLS FUS/ expected the at migrated complexes FUS/TLS-RNA Immunoprecipitated panel). (second complexes protein-RNA detect not did antibodies IgG with coated Beads sequencing. for used were box red the by highlighted Complexes panel). (first (MNase) nuclease micrococcal of concentrations increasing with trimmed and Ab1 with immunoprecipitated brain mouse from complexes protein–RNA TLS ( signal. localization nuclear ZF,L, finger; zinc arginine/glycine; R/G, motif; recognition RNA RRM, signal; export nuclear E, glycine; G, tyrosine; serine, glycine, Q/G/S/Y, glutamine, Ab3. and Ab2 Ab1, antibodies generate to antigens as used ( brain. human 1 Figure t r a  ( genes in known pre-mRNA i h g Supplementary Fig. 1a Supplementary Fig. 1 Fig.

) Overlapping TDP-43 and FUS/TLS clusters clusters FUS/TLS and TDP-43 ) Overlapping ) FUS/TLS binding in human and mouse mouse and human in binding ) FUS/TLS ) Venn diagrams showing genes with TDP-43 TDP-43 with genes showing ) Venn diagrams Positions of mapped reads revealed similar binding sites for RNAssites binding similar revealed reads of mapped Positions Supplementary Fig. 1e Fig. Supplementary Gria3 d Supplementary Fig. 1d Fig. Supplementary c ) Flow chart illustrating reads analyzed analyzed reads illustrating chart ) Flow

c C I ). Genome-wide consistency of identified FUS/TLS-binding FUS/TLS-binding identified of consistency Genome-wide ). Fig. 1 Fig. FUS/TLS RNA targets in mouse and and mouse in targets RNA FUS/TLS ) FUS/TLS (green) and TDP-43 TDP-43 and (green) ) FUS/TLS pre-mRNA. e l Fig. 1 a b ) FUS/TLS protein domains domains protein ) FUS/TLS ). After size selection, sequencing of RNAs immuno sequencing selection, size ). After s e b ) Positional distribution distribution ) Positional b ) Autoradiograph of FUS/ of ) Autoradiograph and 1 Nefl 5 , were re-sequenced and reads were mapped mapped were reads and re-sequenced were b , Supplementary Fig. 1d Fig. Supplementary b Supplementary Supplementary Fig. 1c ), ), resulting in a sub ) RNA. Vertical Vertical ) RNA. ). ). No complexes were immunoprecipitated f ) Percentages ) Percentages Fig. 1 Fig. ) prompted the merging ) of prompted data sets the merging the Fig. 1a Fig. ). Previous TDP-43 CLIP-seq (cross- CLIP-seq TDP-43 Previous ). Fmr1 Nefl d ). The majority of the remainder of remainder the ). majority The RNA. RNA. ) ) pre-mRNA (

,

b

and and

- ). ). Each antibody pre ). a g f d b 175 UV IP 2% 5 Mouse TDP-43 46 50 80 ′ CLIP binding : UTR Fig. 1 Fig. : Mouse FUS 5% Ab1 Human FUSCLIP Mouse FUSCLIP Human fragileXmentalretardationgene GUGGU Mouse fragileXmentalretardationgene 62% MNase Q/G/S/Y-rich region + 1% 166,356 clusters Uniquely mappednonredundantreads in 8,213genes 30% In pre-mRN c 82,358,392 ). Binding ). Binding

+

FUS Ab1 + 10% CLIP binding intron Distal Mouse FU Ab3 10% RNA A 93,005,761 FUS CLIP 51% 4% + - - ­ G-rich region 25% 2 kb + Long ncRNA S 1,620,506 Not inpre-mRNA Exo are conserved in both human and mouse. and human both in conserved are ( overlap significant a in human targets highly representing also brain, RNAwere of in brain (69%) mouse the majority targets the FUS/TLS of annotated protein coding genes ( outside mapped reads) of 1,784,317 (16%, reads only a fraction small ( reads mapped nonredundant 9,373,763 human from 5,594 identified were from genes pre-mRNAs in sites FUS/TLS-binding 44,490 human genome (University of California, Santa Cruz, reference hg18), ( brain mouse from similar showed ( complexes patterns migration protein–RNA FUS/TLS human The efficiently which ( FUS/TLS (Ab1), human 1 immunoprecipitated antibody FUS/TLS using cortex brain RNAs ( mouse 8,213 in reads) 92 of average an and each reads 28 minimally (with levels expression variable and length pre-mRNA for account that thresholds specific Fig. Supplementary 1f (lncRNAs; RNAs noncoding long annotated to mapped P Supplementary Fig. 2c Fig. Supplementary 10,647,369 Proximal – n < 10 FUS/TLS targets were also identified by CLIP-seq from human human from CLIP-seq by identified also were targets FUS/TLS 10% intron CLIP binding Human FU 2 kb 12% IgG + 5% E 54% −37 Pre-miRNA 19% RRM MNase ( 62,067 + + ( Fmr FUS Ab1 Fmr Beads ), illustrating that the FUS/TLS protein–RNA interactomes protein–RNA that the interactomes ), FUS/TLS illustrating advance online publication online advance S 1 1 R/G rich ) Protei ) 4% Ab2 Fig. 1 Fig. + + distribution 4% 58% 0.5 kb n Supernatant Pr Size 15 ZF 1% e , R/G rich 3 1 Post ′ Supplementary Fig. 1a Fig. Supplementary 7 33% UTR d + , we identified 166,356 FUS/TLS-binding sites sites FUS/TLS-binding 166,356 identified we , Supplementary Fig. 2a Fig. Supplementary ). ). ). Using a cluster-finding algorithm with gene- L 0 176 0 377 ). Consistent with our findings in mouse, mouse, in findings our with Consistent ). h e c i Mouse neurofilament

Motif enrichment TDP-43 CLIP FUS CLIPAb3 FUS CLIPAb2 FUS CLIPAb1 GUGGU –500 0 1 2 3 Strict overlap 2,727 genes TDP-43 CLI FUS CLI Glutamate receptor3

–400 Human FUSCLI Supplementary Fig. 2c Supplementary

Distance fromFUSclusterpeak(bp) –300 P

P –200 Supplementary Fig. 2a Fig. Supplementary

6,312 genes

Any cluste –100 ( P Nef nature nature ). After mapping to the the to mapping After ). ( l ) with those obtained obtained those with ) Gria ) gene 0 3 ) gene r 100 Mouse FUSCLI NEUR FUS 8,213genes TDP-43 7,951genes 200 1 kb 200 bp 300 Fig. 1 Fig. OSCI ). ). Notably, 400 0 176 0 377 d P

EN 500 0 0 0 0 32 52 76 70 and and , b C ). ). E © 2012 Nature America, Inc. All rights reserved. the relative size of distal intronic regions in pre-mRNA ( pre-mRNA in regions intronic distal of with size relative accordance in the sites FUS/TLS-binding of fraction for highest accounted the junction, exon-intron nearest the from kb 2 least at in introns. The distal intronic region, defined as the sequences located localized being majority the with transcripts, pre-mRNA in patterns binding FUS/TLS of position the ( defining sequences GUGG at cross-linking reflected that polymorphisms sequence of degree high a harbored snRNA U1 to mapped reads CLIP-seq FUS/TLS ticular, ( transcripts (snRNA) nuclear small ( sites TDP-43 of from sites pre-mRNA along that different regions was distinct visibly binding FUS/TLS ( of binding TLS peak the with ( coincided often motif this ( clusters FUS/TLS human and mouse of ~60% in from GGUG motif reported ( GUGGU motif sequence RNA primary nature nature syndrome ( syndrome mutated in X fragile mental retardation and adult onset ataxia/tremor and human brain of with FUS/TLS exon 4 of the One example of exonic binding in is interaction mouse the conserved ( sequences pre-mRNA to contribution their from expected in binding for enrichment to be what would compared (UTRs) regions and untranslated exonic substantial a also was there Notably, for proper neuronal function, including neurexin 1 and 2 ( 2 and 1 neurexin including function, neuronal proper for TDP-43– and FUS/TLS-binding sites include ( many genes alone that FUS/TLS are or crucialTDP-43 either with compared sites FUS/TLS-binding 3 in localization pro RNA-binding Nova unrelated tein, an of sites the around enriched not 4a Fig. ( versa vice enrichment 1.8-fold a and clusters TLS ( surrounding TDP-43 immediately of binding enrichment 2.2-fold a revealing analysis genome-wide with superimposed, perfectly were sites FUS/TLS-binding and 43– on ( 3 receptor glutamate the binding by exemplified as reads, sequence of peaks positioned, by but clusters were distinct, closely TDP-43–FUS/TLS characterized complex same the in present being proteins FUS/TLS of and TDP-43 fraction least at by overlapping nucle sites one binding with clusters, FUS/TLS and ( tau protein ( 2 and 1 and ( 1 ( dismutase superoxide encoding genes the ing includ disorders, neurodegenerative and other of ALS pathogenesis to 3 the binding FUS/TLS formation spine dendritic in FUS/TLS of role with the consistent RNAs, these of translation the localized in and involved transport is FUS/TLS that suggests preference binding This (corrected ponents of the synapse and molecules residing in neuronal projections com for enrichment identified analysis ontology gene and mRNAs, Fig. 1 Fig. 3b Fig. Supplementary Fig. 1 Fig. Using the HOMER algorithm, we identified a significantly enriched In the mouse brain, 6,312 genes contain both TDP-43 (ref. (ref. TDP-43 both contain genes 6,312 brain, mouse the In 3 the in found was binding FUS/TLS Mouse and human brain FUS/TLS sites showed similar distribution NEFH c e , upeetr Fg 4d Fig. Supplementary ), medium and heavy chains of neurofilament ( of neurofilament chains and heavy ), medium ), its presence was neither necessary nor sufficient for FUS/ for sufficient nor necessary neither was presence its ), b NEUR 2 UBQLN1 0 ). In contrast, FUS/TLS and TDP-43 CLIP reads were were reads CLIP TDP-43 and FUS/TLS contrast, In ). o . In some instances, these overlapping or neighboring neighboring or overlapping these instances, some In . 2 ), the glutamate transporter EAAT2 ( EAAT2 transporter glutamate the ), tide in only 2,727 genes ( genes 2,727 only in tide P Fig. 1 Fig. 1 MAPT ( values: values: Fig. 1 Fig. OSCI Supplementary Fig. 4c Fig. Supplementary ′ UTRs was observed for overlapping TDP-43– and and TDP-43– overlapping for observed was UTRs g Fig. 1c Fig. ). and and ) ( ) EN c P ). The distribution of mouse FUS/TLS-binding FUS/TLS-binding mouse of distribution The ). Supplementary Table 1 Supplementary C = 2.1 × 10 × 2.1 = E UBQLN2 ,

, f c ′ ). FUS/TLS binding was also observed on on observed also was binding FUS/TLS ). Gria3 UTR of several transcripts involved in involved the transcripts UTR of several ). advance online publication online advance in in vitro ) gene ( gene ) ). RNA targets with overlapping overlapping with targets RNA ). −5 ), and the microtubule-associated microtubule-associated the and ), RNA binding or or Supplementary Fig. 3a Fig. Supplementary Fig. 1 Fig. ± 50 bases) the center of FUS/ of center the bases) 50 ). A stronger enrichment for for enrichment stronger A ). P Fig. 1 Fig. ′ = 7.2 × 10 × 7.2 = UTR of 4,272 mouse brain brain mouse 4,272 of UTR P h < 10 < ), consistent with a small small a with consistent ), i ). In other cases, TDP- cases, In other ). ). FMR1 −3451 1 EAAT2 Fig. 1 Fig. 8 ) ) that was present 1 SOD1 −7 9 Supplementary Supplementary . We identified identified We . ; similar to the the to similar ; , respectively). respectively). , gene, which is NEFL e ). Although Although ). ), ubiquilin ubiquilin ), ), the light light the ),

). ). In par Fig. 1 Fig. Fig. 1 Fig. NRXN1 , , NEFM 15) 15) f f ). ). ). ). - - - -

in our RNA-seq data from mouse brain ( brain mouse from data RNA-seq our in literature-curated lncRNAs. Two-thirds (66%) of these contained contained these of (66%) Two-thirds lncRNAs. ­literature-curated 5 at the Fig. 6b ( region intronic this containing transcripts nascent of prevalence higher the of result a likely is introns of beginning the ( elongation scription tran during introns long along deposition FUS/TLS with consistent transcripts nascent mark to proposed been has which pattern, binding sawtooth-like A proteins, such as TDP-43 and RBFOX2 in mouse brain ( brain mouse in RBFOX2 and TDP-43 as such proteins, of patterns the with RNA-bindingCLIP-seq other sharply contrasted seq data, as by illustrated the 1 neuroligin ( CLIP- FUS/TLS human and mouse both in evident was pattern This 3 the short ( being substantially higher at the beginning of long ( density cluster FUS/TLS with kb), (>100 introns long with genes on ( clusters, CLIP FUS/TLS exhibiting about fourfold had more binding sites per region than regions expected candidate these of 40% than for observations our with Consistent mediated decay substrate, most likely reflects an autoregulatory mech is that a of nonsense- an isoform production the with in conjunction pre-mRNA, own its to FUS/TLS of Binding FUS/TLS. from distinct 5a Fig. of 7 intron and 7 exon between junction the at reads CLIP-seq of density high a with 3 alternative this of entirety the with interacted FUS/TLS decay. nonsense-mediated by degradation ( 9 exon in 8, intron not but both mouse and human 7, for brain, intron using specific primers 7 and intron retained that isoform FUS/TLS a presence of the identified We level conservation. high sequence a mammalian and of reads RNA-seq of abundance an has region This 3 mature mRNA, we identified 1,866 previously unannotated 3 of value reads median the above RNA-seq containing regions intronic that Reasoning exons. 3 annotated in density read RNA-seq brain mouse wild-type 3 members protein ribonucleoparticle related proteins, as has been observed for other heterogeneous nuclear cross potential ( gene 1 region breakpoint sarcoma Ewing the of region intronic retained or 3 alternative conserved highly a on pattern binding FUS/TLS of TDP-43 (ref. that to similar anism noae a ete a ito o a atraie 3 alternative an or intron an either as annotated pr was that region a in pre-mRNA own its on binding TLS broad regions of its pre-mRNA targets. This was by exemplified FUS/ peaks ‘sharp’binding edly different from that of TDP-43. In contrast with the more discrete its pre-mRNAs target across was mark mode The FUS/TLS-binding patterns binding distinct have TDP-43 and FUS/TLS ( and Supplementary Fig. 5d Fig. Supplementary APP ′ ′ UTRs or retained introns, we first determined the distribution of of distribution the determined first we introns, retained or UTRs T o rtie itoi rgos ( regions intronic retained or UTR We also identified 843 FUS/TLS-binding sites on 71 of 234 234 of 71 on sites FUS/TLS-binding 843 identified also We The most distinguishing feature The of most binding was distinguishing FUS/TLS observed alternative other with interacts FUS/TLS whether investigate To NRXN2 ′ ) and ataxin 10 ( 10 ataxin and ) end, producing a sawtooth-like pattern across the pre-mRNA. pre-mRNA. the across pattern a sawtooth-like producing end, Supplementary Fig. 6a ) and not of an increased density of the FUS/TLS-binding motif ). Notably, TDP-43 also bound the same region, but at sites sites at but region, same the bound also TDP-43 Notably, ). ′ end of introns ( introns of end Ewsr1 Supplementary Fig. 5a Fig. Supplementary ), ), ) pre-mRNA ( pre-mRNA ) - UBQLN2 regulation between Ewsr1 and FUS/TLS, both closely closely both Ewsr1 and FUS/TLS, between regulation FUS/TLS 2 2 , was observed in long intron–containing genes genes intron–containing long in observed , was 15 ATXN10 Fig. 2 Fig. , 1 Fig. 2 Fig. , , 6 ). of TDP-43, FUS/TLS sites extended along along extended sites FUS/TLS TDP-43, of FUS/TLS pre-mRNA ( pre-mRNA c ′ Supplementary Fig. 5b Fig. Supplementary UTR read density are likely retained in in retained likely are density read UTR ). The FUS/TLS-binding enrichment at enrichment FUS/TLS-binding The ). ), introns, and a gradual decrease toward e ). ). ). This isoform is likely subjected to to subjected likely is isoform This ). ′ UTR or retained intronic region, region, intronic retained or UTR , amyloid beta precursor protein protein precursor beta amyloid , 1 15). We also observed a similar a similar We15). observed also 7 . FUS/TLS Fig. 2 Fig. upeetr Fg 5c Fig. Supplementary Nlgn1 Supplementary Fig. Supplementary a and and ) ) gene ( and and Fig. 2b t r a ′ T ( UTR Supplementary Supplementary Supplementary Supplementary ). This suggests suggests This ). EWSR1 Fig. – C I d

candidate Fig. 2 Fig. ), but not Fig. 2 Fig.

e 2 viously viously b , more more , ′ ′ e l ), ), and UTR UTR UTR UTR

6b d a s ). ). ). ). ). ). ), ),  - - -

© 2012 Nature America, Inc. All rights reserved. number of mapped reads per kilobase of exon, per million mapped mapped the million by per exon, of kilobase determined per reads mapped were of number gene protein-coding annotated each for the to mapped ( genome uniquely mm9 mouse reads nonredundant million ~26 aver of an age sample, each For depletion. FUS/TLS ASO-mediated ing follow mice three from striata of analysis RNA-seq strand-specific treatment. of weeks 2 after FUS/TLS of reduction marked with mice in observed and of respectively, in levels, and normal the striatum cord spinal ( ~20% or ~10 control to RNA and a protein FUS/TLS not depleted but saline, or ASO ASO, FUS/TLS-specific a of infusion 2-week or cord spinal the of levels all including CNS, the ( ventricle ( injection intrastriatal tic stereotac either by mice of CNS adult the or into delivered were genome, saline mouse the in sequences complementary without ASO end of action the through RNA of degradation catalytic direct that (ASOs) tion deple FUS/TLS after changes splicing and expression To determine after altered RNAs ( lncRNAs syndrome ( binding on 3 expressed maternally ( by exemplified as TDP-43, of those from distinct were lncRNAs on sites FUS/TLS-binding of position the targets, RNA protein-coding the for observed we what to Similarly GUGGU. motif FUS/TLS the bin. 1% per clusters of fraction gene per the of (SNR) ratio 5 ( co-transcriptionally. deposition FUS/TLS with consistent pattern a sawtooth-like produce to accumulated elongation of stages different at transcripts nascent of A collection splicing. co-transcriptional and elongation 1 ( neuroligin as such to 2 Figure t r a  biological the from reads sequence combined The (RPKM). reads c Conservation a

′ end of long introns. ( introns. long of end FUS/TLS function in mRNA metabolism was assessed with with assessed was metabolism mRNA in function FUS/TLS FUS/TLS Co-transcriptional during elongation upeetr Fg 8a Fig. Supplementary

n vivo, in Human Mouse CLIP pattern Long introns o Deposition

Fus FUS Sawtooth

C I genous RNase H. An ASO specific for FUS/TLS, a control control a FUS/TLS, for specific ASO An H. RNase genous splicing FUS/TLS binding patterns in mouse and human brain. ( brain. human and mouse in patterns binding FUS/TLS TDP-43 CLIP FUS CLIP FUS CLIP Supplementary Fig. 7 Fig. Supplementary Fig. RNA in a highly conserved region that represents either an alternative 3 alternative an either represents that region conserved a highly in RNA RNA-seq Ipw e l we used single-stranded antisense oligonucleotides oligonucleotides antisense single-stranded used we

) or nuclear paraspeckle assembly transcript 1 ) transcript assembly ( or nuclear paraspeckle 3 b s ). The latter allows ASO distribution throughout throughout distribution ASO allows latter The ). Nlgn1 in vivo in e ) Graph displaying a uniform density of FUS/TLS motif frequency across long and short introns measured by the signal-to-noise signal-to-noise the by measured introns short and long across frequency motif FUS/TLS of density a uniform displaying ) Graph Supplementary Fig. 8b Fig. Supplementary ) in a characteristic sawtooth-like pattern. ( pattern. sawtooth-like a characteristic ) in Fig. 3 Fig. FUS/TLS depletion FUS/TLS ). No acute abnormal phenotype was was phenotype abnormal acute No ). a ). ) or bolus injection into the lateral lateral the into injection bolus or ) Meg3 ), ), imprinted in Prader-Willi 23 ). Expression levels levels Expression ). , 2 4 5kb . Bolus injection injection Bolus . a FUS protein d ) Mouse (light green) and human (dark green) FUS/TLS or TDP-43 (purple) binding binding (purple) TDP-43 or FUS/TLS green) (dark human and green) (light ) Mouse ) Graph showing enrichment of FUS/TLS, but not TDP-43 or RBFOX2, binding at the the at binding RBFOX2, or TDP-43 not but FUS/TLS, of enrichment showing ) Graph Fig. Neat1 c

0 48 0 1023 0 291 3 ) Model for the deposition of FUS/TLS on long introns during transcriptional transcriptional during introns long on FUS/TLS of deposition the for ) Model c - - - - )

domain-containing protein 3 protein ( domain-containing ( protein cyte ( 4 protein interacting Fig. 8a ( cord spinal versus striatum in depletion of FUS/TLS level the with correlated downregulation of degree the Notably, tion PCR (qRT-PCR) of striatal and spinal cord RNA samples ( tion were confirmed for selected RNAs by quantitative reverse ( twofold transcripthan more downregulated were genes 61 contrast, In fold. two than more by changed were genes upregulated the of four Only observed in the different biological replicates ( and lated ( by (defined upregulated were genes 275 that revealed samples ASO–treated trol striatum the in depletion TDP-43 from files replicates following ASO-mediated four biological comparison by ( determined as TDP-43, on dependent those from distinct completely almost was of RNAs a that set affected depletion of ( levels normal of reduction ~10% to RNA a confirmed FUS/TLS encoding gene ( differentially genes the replicates expressed of values RPKM biological of clustering independent hierarchical the using of groupings expected supported by was condition per reads of Pooling condition. either in RPKM of 0.5 threshold the satisfied that genes protein-coding tated 8c ( Fig. ASO control or FUS/TLS with treated replicates Supplementary Tables 2 Supplementary Fig. 8d Fig. Supplementary d b Statistical comparison of RPKM values between FUS/TLS- and con Normalized CLIP signal Human Mouse Supplementary TablesSupplementary 2

0 1 2 3 4 5 NLGN1 Nlgn1 ′ UTR, or a retained intron. ( intron. a retained or UTR, 2 0 z Binding onlongintrons(>100kb) ), as exemplified ), by as channel the exemplified potassium the genes encoding score <−1.96, score <−1.96, ) were used to identify mRNAs encoded by 13,627 anno 13,627 by encoded mRNAs identify to used were ) TDP-43 CLIP FUS CLIP FUS CLIP advance online publication online advance Intronic position(% 4 0 Fox2 CLIPclusters TDP-43 CLIPclusters GUGGU motif FUS CLIPclusterswith FUS CLIPclusters z score >1.96, >1.96, score Mal 6 0 ), and the histone methyltransferase Set and Mynd and Set methyltransferase histone the and ), Supplementary Fig. 8d Fig. Supplementary 8 0 P Kcnip4 < 0.05) following FUS/TLS depletion ( < depletion FUS/TLS following 0.05) ) of similarly obtained RNA pro obtained expression ) of similarly ) 1 and 0 5 P . < 0.05) and 355 genes were downregu were genes 355 and 0.05) < 100 and 3 ), parkin ( parkin ), Smyd3 ). Reduced levels after FUS/TLS deple b ) FUS/TLS bound to long transcripts transcripts long to bound ) FUS/TLS Supplementary Fig. 8e Fig. Supplementary 3 e ). ). Similar expression changes were Normalized motif presence 0 1 2 3 4 5 ) ) ( 2 0 Fig. 3 Fig. Park2 FUS motifpresenceonintrons

). The RPKM ratio of the the of ratio RPKM The ). nature nature 4 0 Intronic position(% e Supplementary Fig. 9 ), myelin and lympho and myelin ), ). 6 0 on shortintrons on longintrons Supplementary Supplementary Supplementary Supplementary NEUR 200kb 8 0 Fus/Tls ). FUS/TLS FUS/TLS ). ) OSCI 0 Fig. 3 ( > ( Fig. Fig. 3 < 100 100 kb) ( 10 kb) EN Fus 0 28 0 307 0 721 e C ). ). d E ­ ------)

© 2012 Nature America, Inc. All rights reserved. with crucial functions in synaptic activity, such as as such activity, synaptic in functions crucial with had more proteins than 600 clusters each and encoding those include genes long Exceptionally pre-mRNA. per sites FUS/TLS-binding 94 RNAs, with the hundred most reduced RNAs containing an average of ment of sites FUS/TLS-binding was found in the most downregulated of bp,51,687 (average or upregulated unaffected were that those than bp) 158,997 of (average longer threefold were most the downregulated were that genes the that revealed depletion FUS/TLS by affected RNAs to ysis contains numerous TDP-43–binding sites of each which long introns, exceptionally of with mRNA genes levels the for maintaining that TDP-43 Weis crucial established previously RNAs long processing in intersect TDP-43 and FUS * replicas. biological three in s.d. represent bars Error tissues. TDP-43–depleted and FUS/TLS- ( both. or TDP-43 FUS/TLS, targeting ASOs ( quadrants. other all to compared downregulated, commonly genes in length intronic increased the and clusters FUS/TLS and TDP-43 of density high the showing graphs Bar diamonds). (yellow directions opposite in regulated genes few and respectively, upregulated, or downregulated similarly were that genes 41 and 45 only with FUS/TLS, or TDP-43 either of depletion following misregulated genes ( gene. downregulated and upregulated each for count cluster Inset, plotted. were genes 100 next the for line) (blue length intron total mean the or line) (green ( depletion FUS/TLS after regulation of degree their on ranked were Genes sites. FUS/TLS-binding ( replicas. biological three in s.d. represent bars Error ASO. control with compared knockdown ( striatum. the in depletion FUS/TLS following (green) downregulated or (red) upregulated ( treatment. ( respectively. ventricle, lateral the or striatum the into ASOs of injection by ( striatum mouse in FUS/TLS of depletion ( cord. spinal and brain in TDP-43 or FUS/TLS either of KD) (knockdown, depletion after targets TDP-43 and FUS/TLS of expression 3 Figure nature nature Nlgn1 f c a e GAPD ) ) qRT-PCR for Total intron length (kb) FUS 100 200 300 400 500 H 0 , , g Nlgn3 upregulated Most ) Scatter plot comparing RNA level alterations following TDP-43 or FUS/TLS depletion. Venn diagrams showing the number of overlapping overlapping of number the showing Venn diagrams depletion. FUS/TLS or TDP-43 following alterations level RNA comparing plot ) Scatter Control

NEUR Changes in the the in Changes a Intrastriatal injection

) and spinal cord ( cord spinal ) and Cluster count d 1,000 1,200 ) RNA-seq analysis identified 355 and 275 genes that were significantly significantly were that genes 275 and 355 identified analysis ) RNA-seq a 200 400 600 800 c and P , of ASOs Striatum ) Immunoblot of FUS/TLS after FUS/TLS ASO, control ASO or saline saline or ASO control ASO, FUS/TLS after FUS/TLS of ) Immunoblot b 0 Dissection of OSCI the striatum < 5.4 × 10 Kcnip4 ) Strategy for for ) Strategy FUS Upregulated Kcnip4 EN Saline , , C regulated Park2

E ( Not

−9

Fig. 3 by advance online publication online advance Downregulated b

) , ,

b

Mal

t

test; f Saline and and and GAPDH h TDP-43 downregulated Spinal cord Fig. Fig. 3 FUS Supplementary Supplementary Tables Smyd3 i ) ) qRT-PCR for f 1 Most FU ). Furthermore, ). an Furthermore, enrich 5 . . Applying the same anal in striatum and spinal cord with FUS FUS with cord spinal and striatum in Saline S Intraventricular injection

0 10 20 30 40 50 60 70 80 90 100 Mean cluster count in introns in count cluster Mean of ASOs FUS KD Nrxn1 Kcnip4 g

Binding sites (×10) Binding sites (×10) the spinalcord Dissection of 180 180 FUS down,TDP-43down 90 90 FUS down,TDP-43up 0 0

FUS FUS 1 1 , , , , TDP-43 KD Nrxn3 TDP-43 Park2

and TDP-43 45 genes 5 genes

2 Intron Intron length length , , ). ).

- -

, Smyd3 FUS

TDP-43 KD d 800 0 400 800 0 400

does not affect the steady-state levels of mRNAs once they reach reach they once mRNAs cytoplasm of the levels steady-state the affect not does FUS/TLS that proposal recent the with consistent targets, 3 FUS/TLS the in seen was binding FUS/TLS in ment reduction TDP-43 after upregulated are that 3 the in observed viously loss. consistent FUS/TLS of are consequence indirect an as upregulation arising levels of expression altered with degree modest infrequent and The sites targets. binding FUS/TLS upregulated most hundred the among sites binding from six RNAs only of on average an with binding genes, of upregulated density lower much a with contrast in was RNAs downregulated on binding FUS/TLS of prominence The + Expression in FUS knockdown

In contrast with the increased number of TDP-43–binding sites pre (log2 RPKM) Intron length (kb) length Intron , , (kb) length Intron 10 12 Expression incontrol(log –15 2 4 6 8 Nrxn3

i down down –12 335 335 Relative mRNA expression Upregulated genes(275) Downregulated genes(335) 8 6 4 2 , ,

Nlgn1 (% of saline) 45 5 100 120 140 160 –9 down 20 40 60 80 283 471 2 0 up f 5 ) Correlation between differentially expressed genes and and genes expressed differentially between ) Correlation P . –6 , , < 0.05, ** < 0.05, Kcnip4 Nkain2 x ** TDP-43 axis) and the mean number of intronic CLIP clusters clusters CLIP intronic of number mean the and axis) FUS KD Control oligonucleotide Saline –3 FUS 2 –15 –12 RPKM) –9 –6 –3 12 15 h 3 6 9 ) Immunoblot in mouse brains treated with with treated brains mouse in ) Immunoblot and and Park2 10 ′ z UTR of genes, such as progranulin ( progranulin as such genes, of UTR score ** P 3 < 0.01, < 0.01, Csmd1 e Smyd3

6 Relative mRNA expression 275 ** up 275

up (% of control oligonucleotide) 100 120 140 in FUS/TLS-, TDP-43–, or both both or TDP-43–, FUS/TLS-, in 21 20 40 60 80 41 9 FUS t 0 test. test. down TDP-43 FUS 283 471 up Nrxn3

Kcnip4 12 z score * FUS +TDP-43KD TDP-43 KD * FUS KDspinalcord FUS KDstriatum Control oligonucleotide 15 ′ 1 Nlgn1 UTR of upregulated upregulated of UTR 5 Binding sites ( 10) Binding sites ( 10) Park2 180 ×180 × , no similar enrich similar no , ** 90 90 FUS up,TDP-43down 0 0 t r a FUS FUS FUS up,TDP-43up

TDP-43 TDP-43 21 genes 41 genes Nkain2 Ma ** Intron Intron l C I length length

Smyd3 Csmd1

e l 0 400 800 0 400 800 *

Grn *

Intron length (kb) length Intron Intron length (kb) length Intron s ), ),  - -

© 2012 Nature America, Inc. All rights reserved. even for these RNAs, TDP-43 and FUS/TLS act in parallel rather rather parallel in act FUS/TLS and TDP-43 RNAs, these for even apse formation and activity; syn for important are that genes in enriched are these (which of genes long expression of levels the on effect greater no had FUS/TLS and TDP-43 both of Depletion loss. FUS/TLS following than loss 43 TDP- following reduced more consistently were transcripts selected ( in proteins both of as knockdown with well samples the as depletion, FUS/TLS or TDP-43 either after regulated down are that genes intron–containing long several for qRT-PCR 10b Fig. Supplementary ( alone ASO either injecting by to achieved similar depletion respectively, the levels, normal of 30% and 10% to reduced were TDP-43 and FUS/TLS for protein and RNA weeks, 2 after and, system ( nervous adult the in simultaneously both depleted we targets, their ( FUS/TLS for longer enrichment introns and sites binding for both TDP-43 and marked a bearing genes downregulated with protein, deple either to of tion response in respectively, downregulated, or upregulated ( genes TDP-43 FUS/ on or TLS dependent are expressed that RNAs of sets distinct 13,627 expression highly revealed all of of changes comparison Systematic TDP-43 and FUS/TLS both on depends RNAs of set A small ( depletion TDP-43 ( depleted was 43 TDP- or FUS/TLS when directions opposite in ( depleted was TDP43 or FUS/TLS either when direction similar in changed ( depletion FUS/TLS after only changed that events splicing of validations RT-PCR ( brain. mouse adult in depletion TDP-43 published previously and depletion FUS/TLS of result a as events splicing alternative changing significantly ( s.d. represent bars error group; per replicas biological three of exclusion) to inclusion of (ratio quantification the show Graphs controls. Fus/Tls from brain embryonic and brain mouse adult in depletion FUS/TLS ASO-mediated following changes splicing alternative of analyses ( indicated. are (anti-regulated) direction opposite and (co-regulated) direction same the in changing from brain embryonic in and brain mouse adult in depletion FUS/TLS following events splicing alternative changing significantly of overlap ( 2 kb. in clusters FUS/TLS contain that microarray sensitive splicing- by detected as depletion, FUS/TLS following included or unchanged excluded, were that exons of percentage Bottom, (top). 2 kb within clusters FUS/TLS contain that exons the of all or spliced, constitutively or (cassette) excluded alternatively of percentage the displaying (right) plots bar and (left) ( brain. mouse in splicing 4 Figure t r a  synergistically. than d

) Venn diagram comparing the overlap of of overlap the comparing diagram Venn ) To explore whether TDP-43 and FUS/TLS act synergistically on on synergistically act FUS/TLS and TDP-43 whether explore To Fus/Tls −/−

C I Supplementary Fig. 10a Supplementary FUS/TLS-dependent alternative alternative FUS/TLS-dependent mice compared with the respective respective the with compared mice c Fig. 3 Fig. −/− ) Semi-quantitative RT-PCR Semi-quantitative ) e l mice. The number of events events of number The mice. b s g g ) Venn diagram showing the the showing ) Venn diagram ), or changed only after after only changed or ), h ). Fig. 3 Fig. e ). – h ) Semi-quantitative Semi-quantitative ) a ) Schematics ) Schematics g ). Levels of expression were confirmed by confirmed were expression of Levels ). ). Only 41 and 45 genes were similarly similarly were genes 45 and 41 Only ). Supplementary Supplementary Table 4 f ). ). ASOs targeting both were injected ), changed changed ), e ), ),

Fig. 3 Fig. a e c Ptk2b hnRNP-D Mapt Junction array EST/mRNA ), ), indicating that, Ttc3 Kcnd3

Ndrg2 defined defined Ddef2 i Mapt ). Of note, the the note, Of ). Ttc3 Control Fig. 3 Fig. Control FUS specific FUS h FUS depletion and and KD Constitutive Unchanged Adult brain TDP Excluded All exons Cassette Included - - - 1. 0. 0.

0 5 0 4 0 6 0 2 0 1 0 4 FUS +TDP Control ing was found ~200 nucleotides upstream of ing upstream was found exons nucleotides following ~200 skipped unchanged were identified. A significant enrichment of remained FUS/TLS that bindthose versus exons regulated the around binding TLS reports recent in as ent, recognition. Although no strong exon rules for FUS/TLS splicing repressing regulation were in appar role direct a having FUS/TLS with ( boundary exon the of kb 2 within sites FUS/TLS-binding have tion deple FUS/TLS following included differentially exons cassette 114 teins, including TDP-43 However,(refs. 15,17). more than 55% of the after depletion of other heterogeneous nuclear ribonucleoparticle pro (374) is relatively small compared with the number of changes observed Notably, the number of FUS/TLS-dependent splicing events identified ( tion deple ASO-mediated FUS/TLS following changed that brain adult in events splicing 374 identified we or arrays, splicing-sensitive included using alternatively of proximity ( exons ‘cassette’ excluded the in enriched are sites FUS/TLS-binding exons, constitutive to relative that, determine to initial test, we used publicly available ref. EST/mRNA in transcript databases (reviewed selection to site splice proposed RNA in been participate has factors, other with association in FUS/TLS, targets RNA its of splicing alternative mediates FUS/TLS P Percent ofexonswithFUSbindingwithin2kb 0 < 0.05, compared to ~38% of unchanged exons; KD Supplementary Fig. 11a Fig. Supplementary 10 Kcnd3 Ndrg2 T ia1 advance online publication online advance WT f 20 Control Embryonic brain FUS knockout KO 30 Co-regulated FUS 0.6 0.3 0.4 1.5

0 0 0 0 2 0 0 4

< 10 < 40 P

TDP WT –76 26 , FUS +TDP P 2 50 KO 7 < 10 < , position-specific preferences for FUS/ for preferences position-specific , and and

60

More inclusion More exclusion More < 0.05 < −76 P Supplementary Tables5 Supplementary , , Ppp3ca χ d b h Dnajc5 Kcnip2 g 2 Sort1 FUS depletion test; test; Ma Adult brain 307

291 g FUS depletion 5 anti-regulated 78 co-regulated Contro nature nature Contro Adult brain Fig. 4 Fig. TDP-43 specific F l 2 anti-regulated 65 co-regulated Anti-regulated F l 83 Fig. Fig. 4 TDP-43 depletion US US a Adult brain NEUR ). Furthermore, Furthermore, ). 67 TD TD Embryonic brain a FUS knockout F P F P ), ), consistent 14). As an an As 14). OSCI US +TD US +TD and and 705 233 EN P P 6 C ). ). E - - - - -

© 2012 Nature America, Inc. All rights reserved. of either FUS/TLS or TDP-43 in adult brain revealed that TDP-43 TDP-43 that revealed brain adult in TDP-43 or FUS/TLS either of dementia temporal of exon inclusion the 10 to are fronto linked that mutations increase MAPT ( 1 Tables respectively protein, FUS/TLS of depletion after (mm9 chr11: in 104,171,848–104,182,664) adult and embryonic brain 10 exon and 104,143,794–104,156,172) chr11: (mm9 4 and 3 exons 4 Fig. human the of 6 variant transcript on 10 of the classical exon 9 of the human mouse the in annotated exon not upstream included to an but genome, alternatively corresponds is that exon 54-bp a of inclusion the dementia frontotemporal in mutated disease astrocytes in expressed is eases, such as the N-myc downstream-regulated gene 2 ( Fig. ( changes splicing FUS/TLS-dependent ( brains adult or embryonic in either loss FUS/TLS after direction same in the altered adult brains, splicing of 65 of these 67 RNAsFUS/TLS-dependent was and embryonic of patterns splicing alternative the in differences tal altered by from depletion FUS/TLS adult brain. also Despite developmen was splicing whose RNAs in occurred ( mice splicing 300 from brains embryonic in events identified we microarrays, splicing-sensitive Using death. perinatal by FUS/TLS full-length of ( loss complete a mice an by confirmed was ing 11b Fig. Supplementary ( loss FUS/TLS following ing in multiple regions around exons included bind significant and exon), the of inclusion FUS/TLS depletion (that is, FUS/TLS promoted by * group. each in replicas biological two least at in s.d. represent bars Error neurons. differentiated in depletion FUS/TLS or 43 ATXN1, KCND2 of ( neurons. differentiated in depletion ( NPCs. in depletion FUS/TLS or TDP-43 after NLGN1 ( NPCs. in depletion TDP-43 or FUS/TLS of ( (shControl). shRNA or (shTDP-43) targeting shRNAs encoding lentiviruses with transduction by followed cells, ES human from neurons and NPCs (EBs), bodies ( cells. ES from derived neurons in targets human 5 Figure nature nature FUS/TLS than alterations splicing of (788) number larger a affected d c upeetr Fg 11c Fig. Supplementary ) ) qRT-PCR of ) qRT-PCR validation of of ) qRT-PCR validation Comparison of depletion Comparison the changes splicing after ASO-mediated these confirmed RNAs selected of RT-PCR Semi-quantitative splic alternative in FUS/TLS for role A PARK2 t test.

12 2 8 c a Supplementary Fig. 11a Fig. Supplementary , in which gene disruption produces produces disruption gene which in , exon 10 is of particular relevance for disease pathogenesis, as pathogenesis, for disease relevance of is particular 10 exon , , ). Splicing arrays also identified increased inclusion of of inclusion increased identified also arrays Splicing ). ), including transcripts implicated in neurodegenerative dis neurodegenerative in implicated transcripts including ), 3 ) Schematic for generation of embryoid embryoid of generation for ) Schematic

NKAIN2, ATXN1, KCND2 NKAIN2, 0 NEUR Alterations of TDP-43 and FUS/TLS FUS/TLS and TDP-43 of Alterations , , , and the microtubule-associated protein tau ( tau protein microtubule-associated the and , and SMYD3 5 FUS/TLS OSCI PARK2 and and ). Identification that FUS/TLS suppresses splicing of of splicing suppresses FUS/TLS that Identification ). Fig. 4 Fig. , , NRXN3 KCNIP4 b 3 EN 1 ) qRT-PCR validation ) qRT-PCR validation , , . (shFUS), or a control a control or (shFUS), SMYD3 b C ). E FUS/TLS

). , , a P NLGN1 after either TDP- either after lysis of of lysis advance online publication online advance < 0.05 by 0.05 < 2 9 ), accompanied accompanied ), and mis-accumulated in Alzheimer’s Alzheimer’s in mis-accumulated and , , NRXN3 ), ), 67 of which and and P or or < 0.01 < 0.01 , , e Fus/Tls Fus/Tls NKAIN2, NKAIN2, MAPT ) ) qRT-PCR TDP-43 IPW TDP-43 , 3 χ 1 . Loss of FUS/TLS resulted in in resulted FUS/TLS of Loss . 2

test; test; Fig. 4 Fig. MAPT

gene (also reported as exon −/− −/−

- -

c and and gene (UCSC, hg18); hg18); (UCSC, gene d b a ES cell Neurons Supplementary Supplementary Supplementary Supplementary NPC relative mRNA expression relative mRNA expression Ndrg2 (% of control) (% of control) 100 120 TDP-43 knockdown FUS knockdown Control Mapt 100 120 140 20 40 60 80 20 40 60 80 0

0 ), which ) gene gene ) FUS FUS Mapt * * EB TDP-43 - - ­ TDP-43

* * NPCs treated with a scrambled shRNA ( a scrambled with treated NPCs to 2 and 15% of their normal levels, respectively, when compared that with revealed qRT-PCR human (shRNAs). RNAs hairpin short encoded (ES) ( cells stem embryonic human from generated (NPCs) pro cells neural genitor of differentiation by neurons human in FUS/TLS and We tested the dependence of long intron–containing genes on TDP-43 neurons differentiated human in RNAs of Alterations receptor Nogo protein that inhibits axonal regeneration through interaction with the ( gene glycoprotein myelin-associated the of 12 exon is TDP-43 and FUS/TLS between events splicing regulated ( exons affected the of kb 2 within sites TDP-43–binding of despite TDP-43, loss the after found ( ( gene channel voltage-gated potassium the of 6 exon and granules, stress RNA-containing of component central a is protein encoded of whose 5 exon Conversely, depletion. FUS/TLS or TDP-43 either ( PCR RT- semi-quantitative by validated were targets RNA FUS/TLS and ( direction same the in excluded or included differentially were which of 94% depletion, or TDP-43 FUS/TLS following regulated that were events commonly (374) ( Fig. 4 Fig. Lentivirus: Fig. 5 Fig. Fig. 4e Fig. Kcnd3 Neural rosette Fig. Fig. 4 f FUS/TLS ). FUS/TLS-dependent changes in splicing of of splicing in changes FUS/TLS-dependent ). e c Neurons NPC

a relative mRNA expression d relative mRNA expression ) were more included after loss of TDP-43 or FUS/TLS FUS/TLS or TDP-43 of loss after included more were ) ) and depletion of TDP-43 or FUS/TLS using TDP-43 FUS/TLS of or depletion and ) – shTDP-43 and (% of control)

h (% of control) 3 100 120 140 140 120 100 2 20 40 60 80 ). Exon 8 of of 8 Exon ). 20 40 60 80 ( 0 ( 0 Fig. 4 Fig. FUS +FGF-2 Supplementary TableSupplementary 6 PARK2 PARK2 Fig. 4 Fig. shControl * * ) ) and * *

g SMYD3 SMYD3 and and d RNA * ). Splicing alterations of selected TDP-43 selected of alterations Splicing ). TDP-43 NPC * * Ndrg2 Fig. 4 Fig.

Supplementary Table 6 Supplementary NRXN3 NRXN3 * * * showed increased exclusion after after exclusion increased showed e ( NLGN1 NLGN1 ). A rare example of differently differently of example rare A ). TARDBP – FGF-2 shFUS * * * * Fig. 5 Fig. NKAIN2 NKAIN2 ). ). We 83 identified splicing * * Mag * ) ) mRNAs were reduced * b ATXN1 shControl

). Of the target genes genes target the Of ). ATXN1 ), which encodes a encodes which ), * t r a * * Neurons *

KCND2 RNA KCND2 Mapt ). * * C I KCNIP4 lentiviral- were not not were shTDP-43 IPW * * * e l * Tia1 s  - ,

© 2012 Nature America, Inc. All rights reserved. a a one carrying patients: ALS symptomatic three from fibroblasts mary pri in transcripts intron–containing long of levels mRNA tested we neurons human TDP-43 differentiated in 13 ( Fig. examined we that genes the of intron–containing seven long all of downregulation revealed RNAs resultant the of ( shRNAs independ two lentiviral-encoded of ent either using depleted was TDP-43 endogenous individual, from an generated unaffected cells stem (iPS) pluripotent Similarly, into after ( differentiation a of lineage pan-neuronal induced respectively controls, of 33% and 23 to reduced were TDP-43 or FUS/TLS after cells ES human from differentiated rons and targets pre-mRNAs of these intron long of levels the sustaining in proteins both firming an intersecting and conserved role for ( TDP-43 or FUS/TLS of either in Prader-Willi ( ( 1 ( ( 1 target ciated acting 2 or T-cell lymphoma breakpoint asso ( ( SET and MYND domain-containing protein 3 intron– long ( parkin for pre-mRNAs containing NPCs, in expressed reliably loss. TDP-43 by altered be to expected not were levels whose and target a TDP-43 not is which staining, tubulin of levels normal showed (>80%) cells inclusion–bearing TDP-43 of majority the contrast, In staining. SMYD3 or Parkin KCNIP4, of levels reduced had inclusions TDP-43 with neurons of 70%) (~60– majority the that revealed individuals 3 control and patients sALS 11 of a total from neurons motor individual in levels tubulin or ( tubulin. of levels normal contained levels, Parkin reduced had and aggregates TDP-43 contained which left, the on cell the that Notice (red, (green, Parkin for sample autopsy cord spinal a sALS from ( in highlighted n SMYD3. or Parkin KCNIP4, of levels decreased markedly had inclusions TDP-43 bearing Neurons localization. cytoplasmic mainly expected, the showed SMYD3 and Parkin KCNIP4, aggregation, TDP-43 without k a (red, TDP-43 ( patients (sALS) ALS sporadic ( individuals non-ALS control, from samples autopsy cord spinal in localization ( patients. ALS sporadic from neurons motor in targets RNA 6 Figure t r a  in levels detectable in expressed targets the of One mutation. p t KCND2 NLGN1 SMYD3 – – and and

) Quantification of KCNIP4, Parkin, SMYD3 SMYD3 Parkin, KCNIP4, of ) Quantification FUS/TLS – o e After After identifying targets affected by the loss of TDP-43 or FUS/TLS, ATXN1 s ). In both control and sALS motor neurons neurons motor sALS and control both In ). (green, Parkin ), ) Triple immunofluorescence for images images for ) Triple immunofluorescence r ). The merged images are shown in in shown are images merged The ). o – are higher magnifications of the areas areas the of magnifications higher are ), confirming a dependency of long pre-mRNA levels on on levels pre-mRNA long of dependency a confirming ),

containing genes in human neuronal progenitors. Reduction Reduction progenitors. neuronal human in genes containing C I ), Na ), and the noncoding RNA imprinted ), ), neurexin 3 ( Reduction of TDP-43 and FUS/TLS FUS/TLS and TDP-43 of Reduction ), ), potassium voltage-gated channel R521G e l a + b p – a /K , , ), tubulin (blue, (blue, tubulin ), o – c ) and either KCNIP4 (green, (green, KCNIP4 either ) and o NKAIN2 IPW mutation and two carrying the same same the carrying two and mutation s + , , ) Immunofluorescence ) Immunofluorescence transporting ATPase inter g f , , – h j ) ) were reduced after loss ) or SMYD3 (green, (green, SMYD3 ) or , , NRXN3 l and and or or m b TCBA1 – , respectively. , respectively. q ), ), neuroligin 1 e Fig. 5 Fig. ) and TDP-43 TDP-43 ) and , g a KCNIP4 – , d f j , upeetr Fg 13 Fig. Supplementary , , , k l PARK2 ), ataxin ataxin ), – e ) ) or c o , , ), con ), s. ) for ) for i

, ,

j were also observed in neu in observed also were , in in vitro ), ), - - -

. t Supplementary Supplementary Sporadic ALS SMYD3/TDP-43/Dapi PARK2/TDP-43/Dapi KCNIP4/TDP-43/Dapi Percentage of neurons with TDP-43 Fig. k a p 50 reduced KCNIP4, PARK2, f

SMYD3 or tubulin µ 100 m ). Assay Assay ). 10 20 30 40 50 60 70 80 90

5a 0 PARK2 , G298S d No TDP-43 aggregate Control , n e =97 ). ). - - -

KCNIP4 s whether TDP-43– and FUS/TLS-dependent RNAs from genes with with genes from RNAs FUS/TLS-dependent and TDP-43– whether staining nuclear of loss a by accompanied neurons, sporadic ALS patients, aggregates of cytoplasmic of TDP-43 accumulate in motormajority vast the in but, rare, extremely are mutations FUS/TLS carrying Patients disruption. TDP-43 or FUS/TLS either ( targets common of set small this of levels the that predict data Our neurons motor patient ALS in altered pre-mRNAs Long conclusive. more prove ultimately may neurons motor cell–derived iPS using studies further that suggests and experiments these from conclusions firm drawing from us prevented individuals control other from lines last of fibrob the gene amidst different variability expression hypothesis, (data not shown). Although this preliminary evidence agreed individual with our non-ALS a from fibroblasts control with compared lasts fibr Supplementary Tables 2 2 Tables Supplementary 50 aggregates TDP-43 µ n o =35 m blast cells, cells, blast q b g l advance online publication online advance No TDP-43 aggregate Tubulin n =28 KCND2 PARK2 3 s aggregate TDP-43 n =78 , was substantially reduced in mutant fibrob reduced , substantially was r s and No TDP-43 aggregate n Sporadic ALS =94 3 m TDP-43 h c ) will be reduced in neurons with with neurons in reduced be will ) SMYD3 s aggregate TDP-43 n =24

nature nature s s No TDP-43 aggregate n =22 2 Tubulin 0 , Merge NEUR 3 s . To determine To . determine i d o j e n aggregate TDP-43 n OSCI =49

s EN C E - - © 2012 Nature America, Inc. All rights reserved. ( harbor numerous intronic binding sites for( both FUS/TLS TDP-43 or TDP-43 and either FUS/TLS of loss the after downregulated FUS/TLS pathways in the regulation of a subset of transcripts that were and 78 gene regulation and splicing events in common, respectively. levelsand processing of largely of distinctsets RNAs, and hadonly 86 changed. We found that FUS/TLS and TDP-43 affected the expression els of >600 mRNAs were altered and the splicing patterns of >350 were FUS/TLS leads to a broad misregulation of RNA processing, as the of lev depletion that demonstrate also results Our pre-mRNAs.long on largely distinct from those of TDP-43, including a sawtooth-like pattern thousands of mouse and human brain pre-mRNAs at positionsgenesis of thatALS and wereFTLD. We identified extensive FUS/TLS binding on CNS for TDP-43 and FUS/TLS, proteins that are involved in the patho ing patterns and roles in gene regulation and alternative splicing in the Our study provides a genome-wide, systematic comparison of the bind DISCUSSION ( TDP-43 aggregates was not altered in the large majority (85%) of motor neurons carrying protein that was not predicted to be regulated by TDP-43( or FUS/TLS, SMYD3 for staining normal had with TDP-43 inclusions, whereas 95% of motor non-affected neurons neurons motor of 58% in reduced also was SMYD3 protein plasmic ( levels parkin cytoplasmic strong neurons, motor 95% whereas of motor neurons without TDP-43 displayed aggregates inclusion–containing TDP-43 of 70% in reduced ( fluorescence KCNIP4 of motor 71% neurons with contrast, TDP-43 In aggregates were patients. characterized by ALS reduced sporadic and individuals control in both in observed TDP-43 aggregates identified without of motor neurons 92% was KCNIP4 of staining cytoplasmic granular strong ( inclusions cytoplasmic KCNIP4 parkin, or and SMYD3 fluorescence the a strong Nevertheless, correlation was apparent in reduction between duced a reproducible immunofluorescent staining of nuclear TDP-43. With the conditions used, ( neither of the TDP-43 inclusions antibodies tested TDP-43 pro primarily recognize both TDP-43 of that part C-terminal the for specific antibody clonal with either a polyclonal antibody forspecific performed phospho–TDP-43 or was a mono cords spinal of staining FUS/TLS. or Immunofluorescence TDP-43 on dependent is maturation whose mRNAs of pre- long by encoded SMYD3) either and (PARK2, KCNIP4 proteins three and TDP-43 for co-labeling by individuals control 3 patients and ALS sporadic 11 from samples autopsy in neurons motor individual assayed we Consequently, neurons. affected less or more from RNAs of pooling by confounded be might function TDP-43 of sis of altered expression of long intron–containing genes following loss patient motor neurons ( A5 Ephrin of exception the with significance, statistical reached none although transcripts, these of each in tions ( factor ( activator secretion calcium-dependent (including genes containing 1,000 motor approximately neurons of from 12 ALS patients pooling and microdissection laser after obtained reanalyzed initially of RNAsanalysis we microarray from a published alterations expression ALS, sporadic in affected are introns long nature nature encode protein products that are crucial for normal neuronal function Figs. Figs. 2 Nevertheless, our evidence identifies intersection of the TDP-43 and in pathology TDP-43 of heterogeneity well-recognized the Given FGF14 and NEUR

3 ), ), contain exceptionally long introns ( OSCI ) and Ephrin A5 ( A5 Ephrin and ) EN Fig. 6p Fig. C 3 4 E Fig. , , we that analy reasoned a microarray-based Fig. advance online publication online advance –

6a 6 t ). and and PARK2 – EFNA5 e , Fig. t ). Similarly, parkin was strongly strongly was parkin Similarly, ). Supplementary Fig. 15 Fig. Supplementary Fig. 6k Fig.

, , 6f )) revealed a trend for reduc for trend a revealed )) KCNIP4 – CADPS 3 j upeetr Fg 14 Fig. Supplementary , 3 – t . . Analysis of long intron– o ). The nuclear and cyto and nuclear The ). , t ). Notably, tubulin, a tubulin, Notably, ). P = P

, , ), fibroblast growth growth fibroblast ), presence presence of TDP-43 Figs. SMYD3 0.0123).

2 2 and , ,

). Indeed, Indeed, ). NLGN1 Fig. Fig. 3) , and 3 ). ). ), ), ------, dicts that a combined reduction of multiple targets contributes to, contributes targets multiple of reduction combined a that dicts pre patients ALS from neurons motor affected in altered are targets models mouse and patients function human in neuronal compromised to leads individually KCNIP4 or parkin either of loss the that given Furthermore, pathogenesis. ALS to contributes FUS/TLS or TDP-43 of function of loss that supports ( pre-mRNAs long by encoded teins by of of loss pro TDP-43 is accompanied mis-accumulation plasmic cyto where cords, spinal ALS sporadic in neurons motor individual in case the indeed is this that Wefound proteinopathies. FUS/TLS and TDP-43 in death neuronal to contributes RNAs levels target the these of of reduction disease, result in a aggregation as and/or FUS/TLS mutation or of TDP-43 either of interruption functional ( 13 Fig. cells human Supplementary in FUS/TLS or TDP-43 on pre-mRNAs intron–containing long of dependency the confirm cells iPS and ES in a analysis previous used cells) (293T line cell ized are appreciably by (as expressed revealed RNA-seq brain data) in an immortal mouse adult the in depletion TDP-43 or FUS/TLS either following downregulated are RNAsthat 45 the of none example, For not be identifiable by similar analyses performed in non-neuronal cells. ( (microarray, RNAseq and CLIPseq). and RNAseq (microarray, codes. Accession the o in version available are references associated any and Methods M TAF15 in alterations FTLD. and ALS mediate by to shown recently were that proteins ing affected also are or 47,48) (refs. RNA/DNA EWSR1 two additional 49,50), (refs. bind uncovered we that pathways FUS/TLS and TDP-43 intersecting the whether determine there for recruitment its responsible may be regions promoter to complementarity with ncRNAs on FUS/TLS of complex TFIID the and II polymerase RNA with FUS/TLS of association the with junction single- cells HeLa with human in regions interacts promoter in DNA FUS/TLS stranded that observation recent very The of RNAs. deposition nascent intron–containing of long sites the on multiple FUS/TLS mediates mechanism similar a that conceivable cytoplasm the to transport mRNA lowing fol interaction this maintaining by RNAs those of decay regulating thereby mRNAs, nascent onto deposited co-transcriptionally be can elongation. transcriptional transcripts nascent to binding for expected is as pattern, sawtooth-like a in introns long (ref. SC-35 protein, RNA-binding another for shown been has what to similar elongation, to bound introns these may transcription ecules facilitate splicing events. Alternatively, multiple TDP-43 and/or mol FUS/TLS ensures of the splicing normal that long introns, preventing unproductive scaffold protein a as serve may introns long TDP- to of FUS/TLS or binding 43 that is these possibility of attractive An levels proteins. two the for the identical be not may sustains or may and sites unknown remains ­transcripts intronic multiple on binding and proteinopathies. FUS/TLS TDP-43 in death neuronal causes, singlehandedly than rather Supplementary Table et Analyses in progenitors and 1-month-old neurons derived from from derived neurons 1-month-old and progenitors in Analyses A recent study found that proteins recruited to promoter regions regions promoter to recruited proteins that found study recent A FUS/TLS or TDP-43 which by mechanism molecular exact The h ods f the pape the f 41). In fact, our observation that FUS/TLS bound along along bound FUS/TLS that observation our fact, In 41). Gene Expression Omnibus: series series Omnibus: Expression Gene r 22 . , 4 2

4 , supports the idea that it may be involved in in involved be may it that idea the supports , 4 ). These distinct attributes of target genes can ). Our findings predict that, in cells with with cells in that, predict findings Our ). 5 , supports this view. Alternatively, binding binding Alternatively, view. this supports , Fig. Fig. 36– 6 ). Our combined evidence evidence combined Our ). 4 0 4 4 , our finding that these these that finding our , 3 6 . Mindful of this, it is is it this, of Mindful . . It will be necessary to necessary . be It will t r a 3 5 . Fig. Fig. C I GSE4065 4 4 , in con in , online online e l 5 and and s 3  ------

© 2012 Nature America, Inc. All rights reserved. 18. 17. 16. 15. 14. 13. 12. 11. 10. 9. 8. 7. 6. 5. 4. 3. 2. 1. r at online available is information permissions and Reprints Published online at The authors declare no competing interests.financial and D.W.C. wrote the manuscript. G.W.Y. and D.W.C. the designed experiments. C.L.-T., M.P., A.Q.V.,K.R.H., G.W.Y. the ASO experiments. J.R. provided the ALS patient tissues. C.L.-T., M.P., K.R.H., Fus/Tls splice-junction microarray analyses. G.G.H. provided the brain samples from the antibody Ab3.FUS/TLS-specific J.P.D. and L.S. conducted the preliminary and T.Y.L. conducted the bioinformatics analysis. S.-C.L. developed the polyclonal C.L.-T., M.P., A.Q.V., M.B. and K.M.C. performed the S.C.H. experiments. K.R.H., G.W.Y. Fellowship. P. G.W.Y. Research Alfred Sloan the of recipient a is to RB3-05009) and (RB1-01413 Medicine Regenerative for Institute California the and GM084317) and (HG004659 Health of Institutes National US the from grants by supported partially was and Stroke, and Disorders Neurological of Institute National US the from R01NS075449 number grant by supported also was work This Fellowship. Research Graduate Foundation Science National US a by funded is S.C.H. Research. Cancer for Institute Ludwig the from support D.W.C. salary receives Association. Sclerosis Lateral Amyotrophic the from fellowship post-doctoral Milton-Safenowitz the and Association Dystrophy Muscular the Award from Development Career a of recipient the C.L.-T. is Organization. Program Science Frontier Human international the from fellowship M.P.). long-term to a of M.P. recipient the is K99NS075216 D.W.C. to and (R37NS27036 Health of Institutes National US the from grants by supported was work This project. this on suggestions and comments critical for Pharmaceuticals Isis at team the and laboratories Yeo Cleveland and the of members the as well as support, Jr. Ares generous M. for sequencing, Illumina the with help technical for Ye,Z. U. Kuan, S. Edsall, WagnerL. and Ren’sB. of especially members laboratory, thank to like would authors The Note: Supplementary information is available in the t r a 1 COM AUTH Acknowledgmen e 0 p

r

Lerga, A. S.C. Huelga, Tollervey, J.R. Polymenidou,M. FUS/TLS: and TDP-43 D.W. Cleveland, & M. Polymenidou, C., Lagier-Tourenne, I.R. Mackenzie, D.G. Munoz, Woulfe, J., Gray, D.A. & Mackenzie, I.R. FUS-immunoreactive intranuclear inclusions M. Neumann, C. Vance, T.J.Kwiatkowski, J. Sreedharan, E. Kabashi, M.A. Gitcho, Polymenidou, M. T.Arai, M. Neumann, in FUS/TLS and TDP-43 of role the D.W.Understanding Cleveland, & S. Cruz, Da factor TLS. factor proteins. hnRNP by splicing alternative of TDP-43. by regulation TDP-43.neuronalvulnerabilityof lossfrom (2010). R46–R64 emerging roles in RNA processing and neurodegeneration. and processing RNA in roles emerging and severity disease with correlating mutation. patterns distinct two mutations: FUS with Neuropathol. Acta disease. neurodegenerative in pathology. 6. type sclerosis lateral amyotrophic sclerosis. lateral amyotrophic familial sclerosis. sclerosis. lateral amyotrophic Neurol. Ann. Res. Brain Commun. Res. Biophys. sclerosis. lateral amyotrophic and degeneration lobar frontotemporal in sclerosis. lateral amyotrophic and beyond. and ALS i n t P s O −/− E / i n TI R R C I d mice. M.P., C.L.-T., C.M., E.W., A.S.K., A.W., S.F. and C.F.B. conducted CON e et al. et NG x et al. Science . Acta Neuropathol. Acta

Brain h t al. et 1462 e l FI J. Biol. Chem. Biol. J. t

m t al. et TDP-43 is a component of ubiquitin-positive tau-negative inclusions tau-negative ubiquitin-positive of component a is TDP-43 et al. et TRIBUTI 63 t al. et et al. et al. et Identification of an RNA binding specificity for the potential splicing N l et al. et t al. et . t al. et

, 535–538 (2008). 535–538 , h uain i FS a RA rcsig rti, as familial cause protein, processing RNA an FUS, in Mutations A t al. et , 3–15 (2012). 3–15 , 132 et al. s et al.et t

et al. et

Curr. Opin. Neurobiol. Opin. Curr. t Integrative genome-wide analysis reveals cooperative regulation cooperative reveals analysis genome-wide Integrative ADP uain i idvdas ih prdc n familial and sporadic with individuals in mutations TARDBP NC 319 118 p Characterizing the RNA targets and position-dependent splicing A new subtype of frontotemporal lobar degeneration with FUS with degeneration lobar frontotemporal of subtype new A D-3 35 mtto i fmla mtr ern disease. neuron motor familial in mutation A315T TDP-43 TDP-43 mutations in familial and sporadic amyotrophic lateral amyotrophic sporadic and familial in mutations TDP-43 : bqiiae TP4 i fottmoa lbr degeneration lobar frontotemporal in TDP-43 Ubiquitinated ts , 2922–2931 (2009). 2922–2931 , / U ptooy n aohlc nlso bd disease. body inclusion basophilic in pathology FUS / Misregulated RNA processing in amyotrophic lateral sclerosis. Longpre-mRNA depletion missplicingRNAand contribute to ahlgcl eeoeet i aytohc aea sclerosis lateral amyotrophic in heterogeneity Pathological IA w , 1668–1672 (2008). 1668–1672 , , 617–627 (2009). 617–627 , Nat. Neurosci. Nat. Mutations in the FUS/TLS gene on chromosome 16 cause 16 chromosome on gene FUS/TLS the in Mutations

w ON 351 L w

I . 276 n N S

, 602–611 (2006). 602–611 , 122 a Nat. Genet. Nat. T Brain Pathol. Brain t u , 6807–6816 (2001). 6807–6816 , E r R e , 87–98 (2011). 87–98 , Science . E c om/doifinder/10.1038 STS Science

Science 14

21 , 452–458 (2011). 452–458 ,

40 Cell Rep. Cell 314 Nat. Neurosci.Nat.

, 904–919 (2011). 904–919 , 20

, 572–574 (2008). 572–574 , 323

323 , 589–597 (2010). 589–597 , , 130–133 (2006). 130–133 , online version of the pape , 1208–1211 (2009). 1208–1211 , , 1205–1208 (2009). 1205–1208 ,

1 , 167–178 (2012). 167–178 ,

14 http://www. /nn.323 Hum. Mol. Genet. Mol. Hum. , 459–468(2011)., 0 .

n

a t Biochem. u r r . e . c

o 19 m , /

31. 30. 29. 28. 27. 26. 25. 24. 23. 22. 21. 20. 19. 50. 49. 48. 47. 46. 45. 44. 43. 42. 35. 34. 33. 32. 41. 40. 39. 38. 37. 36.

Hutton, M. Hutton, C. Mitchelmore, T.,Okuda, T.Miyata, & K. Kokame, family NDRG of patterns expression Differential G.G. Hicks, Rogelj, B. S. Ishigaki, C. Colombrita, H.B. Kordasiewicz, R.A. Smith, Ameur, A. C. Zhang, S.C. Ling, R. Fujii, emn, M. Neumann, J. Couthouis, J. Couthouis, Ticozzi, N. X. Wang, A. Bertolotti, TLS/FUS J.L. Manley, & H.J. Bussemaker, T., Coady, T.R., Riley, A.Y., Tan, Single-molecule R.H. Singer, & C.C. Query, A., Moldon, D.R., Larson, T., Trcek, Y. Wada, J.I. Hoell, A. Bodansky, S.J. Rabin, Myelin-associated S.M. Strittmatter, & T. GrandPre, A., Fournier, B.P., Liu, Lin, S., Coutinho-Mansfield, G., Wang, D., Pandit, S. & Fu, X.D. The splicing factor splicing The X.D. Fu, & S. Pandit, G., D., Coutinho-Mansfield, Wang, S., Lin, S. Massone, Morohashi, Y. M.S. Goldberg, J.M. Itier, T. Kitada, the inherited dementia FTDP-17. dementia inherited the Dis. Neurobiol. FUS/TLS. with complexes TDP-43 promote lcpoen s fntoa lgn fr h Ng-6 receptor. Nogo-66 the for ligand functional a as glycoprotein (2008). system. nervous central the in proteins perinatal and death. instability chromosomal of levels high activation, and development brain. the in splicing transcriptions. and events splicing cells. motoneuron-like in fate post-transcriptional their regulate differently and RNAs messenger cytoplasmic synthesis. Huntingtin of (2012). repression transient by Invest. Clin. J. brain. human the (2011). 1435–1440 in splicing co-transcriptional controls. combinatorial its and (2010). 13318–13323 mGluR5activation regulatesand spinemorphology. mutations. FUS with sclerosis lateral amyotrophic from pathology FUS with FTLD distinguish sclerosis. lateral amyotrophic genes. Genet. gene for familial amyotrophic lateral sclerosis. transcription. inhibit to 18 complexes. II polymerase RNA and TFIID hTAFII68,of subunits and family,TET and the EWS of members two between interactions II: polymerase RNA (2012). 6030–6035 elements. response DNA single-stranded via transcription gene target regulates sarcoma) in liposarcoma/fused in (translocated 147 promoter-regulatedreveal measurements decay mRNA yeast. in stability mRNA USA Sci. Acad. Natl. Proc. (2008). 819–826 spread. disease (2010). of columns independent neuron motor lower the in distributed widely and frequency low are ALS biology.adhesion cell-matrix altered and (2002). 1190–1193 SC35 has an active role in transcriptional elongation. transcriptional in role active an has SC35 Biol. Cell J. channel-interacting protein contributing to brain complexity and neurodegeneration. Kv4. subunit channel potassium voltage-gated and 2 presenilin with interacting protein EF-hand neurons. dopaminergic of mouse. the in neurotransmission parkinsonism. Biol. Mol. Struct. Nat. , 1489–1497 (1998). 1489–1497 , , 1484–1497 (2011). 1484–1497 ,

Nat. Genet. Nat. Proc. Natl. Acad. Sci. USA Sci. Acad. Natl. Proc. 156B advance online publication online advance t al. et et al. et et al. et al. et al. et t al. et et al. et et al. Brain t al. et et al. et

t al. et t al. et et al. et 193 t al. et t al. et , 285–290 (2011). 285–290 , et al. et t al. et J. Biol. Chem. Biol. J. et al. et et al. et al. et t al. et h RA idn poen L i tasoae t dnrtc pns by dendritic spines translocated to is TLS protein binding RNA The t al. et Nature

Induced ncRNAs allosterically modify RNA-binding proteins in proteins RNA-binding modify allosterically ncRNAs Induced Widespread binding of FUS along nascent RNA regulates alternative et al. et Total RNA sequencing reveals nascent transcription and widespread t al. et 116 et al. et Mutational analysis reveals the FUS homolog TAF15 as a candidate 16 Integrative modeling defines the Nova splicing-regulatory network splicing-regulatory Nova the defines modeling Integrative Mutations in the parkin gene cause autosomal recessive juvenile recessive autosomal cause gene parkin the in Mutations wv o nset rncito o atvtd ua genes. human activated on transcription nascent of wave A

, 851–866 (2011). 851–866 , L-soitd uain i TP4 ices is tblt and stability its increase TDP-43 in mutations ALS-associated Association of missense and 5 and missense of Association t al. et Antisense oligonucleotide therapy for neurodegenerative disease. neurodegenerative for therapy oligonucleotide Antisense 134 Sporadic ALS has compartment-specific aberrant exon splicing exon aberrant compartment-specific has ALS Sporadic N tres f idtp ad uat E fml proteins. family FET mutant and wild-type of targets RNA RNA polymerase III drives alternative splicing of the potassium the of splicing alternative drives III polymerase RNA oiindpnet U-N itrcin rglt alternative regulate interactions FUS-RNA Position-dependent , 48–58 (2004). 48–58 ,

Molecular cloning and characterization of CALP/KChIP4, a novel TDP-43 and ubiquitinated cytoplasmic aggregates in sporadic sporadic in aggregates cytoplasmic ubiquitinated and TDP-43 A yeast functional screen predicts new candidate ALS disease ALS candidate new predicts screen functional yeast A akn ee nciain les eair n dopamine and behavior alters inactivation gene Parkin W, u nt W-L-, s soitd ih oh FI and TFIID both with associated is EWS-FLI-1, not but EWS, Fus deficiency in mice results in defective B-lymphocyte indefective results mice in deficiency Fus 24 , 2290–2296 (2006). 2290–2296 , vlaig h rl o te U/L-eae gn ES1 in EWSR1 gene FUS/TLS-related the of role the Evaluating t al. et TDP-43 and FUS RNA-binding proteins bind distinct sets of sets distinct bind proteins RNA-binding FUS and TDP-43 Parkin-deficient mice exhibit nigrostriatal deficits but not loss not but deficits nigrostriatal exhibit mice Parkin-deficient E poen TF5 n ES r slcie akr that markers selective are EWS and TAF15 proteins FET

, 2595–2609 (2011). 2595–2609 , Sci. Rep. Sci. 392 , 175–179 (2000). 175–179 ,

DG: nvl lhie’ dsae soitd protein. associated disease Alzheimer’s novel a NDRG2: 18 Nature Sustained therapeutic reversal of Huntington’s disease disease Huntington’s of reversal therapeutic Sustained J. Biol. Chem. Biol. J. , 1428–1431 (2011). 1428–1431 , , 605–608 (1998). 605–608 , J. Biol. Chem. Biol. J.

106 277 Hum. Mol. Genet. Mol. Hum.

Science 454 2 , 18357–18361 (2009). 18357–18361 , , 14965–14975 (2002). 14965–14975 , , 603 (2012). 603 , Hum. Mol. Genet. Mol. Hum. Nature

Sci. Rep. Sci. 108 , 126–130 (2008). 126–130 , morp. aea Scler. Lateral Amyotroph.

, 20881–20890 (2011). 20881–20890 ,

329

287 Hum. Mol. Genet. Mol. Hum. 278 . itce. Cytochem. Histochem. J. 393 , 439–443 (2010). 439–443 ,

, 15635–15647 (2012). 15635–15647 , Am. J. Med. Genet. B. Neuropsychiatr. 2 , 43628–43635 (2003). 43628–43635 , , 702–705 (1998). 702–705 , rc Nt. cd Si USA Sci. Acad. Natl. Proc. , 529 (2012). 529 , rc Nt. cd Si USA Sci. Acad. Natl. Proc.

′ 21

-splice-site mutations in tau with tau in mutations -splice-site Curr.Biol. nature nature , 2899–2911 (2012). 2899–2911 ,

a. tut Ml Biol. Mol. Struct. Nat. 12 , 2277–2291 (2003). 2277–2291 , Nat. Struct. Mol. Biol. Mol. Struct. Nat. Neuron

19

15 NEUR , 313–328 (2010). 313–328 ,

, 587–593,(2005). 74

11 , 1031–1044 1031–1044 , Science Mol. Cell Biol. Cell Mol. 56 OSCI , 321–327 321–327 , 175–182 , EN

109 297 107

Cell 15 18 C cis E , , , , ,

© 2012 Nature America, Inc. All rights reserved. mouse DM1mouse 1:2,000), 10782, (Proteintech,TDP-43 to antibody rabbit 1:2,000), to diluted (serum FUS/TLS of 128–140 acids amino rabbitto antibody1:500), sc-47711, 1:5,000),mouse antibody to FUS/TLS (Santa Biotechnology, Cruz clone 4H11, bodies, we used rabbit antibody to FUS/TLS (Bethyl Laboratories, A300-302A, formed in a least three mice for each treatment group ( Antibodies for immunoblots. are available in sequences designed using Primer3 software ( ples. Inter-group differences were assessed by two-tailed Student’s were expressed as a percentage of the average expression of saline-treated sam least two of the following control genes: system software (Bio-Rad; version 2.1). Expression values were normalized to at PCR detection system (Bio-Rad). Analysis was performed using the iQ5 optical multicoloriQ5 real-timethe on (Bio-Rad)supermix SYBRgreen iQ theusing control ASO, or ASO to either FUS/TLS or TDP-43) and two technical replicates forgroupmiceeachgenes 3–5tested performedwith was(treated saline,with specific primers and 5 normalization control. cDNA synthesis and amplification were performed using cler ABI Prism 7700 (Applied Biosystems), with the cyclophilin formedA ( using the Express One-Step SuperScript kits (Invitrogen) and thermocy biological replicates per group. qRT-PCR for mouse betweenproducts with included and excluded exons were averaged from three NationalInstitutes(USImageJ softwareIntensityHealth). with of ratios tified on 10% polyacrylamide gels and stained with SYBR gold (Invitrogen) and quan treated with either a control or FUS/TLS mice ASO. three Isoform from productscDNA Invitrogen)were separated III, (Superscript transcribed reverse and ing, we carried out RT-PCR amplification (24–27 cycles) using poly-A–selected RT-P cycles. 100 for platform 2000 library RNA-seq constructed each of pmol 8 We sequenced (TDP-43). AAGGCttcatattgtACTTT (FUS/TLS); CCTGGttatttcccaTGAGC con 2 nucleotides taining capitalized with gapmers are ASOs used The was genome comparison. for mouse (125 the in dose target same known any the without with ASO control injected of group control A striatum. the into 50 or with mixed was ASO TDP-43 of in frozen and dissected were 1 respectively, cord, spinal the or area cortex adjacent and striatum and killed, were they until 2 weeks for effects mice adverse C57Bl/6 female 8–10-week-old of 300 or 75 10 or 2 either of injections FUS/TLS To deplete using (Invitrogen). Trizol extracted were proteins and RNAs and 18.5 day embryonic at lected from Brains 86-23). NIH Publication of and Human Health Services, (Department at San Diego of of and Institutional Care University California Usethe Animal Committee the by approved protocol a using accomplished were cedures of AS Injections brain of one mouse was used. the libraries, CLIP-seq three the of each For 128–140. acids amino spanning Cruz (Santa FUS/TLS Biotechnology, clone of and 4H11, sc-47711), Ab3, a rabbit antibody to a peptide terminus C the for specific antibody monoclonal proteins,human the FUS/TLS mouse and Ab2,the between a mouse identical almost are which company’sdescription), the (per FUS/TLS human of 1–50 acids amino for specific A300-302A) Laboratories, (Bethyl antibody rabbit a platform for 36 cycles linking. CLIP-seq libraries were andconstructed onsequenced the Hiseq 2000 100 size pore of strainer cell a using dissociated were specimens cortical autopsy human frozen or mice C57Bl/6 CL ONLINE MET doi:10.1038/nn.3230 (Abcam, AB8245, 1:10,000).

ml of Trizol (Invitrogen). For the double knockdown experiment, 75 75 experiment, knockdown double the For (Invitrogen). Trizol of ml IP-seq library preparation and sequencing. C Fus/Tls R and qRT-P ′ -O-(2-methoxy)ethyl modifications with sequences as follows: follows: as sequences with modifications -O-(2-methoxy)ethyl µ g of ASOs, respectively, into the striatum or the right ventricle ventricle right the or striatum the into respectively, ASOs, of g α −/− antibody to tubulin (1:10,000) and mouse antibody to GAPDHantibodyto mouse andtubulin(1:10,000) antibodyto Supplementary Table 7 mice, in which the the which in mice, H O ODS in mice mice in C 1 R of R ′ 5 FAM, 3 . Three different antibodies to FUS/TLS were used: Ab1, N CN µ A from mice samples. ′ l of ASO solution, corresponding to a total of of total a to corresponding solution, ASO of l Immunoblots for FUS/TLS or TDP-43 were per TAMRA–labeled probes. qRT-PCR for all other S and S R and µ Fus/Tls m (BD Falcon) before ultraviolet cross- ultraviolet before Falcon) (BD m . Actb N µ g of FUS/TLS ASO before injection injection before ASO FUS/TLS of g A-seq library preparation. library A-seq in vivo in 1 5 , http://frodo.wi.mit.edu/primer3 . Mice were monitored for any any for monitored were Mice . Actg1 gene is interrupted is gene Brains from 8-week-old female , we performed stereotactic stereotactic performed we , Tardbp and To validate alternative splic Fig. Rsp9 and 3 ). For primary anti . Expression values Fus/Tls 5 1 on the HiSeq HiSeq the on Ppia t 2 test. Primer 8 , were col were , ) gene as a were per All pro All

µ µ g) g) / g ------) Differentially expressed genes computed from RPKM against a nucleotide frequency–matched background of all pre-mRNAs,regions surroundingusing thethe center of CLIP clusters to identify strand-specific motifscluster divided by the total CLIP reads overlapping this region. We used 150-nt reads, as well as a local frequency using a CLIP genomic total by divided size genome using frequency genomic a lated calcu pre-mRNAs,annotatedwe of outside clusters CLIP Todeterminecies. sufficient reads to satisfy a bonferroni-corrected clusterhad significant A it. in reads CLIP total pre-mRNAthe by particular a of size dividingtheby calculated was frequencygene-specific a Second, them. dividingpre-mRNAstotallengthof theall bytotalreadsCLIPthemapping in to determine a reads were calculated using a Poisson distribution using two differenttification was frequenciesperformed as previously described CL scriptase step, as a result of protein adducts left by ultraviolet cross-linking sites provided evidence of incorrect base incorporation during the reverse tran Mismatchnucleotide. per coverage read cumulative as plottedwere sequence sequences rather than the genome, and reads mapped to the U1 snRNAstream referenceanalysis. The entire trimmed library was down forkept mapped was alignment scoring best againstthe onlybut multiplelocations, to reference snRNA identical sequences were collapsed into a single read. Reads were allowed to map sion 0.12.2), with parameters −q −p 4 −e 100 −a −m 10 −best–strata. Reads with before mapping to the repeat-masked mouse genome (mm9) using bowtieremoved (ver runs homopolymeric and trimmed sequences adaptor had libraries CL harboring the reprogramming factors c-Myc, Klf4, Oct4 and Sox2 and replated fibroblasts from a non-ALS individual were subjected to infection by culturedretroviruses for an additional 4 weeks without FGF-2. To generate iPS cells, human mature into ­neurons, 10 NPCs derived rosette the differentiate To FGF-2. and B27-RA enzymaticallydissociatedandre-plated GlutaMAX DMEM/F12N2, +with in ml ng (20 FGF-2 and N2 with supplemented GlutaMAX + DMEM/F12 and plates laminin-coated N2 for 1 week as embryoid bodies, before being transferred onto polyornathine/ on ultra low–attachment plates in DMEM/F12 + GlutaMAX supplemented with removed by treatment with collagenase IV (Sigma). Whole colonies were grown with mTeSR1 growth media (Stem Cell Technologies) at 37 °C and 5% CO described previously as progenitors and differentiated into mature neurons tentusingembryonic stem cells (HUES6 line) and iPS cells were ainduced into neural pan-neuronal protocol G unchanged by FUS/TLS ( n detected cassette exons promoted by FUS/TLS (skipped following knockdown, ously reported (ref. 0.5 > scoreseparation a with events filtering described, previously as analysis. microarray Splicing-sensitive intronic regions (1,866 regions) from non-expressed ones. chosen as the cutoff (natural log RPKM > 2) that we used to3 distinguish expressed events, RPKMs of individual regions, not entire genes, were calculated forsaline-treated samples.known To identify putative intron-retention/alternative 3 changedcontrolin also oligo–treated they analysis,as samples compared with a R and RNA-seq reads were analyzed the same way. entire gene to normalize for differing bin sizes. Motifs (defined from HOMER) tobinthis cluster thisequaltofractionthe represents versus clusters all on the intron.cluster,the Forofeachlength addedthe dependent onbinscorewas a 1%-resolutionintrona tocluster a assigning clustersby CLIP distributionsof software HOMER ′ = 63), repressed by FUS/TLS (included following knockdown, following (included FUS/TLS by repressed 63), = N UTRs and their distribution was plotted. The most frequent RPKM value was eneration of z score > 1.96 ( 1.96 > score IP-seq cluster identification, motif and pattern analysis. IP-seq and R and IP-seq -e aayi ad noain f addt ito rtnin events. retention intron candidate of annotation and analysis A-seq µ M inhibitor ROCK (Y27632) was added to the media for 2 d and 1 N P 7 , overlapping FUS/TLS CLIP-seq clusters around splicing array– value. First, a transcriptome-wide frequency was calculated by P N C P 5 A-seq read mapping. read A-seq 5 < 0.05). A subset of 17 genes was excluded from further further from excluded was genes 17 of subset A 0.05). < s and neurons from pluripotent stem cells. . To visualize sawtooth-like patterns, we determined intron −1 58 ). After 1 week, neural rosettes were manually picked,manually were rosettes neural week, 1 After ). n , 5 = 3,664). 9 57). An RNA splicing map was produced as previ asproduced was RNAmapsplicing An 57). . Stem cells grown on matrigel-coated plates (BD) platesmatrigel-coated on grown cells Stem . Reads from CLIP-seq and RNA-seq and CLIP-seq from Reads Microarray analysis was performed performed was analysis Microarray ± 1-kb region surrounding a potential P 15 q value <10 value >0.05 and an absolute an and >0.05 value , 17 nature nature , 53 5 , 6 5 values were defined by 4 . Significant clusters of −4 CLIP cluster iden NEUR for both frequen Human pluripo n OSCI = 114) or 114) = ′ 2 EN UTR were 5 2 . C E ------

© 2012 Nature America, Inc. All rights reserved. eis preblzd y meso i 02 Tio X10 (vol X-100 Triton 0.2% in immersion by The permeabilized wash tubulin. ethanol series, or descending a SMYD3 through KCNIP4, rehydrated deparaffinized, Parkin, were either slides to one antibody an and using TDP-43 patients control to or SALS from lum sections cord spinal paraffin-embedded bar formalin-fixed, samples. on autopsy immunofluorescence double human from Immunofluorescence optimal cutting temperature medium (Tissue Tek, 4583) and frozen. stored at 20–25 °C. For biochemical analysis, tissue segments were embedded in neutralbuffered10%formalin (wt in post-mortem interval of 4 h. For histological studies, gross segments were fixed hospice. Autopsies were performed immediately following death, with anthe hospital’s average critical care unit when life support was withdrawn, or patients on Controltissues were obtained from non-neurologically affected patients out of tion sis was confirmed post-mortem through histologic analysis of TDP-43 aggrega patients who met from the obtained modified were El Escorial tissues criteria ALS for The definite process. Act-compliantALS Accountability archived via an institutional review board and Health Insurance Portability and processing. and acquisition tissueHuman SMYD5 entiated mature neurons. Gene expression values were normalized using either TLS-depletedand TDP-43–depleted human neuralprogenitor cellsand differ FUS/ control,from cDNA reverse-transcribed polyA-selected,from triplicate in (AppliedBiosystems)Mix Master SYBR-GreenFast PCR with Biosystems) rons. qRT-P after infection. ofqRT-PCR quantitationand concentrations viral GFP previously as lentivirus produce described to used was system vector pLKO.1 the in (TRCN0000016038 and TRCN0000016041) and T L propagated separately on Matrigel-coated plates and mTeSR1 growth media. phase-bright phology, including well-defined borders, high nuclear to cytoplasmic ratio,mor andcell–like ES with Colonies d. 5 after fibroblasts embryonic mouse onto nature nature niia shR entiviral L S. 2 . The efficacy of the lentivirus was tested by infection of NPCs at varying varying at NPCs of infection by tested was lentivirus the of efficacy The . , Lentiviral shRNA constructs (Open Biosystems) toward toward Biosystems) (Open constructs shRNA Lentiviral qRT-PCR was performed on 7900HT Fast Real-time PCR system (Applied 3 . Mutations in C , R in human neuronal progenitor cells and differentiated mature neu BMI 5 NEUR 3 . The. control was pLKO.1used virus containing shRNAa targeting

characteristicswere isolated weeksafterof4 reprogramming and or OSCI N GAPDH –eitd elto o hmn T human of depletion A–mediated SOD1 EN levels. C , E C9orf72 , TDP-43 / vol) for 14 d, embedded in paraffin, andparaffin, in embedded d,vol)for 14 and Human CNSs were obtained and obtained were HumanCNSs FUS/TLS FUS/TLS TDP-43 genes were excluded. (TRCN0000010450) D and We performed performed We -3 n FUS/ and P-43 6 0 FUS/TLS . The diagno / o) for vol) TDP-43 5 d 5 ------

53. 52. 51. motor neurons determined by two independent investigators. tubulin.orresultThe presented in inclusion and the qualitative difference of staining for Parkin, SMYD3, KCNIP4 TDP-43of absence orpresence the upon classifiedneurons countedandwere Motor system. microscope confocal Ti Eclipse Nikon a using performed was solved in 70% ethanol for 10 s, and rinsed twice in PBS.autofluorescence-masked VisualizationSudan0.1%Black (volin and imaging were stained using 10 mouse) were diluted directly in PBS and applied at a 1:500 dilution ratio. Slides 488–conjugated donkey antibody to goat or Cy5-conjugated donkey antibody to Alexa rabbit,to Cy3-conjugatedantibodymouse, donkey to antibody donkey Cy3-conjugated rabbit, to antibody goat 488–conjugated (Alexa antibodies and tubulin (Sigma Aldrich, T8660, mouse monoclonal IgG, 1:1,000). Secondary polyclonal IgG, 1:200), SMYD3 (Abcam, ab16027, rabbit polyclonal IgG, 1:200) (R&D, AF1438, goat polyclonal IgG, 1:50), KCNIP4 (Sigma, HPA022862, rabbit house FL4 antibody, mouse monoclonal IgG, dilution of ascites 1:1,000), Parkin in- 1:50; IgG, polyclonalphospho–TDP-43TIP-PTD-P01,rabbit (CosmoBio, TDP-43 antibodies: primary following the of one with overnight incubated in a Dako Pascal pressure chamber. After washing and blocking the slides were wasperformed inScyTek Citrate Buffer pH6.0(CBB999) for 20minat 125°C 10 min, and washed twice in phosphate-buffered saline (PBS). Antigen retrieval 57. 56. 55. 54. 60. 59. 58.

Yeo,G.W. Mapping R.B. Darnell, & C. Zhang, D. Parkhomchuk, Sugnet, C.W.Sugnet, and Mapping B. Wold, & L. Schaeffer, K., McCue, B.A., Williams, A., Mortazavi, S. Heinz, D.G. Zisoulis, Brooks, B.R., Miller, R.G., Swash, M. & Munsat, T.L. El Escorial revisited: revised revisited: Escorial T.L.El Munsat, & M. Swash, R.G., Miller, B.R., Brooks, M.C. Marchetto, G.W. Yeo, nucleotide resolution from HITS-CLIPnucleotidedata.resolutionfrom DNA. complementary (2008). RNA-Seq. by transcriptomes mammalian quantifying identities. cell B and macrophage for Cell Mol. required elements cis-regulatory prime in sites cells. (2009). stem in interactions RNA-protein Other Motor Neuron Disord. Neuron Motor Other sclerosis. lateral amyotrophic of diagnosis the for criteria cells. stem pluripotent (2010). induced human using syndrome progenitors. neural and cells microarrays. splicing by Caenorhabditis elegans Caenorhabditis

38 et al. et t al. et t al. et , 576–589 (2010). 576–589 , et al. et t al. et An RNA code for the FOX2 splicing regulator revealed by mapping by revealed regulator splicing FOX2 the for code RNA An ipe obntos f ieg-eemnn tasrpin factors transcription lineage-determining of combinations Simple Alternative splicing events identified in human embryonic stem embryonic human in identified events splicing Alternative t al. et t al. et Unusual intron conservation near tissue-regulated exons found exons tissue-regulated near conservation intron Unusual µ opeesv dsoey f noeos roat binding Argonaute endogenous of discovery Comprehensive g ml Nucleic Acids Res. Acids Nucleic mdl o nua dvlpet n tetet f Rett of treatment and development neural for model A Transcriptome analysis by strand-specific sequencing of sequencing strand-specific by analysis Transcriptome PLOS Comput. Biol. Comput. PLOS −1

DAPI in ddH 1 PLOS Comput. Biol. Comput. PLOS , 293–299 (2000). 293–299 , . Nat. Struct. Mol. Biol. Mol. Struct. Nat. Figure n vivo in

37 a. tut Ml Biol. Mol. Struct. Nat. 6 2 Nat. Biotechnol.Nat. 0 for 10 min, rinsed twice in ddH is the averagethe iscategory forofeach , e123 (2009). e123 ,

rti-N itrcin a single- at interactions protein-RNA 2 , e4 (2006). e4 ,

3 , 1951–1967 (2007). 1951–1967 , / vol,Fisher, BP109-10)dis a. Methods Nat.

17 Amyotroph. Lateral Scler.Lateral Amyotroph. , 173–179 (2010). 173–179 , doi:10.1038/nn.3230 29 Cell , 607–614 (2011).607–614 ,

143

16

5 527–539 , 130–137 , 621–628 , 2 0, -