Characterizing the RNA targets and position-dependent splicing regulation by TDP-43 : Nature Neuroscience : Nature Publishing Group

TDP-43 is a predominantly nuclear RNA-binding protein that forms inclusion bodies in frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). The mRNA targets of TDP-43 in the human brain and its role in RNA processing are largely unknown. Using individual nucleotide-resolution ultraviolet cross-linking and immunoprecipitation (iCLIP), we found that TDP-43 preferentially bound long clusters of UG-rich sequences in vivo. Analysis of RNA binding by TDP-43 in brains from subjects with FTLD revealed that the greatest increases in binding were to the MALAT1 and NEAT1 noncoding RNAs. We also found that binding of TDP-43 to pre-mRNAs influenced alternative splicing in a similar position-dependent manner to Nova proteins. In addition, we identified unusually long clusters of TDP-43 binding at deep intronic positions downstream of silenced exons. A substantial proportion of alternative mRNA isoforms regulated by TDP-43 encode proteins that regulate neuronal development or have been implicated in neurological diseases, highlighting the importance of TDP-43 for the regulation of splicing in the brain. Introduction TAR DNA binding protein (TDP-43) is a predominantly nuclear protein that has been reported to regulate transcription, alternative splicing and RNA stability1, 2. TDP-43 has two RNA-recognition motif (RRM) domains, which bind UG repeat sequences with high affinity3. Mislocalisation and aggregation of TDP-43 have been implicated in the pathogenesis of ALS and FTLD. Mutations in the gene that encodes TDP-43 are associated with 1–4% of familial and sporadic ALS and rare cases of FTLD, and confer toxicity4, 5. The pathology of ~60% of FTLD and ~90% of ALS cases are characterized by the formation of cytoplasmic inclusion bodies in neurons and astroglia that are rich in ubiquitinated, hyperphosphorylated C-terminal fragments of TDP-43 (refs 5,6). Neurons that contain cytoplasmic TDP-43 inclusions often have markedly reduced TDP-43 staining in the nucleus5, 6. Thus, TDP-43 inclusions might have a toxic gain of function in the cytoplasm or sequester nuclear TDP-43 (or both), disrupting its role in RNA processing. TDP-43 cleavage products in the cytoplasmic inclusions have been shown to alter the splicing of a TDP-43–regulated exon in minigene studies, indicating that they can interfere with wild-type TDP-43 function7. The toxicity of TDP-43 overexpression depends on it retaining its RNA binding function in yeast8, Drosophila and chick9. Only a handful of RNAs regulated by TDP-43 have been identified. To understand the function of TDP-43, and its dysfunction in disease, we have comprehensively identified the RNA sites that interact with TDP-43 in the human brain and characterized changes in RNA that follow reduced TDP-43 expression. To identify RNA targets on a global scale, we used individual-nucleotide resolution CLIP (iCLIP), which captures truncated cDNAs and quantifies them using a random barcode contained within the primer that is used for reverse Characterizing the RNA targets and position-dependent splici... http://www.nature.com/neuro/journal/v14/n4/full/nn.2778.html 1 of 17 03/06/2011 14:57 transcription10. Duplicate sequences that shared the same random barcode were removed from analysis to avoid any apparent binding-site bias due to PCR artifacts. In this study, we sought to identify the RNAs that were bound by TDP-43 in healthy brain tissue and that from subjects with FTLD with TDP-43 inclusions (FTLD-TDP). Although this was the first study of protein-RNA interactions in post-mortem human brain, we identified many RNA binding sites using these samples. Most binding mapped to introns, long noncoding (nc)RNAs and intergenic transcripts, which also had the greatest enrichment of UG-rich motifs. We characterized the changes in alternative splicing that followed TDP-43 knockdown in neuroblastoma cells and identified splicing changes in 158 alternative cassette exons. Analysis of TDP-43 iCLIP cross-link sites in the vicinity of these exons provided insights into the mechanisms by which TDP-43 splicing is regulated. The exons regulated by TDP-43 are enriched in genes involved in neuronal development and those implicated in a range of neurodegenerative diseases. Results Comparing RNA targets of TDP-43 in healthy and FTLD brains We purified TDP-43 in complex with its direct RNA targets from ultraviolet cross-linked cells and analyzed it on SDS-PAGE gel. The size of the dominant TDP-43–RNA complex in the low and high RNase conditions corresponded to a single TDP-43 molecule bound to the RNA; however, a complex corresponding to two TDP-43 molecules bound to the RNA was also present, particularly under low RNase conditions (Fig. 1a). This result agreed with the earlier finding that TDP-43 binds RNA as a homodimer2. Figure 1: Comparison of TDP-43 RNA binding in brain tissue from subjects with and without FTLD-TDP. Characterizing the RNA targets and position-dependent splici... http://www.nature.com/neuro/journal/v14/n4/full/nn.2778.html 2 of 17 03/06/2011 14:57 (a) To validate the specificity of the antibody to TDP-43 for iCLIP, we isolated the 32P-labeled RNA bound to TDP-43 gel from control (Ctr) or knockdown (KD) HeLa cells in the presence or absence of ultraviolet (UV) cross-linking or antibodies to TDP-43. We used high and low RNase concentrations to confirm the presence of RNA bound to TDP-43. Arrows, positions on gel corresponding to the size of TDP-43 monomer or dimer. TDP-43 western analysis of input extracts confirmed TDP-43 knockdown, and GAPDH was used as a loading control. For full image, see Supplementary Figure 1a. (b) The proportion of cDNAs (out of all cDNAs that mapped to human genome) from the TDP-43 iCLIP experiments in the four types of samples that mapped to different RNA regions. (c) The proportion of cDNAs that mapped to different types of ncRNAs. (d) The proportion of cDNAs that mapped to individual RNAs with at least 10 cDNAs in any experiment. Red, RNAs with the largest significant change between samples from control subjects and those with FTLD-TDP (difference in proportion of cDNAs > 0.1% and P < 0.05 by Student's t test, one-tailed, unequal variance). The long ncRNA MEG3 (maternally expressed 3) with the largest decrease in TDP-43 binding in FTLD-TDP is also marked. (e) Real-time PCR analysis of transcripts with largest TDP-43 iCLIP changes in total RNA isolated from control brain samples and samples from subjects with FTLD-TDP. (f) iCLIP cDNA counts for TDP-43 cross-link positions in NEAT1 in experiments from SH-SY5Y and hES cell lines, and healthy and FTLD-TDP tissue. Blue bars, sequences on the sense strand of the genome; height of bars corresponds to cDNA count. Positions of significant cross-link clusters (XL cluster) are shown on top; RNA sequence underlying the two main clusters is shown below (pink, UG repeats). Data show mean ± s.d. proportion of cDNAs that map to NEAT1 transcript in each experiment (out of all cDNAs mapping to the human genome). (g) iCLIP cDNA counts for TDP-43 cross-link positions in EEAT2. Orange bars, sequences on the antisense strand of the genome. Data show mean ± s.d. proportion of cDNAs that map to EEAT2 3′ UTR. The RNA sequence underlying the peak cross-linking sites is shown below; pink, UG repeats. We carried out iCLIP experiments with human cortical tissue from post-mortem brain samples from three cognitively normal (healthy) subjects and three subjects with sporadic FTLD-TDP, as well as from embryonic stem (hES) cells and SH-SY5Y neuroblastoma cells (Supplementary Table 1). When we allowed a maximum of one nucleotide mismatch, 17 million sequence reads aligned as single hits to the human genome. After eliminating PCR amplification artifacts by removing sequences that truncated at the same nucleotide in the Characterizing the RNA targets and position-dependent splici... http://www.nature.com/neuro/journal/v14/n4/full/nn.2778.html 3 of 17 03/06/2011 14:57 genome and shared the same random barcode, we identified 3.7 million reads representing unique cDNA molecules (Supplementary Table 2). Genomic annotation of the cDNA sequences showed that most TDP-43 binding occurred in introns—74% in the cell lines and 58% in brain samples (Fig. 1b). The decreased proportion in brain samples, and corresponding increase in exonic sequences, might be because intronic RNA is more sensitive to degradation in postmortem samples. About 5% of iCLIP cDNAs mapped to long ncRNAs in all samples (Fig. 1c). Samples from subjects with FTLD-TDP showed increased binding of TDP-43 to small nucleolar (sno)RNAs, small nuclear (sn)RNAs, ribosomal (r)RNAs and telomeric ncRNAs (Fig. 1c). In total, we found cross-link sites in 2,139 ncRNA genes in control samples and 2,702 in samples from subjects with FTLD-TDP . The high proportion of intronic cDNA sequences indicated that the iCLIP data mainly represented nuclear RNA targets of TDP-43. We quantified the cross-linked complexes that were obtained using the iCLIP procedure from nuclear and cytoplasmic fractions of brain tissue from healthy subjects and those with FTLD-TDP. Only 6.5% of the total TDP-43–RNA complexes were obtained from cytoplasmic fractions of brain tissue, with no significant difference between tissue from healthy subjects and those with FTLD-TDP (Supplementary Fig. 1b). Analysis of the RNAs bound by TDP-43 in the two fractions in SH-SY5Y cells by iCLIP showed 34% 3′ UTR binding in the cytoplasm, 3.2% in the nuclear fraction and 3.8% in total cell extract (Supplementary Fig. 1d and Fig. 1b). As a control, we analyzed cross-link complexes with TIAL1, a protein that shuttles between the nucleus and cytoplasm. We obtained 31.5% of total TIAL1–RNA cross-linked complexes from cytoplasmic fractions of healthy brain tissue and samples from subjects with FTLD-TDP (Supplementary Fig. 1b). Thus, even though the TIAL1 analysis showed that iCLIP can efficiently purify proteins bound to cytoplasmic RNAs, our analysis indicates that more than 93% of TDP-43 iCLIP data presented here represents interactions with nuclear RNAs in brain tissue from both healthy subjects and those with FTLD-TDP. To study the changes in TDP-43 binding to individual transcripts in FTLD-TDP, we analyzed the proportion of cDNAs that mapped to individual co-expressed genomic regions in the different brain samples. We found a significant (P < 0.05 by Student's t-test, one-tailed, unequal variance) and greater than 0.025% change in the proportion of cDNAs between control brain tissue and tissue from subjects with FTLD-TDP in 4 ncRNAs, 3′ UTRs of 7 transcripts and introns of 48 transcripts (Supplementary Table 3). The fact that introns had the greatest number of changes indicates that the changes detected by iCLIP mainly reflect changes in RNA interactions that occur in cell nuclei. Four transcripts had a significant and greater than 0.1% change in the proportion of cDNAs (P < 0.05; Fig. 1d). Analysis of the abundance of these transcripts by RNA-seq and real-time PCR showed that the change in TDP-43 binding agreed with a similar extent of change in transcript expression (Fig. 1e and Supplementary Fig. 2). The most significant increase in TDP-43 binding in FTLD-TDP was observed in nuclear paraspeckle assembly transcript 1 (NEAT1) and metastasis-associated lung adenocarcinoma transcript 1 (MALAT1, also known as NEAT2), which are transcribed from proximal loci on chromosome 11. Whereas NEAT1 was previously identified as a 4-kb ncRNA11, we detected TDP-43 binding along the length of a 18-kb ncRNA, with the peak binding in a tandem UG-repeat sequence close to the 3′ end (Fig. 1d,f). The primary binding sites in these two ncRNAs contained long UG repeats, and were identified in a highly reproducible manner (Fig. 1f and Supplementary Fig. 1f). The neurexin 3 (NRXN3) transcript and the glial excitatory amino acid transporter-2 (EAAT2, also called SLC1A2) had significantly decreased proportions of cDNA in tissue from subjects with FTLD-TDP (Fig. 1d,g). EEAT2 is necessary for glutamate clearance at the synapse, a process that prevents glutamate excitotoxicity12. Characterizing the RNA targets and position-dependent splici... http://www.nature.com/neuro/journal/v14/n4/full/nn.2778.html 4 of 17 03/06/2011 14:57 TDP-43 binds to clusters of UG-rich sequences To compare the RNA sequence specificity of TDP-43 in the different iCLIP experiments, we assessed the enrichment of all possible pentamers within 30 nucleotides on either side of all cross-link sites. In all iCLIP datasets, the most significantly enriched pentamers were GUGUG and UGUGU (Fig. 2a and Supplementary Fig. 3a–c). The pentamer enrichment scores were correlated between samples from subjects with and without FTLD-TDP (r = 0.91; Fig. 2a). Figure 2: TDP-43 binding motif analysis. (a) z-scores of pentamer occurrence within the 61-nt sequence surrounding all cross-link sites (−30 nucleotides (nt) to +30 nt) for iCLIP of healthy samples and samples from subjects with FTLD-TDP. The sequences of the two most enriched pentamers and the Pearson correlation coefficient (r) between the two samples are given. (b) Enrichment of cross-linking compared to randomized data in UG repeats of different lengths in TDP-43 iCLIP experiments on brain samples from subjects without (blue) and with FTLD-TDP (red), and CELF2 experiments in healthy brain (green). (c) Enrichment of cross-linking compared to randomized data on UG repeats of different lengths in TDP-43 iCLIP experiments from SH-SY5Y and hES cells. Dark gray, all cross-link sites; light gray, only sites that mapped to cross-link clusters. (d) Analysis of positions of UGUGU enrichment compared to randomized data around TDP-43 cross-link sites. Color code as in b. (e) Analysis of positions of UGUGU enrichment compared to randomized data in TDP-43 iCLIP experiments from SH-SY5Y and hES cells. Color code as in c. (f) TDP-43 cross-linking in its own transcript (TARDBP). Replicate experiments are summed and shown in a single track. The RNA sequence underlying the peak cross-linking site is shown; pink, UG and GU dinucleotides. Characterizing the RNA targets and position-dependent splici... http://www.nature.com/neuro/journal/v14/n4/full/nn.2778.html 5 of 17 03/06/2011 14:57 We analyzed the relationship between TDP-43 binding and UG repeat length by monitoring the frequency of cross-linking in the last 10 nucleotides (nt) of UG tandem repeats compared to randomized positions. An example of a UG tandem repeat can be seen in the NEAT1 ncRNA (Fig. 1f). Enrichment of TDP-43 crosslinking to UG repeats increased up to a repeat length of 15 nt, in a manner that was similar in samples from subjects with and without FTLD-TDP (Fig. 2b,c). For comparison, we performed iCLIP with CELF2, which also binds UG-rich motifs in the human brain (Supplementary Fig. 3c). In contrast to TDP-43, binding of CELF2 to UG repeats increased only up to a length of 10 nt, and dropped once the repeats became longer than 15 nt (Fig. 2b). This indicates that TDP-43 has a stronger preference for tandem UG repeats than does CELF2. Proteins that interact with multiple identical sequences on RNA often allow spacing between different binding sites; for instance, NOVA proteins bind clusters of a minimum of three YCAY tetramers, but can tolerate variable spacing between these13. To analyze whether TDP-43 can similarly bind to more dispersed UG-rich motifs, we monitored the frequency of UGUGU motifs over a 200-nt region surrounding the TDP-43 cross-link sites compared to randomized positions. UGUGU was enriched sevenfold at the TDP-43 cross-link sites, and remained twofold enriched even at a distance of 100 nt from the cross-link sites (Fig. 2d,e). One cause for this phenomenon may be a tendency of TDP-43 to bind to long UG-rich RNA regions, such as the 90-nt region in the 3′ UTRs of TARDBP mRNA, where TDP-43 binds to autoregulate its mRNA levels14, 15 (Fig. 2f). By contrast, even though UGUGU is fourfold enriched at the CELF2 cross-link sites, we found no such enrichment at a distance of 100 nt (Fig. 2d). This indicates that TDP-43 has a unique capacity to recognize dispersed clusters of UG-rich motifs, or to spread its RNA binding to positions proximal to the UG-rich motifs. To specifically analyze the high-affinity RNA binding sites of TDP-43, we grouped all data and searched for cross-link sites clustered with a maximum spacing of 15 nt that contained a substantially greater cDNA count than randomized positions (false discovery rate < 0.05). We identified 111,691 such cross-link clusters. The reproducibility of cross-linking within these clusters between different samples increased with the number of iCLIP cDNA sequences that mapped to a cluster (Supplementary Fig. 3d–g). For instance, 76% of clusters with summed cDNA counts of five or more in brain from healthy subjects were similarly bound in brain from subjects with FTLD (Supplementary Fig. 3d). Clustered cross-link sites were highly enriched in UG tandem repeats, with increased enrichment at repeat lengths up to 30 nt (Fig. 2c). UGUGU was 18-fold enriched at the TDP-43 cross-link sites, and remained fivefold enriched even at a distance of 100 nt from the cross-link sites (Fig. 2e). The increased incidence of UG-rich motifs at the cross-link clusters confirms that these clusters are the likely high-affinity binding sites of TDP-43. To compare TDP-43 binding to different types of RNAs, we analyzed the enrichment of the UGUGU motif at the cross-link sites. The rRNAs, snRNAs, tRNAs and snoRNAs had the lowest UGUGU enrichment, indicating that cross-linking in these RNAs mainly reflected low-affinity, transient interactions (Supplementary Fig. 4). We found threefold enrichment in 5′ UTRs and open reading frames (ORFs), and fourfold UG enrichment in 3′ UTRs, micro (mi)RNA precursors and telomeric transcripts (Supplementary Fig. 4). UG was similarly enriched in the 3′ UTRs when isolated with nuclear or cytoplasmic mRNA, indicating that TDP-43 binds to RNA in a sequence-specific manner in both the nucleus and the cytoplasm (Supplementary Fig. 1e). There was sevenfold enrichment of UGUGU pentamers in introns, long ncRNAs and intergenic RNAs, which together contain most TDP-43 binding sites (90% in cell lines and 76% in brain samples; Fig. 1b,c). The high UG enrichment in intergenic RNAs is particularly notable, indicating that high-affinity TDP-43 interactions occur with a large number of unannotated transcripts. Together, the iCLIP results indicate that TDP-43 binds pre-mRNAs and several types of ncRNAs in a sequence-specific manner. The TDP-43 RNA splicing map Characterizing the RNA targets and position-dependent splici... http://www.nature.com/neuro/journal/v14/n4/full/nn.2778.html 6 of 17 03/06/2011 14:57 As most iCLIP cDNAs mapped to introns, we reasoned that TDP-43 might have an important role in splicing regulation. Therefore, we used Affymetrix high-resolution splice-junction microarrays to evaluate splicing changes in TDP-43 knockdown SH-SY5Y cells. Analysis of splicing isoform reciprocity using the ASPIRE3 software identified 158 splicing changes in alternative cassette exons and 71 other types of splicing changes (¦ΔIrank¦≥1 where I is exon inclusion) out of 30,154 alternative exons that were detected by the microarray. We used RT-PCR and capillary electrophoresis to further assess 40 of the cassette exons. For 32 splicing events, primer pairs generated both isoforms, and 28 of these reproduced the direction of the observed splicing change, resulting in an 87% (28/32) validation rate of the microarray data (Supplementary Table 4). To study how TDP-43 binds pre-mRNAs to regulate splicing, we visualized the positions of cross-link clusters in the form of an RNA splicing map, where the cross-link sites are plotted within 500 nt of the silenced (blue clusters) or enhanced (red clusters) cassette exons and the flanking exons (Fig. 3). The cross-link clusters identified three regions on the RNA splicing map where TDP-43 binding was mainly restricted to silenced or enhanced exons (Fig. 3). Binding in region 1, within the alternative exons or 0–150 nt upstream, was present at 11 silenced and 1 enhanced exon (group S1). Binding in region 2, 150–500 nt downstream of the alternative exons, was present at 7 silenced and 1 enhanced exon (group S2). Five of the silenced exons contained multiple cross-link clusters that spanned a sequence of 100 nt or longer in this region. This indicates that 'deep' intronic binding involving multiple dispersed binding sites might contribute to the ability of TDP-43 to silence exon inclusion. Finally, binding in region 3, 0–150 nt downstream of the alternative exons (but not in region 1 or 2), was present at 4 enhanced exons (group E). Figure 3: The RNA splicing map of TDP-43. Map of cross-link clusters at positions within 500 nt of alternative exons and flanking exons. Cassette exons with ΔIrank > 1 (enhanced exons, red clusters) or ΔIrank < −1 (silenced exons, blue clusters) with at least one cross-link cluster in these regions are shown. The exons are grouped by sequential analysis of cross-link cluster positions in three regions: group S1 is identified by clusters in region 1, 150 nt upstream of the exon and within the exon; group S2 by clusters in region 2, 150–500 nt downstream of the exon; and group E by clusters 0–150 nt downstream of the exon. To evaluate the proportion of TDP-43 iCLIP targets that changed splicing after knockdown, we analyzed Characterizing the RNA targets and position-dependent splici... http://www.nature.com/neuro/journal/v14/n4/full/nn.2778.html 7 of 17 03/06/2011 14:57 splicing of exons with cross-link clusters in the three regions defined by the RNA splicing map. We used RT-PCR to evaluate splicing of 41 putative alternative exons that had highest number of cDNAs mapping to these regions. Of these exons, 29% (12/41) showed no evidence of alternative splicing in SH-SY5Y cells (single band detected by RT-PCR). Of the remaining exons, 79% (23/29) changed splicing after knockdown, indicating that iCLIP data alone has high predictive value (Supplementary Table 4 and Supplementary Fig. 5b). We also assayed splicing of 12 alternative exons that had UG-rich regions within 200 nt of the alternative exons, but were not identified by microarray or iCLIP. Splicing changes in 5 of these exons were validated by RT-PCR (Supplementary Table 4). Similar to the iCLIP binding, UG-rich motifs upstream of the exon or dispersed motifs downstream of the exon were associated with silencing, whereas proximal UG-rich motifs downstream of the exon were associated with enhancing the function of TDP-43 (Supplementary Fig. 5c). TDP-43 regulates mRNAs involved in neuronal development We also found TDP-43 cross-link clusters in introns of 7,499 protein-coding genes and 3′ UTRs of 1,172 genes. Sixty-seven percent of the genes with 3′ UTR clusters (785/1,172) also had intronic clusters. For instance, two cross-link clusters were present in the intron downstream of the alternative exon 11 and three clusters in the 3′ UTR of myocyte enhancer factor 2D (MEF2D) mRNA (Fig. 4a). We identified a 29% decrease in inclusion of the MEF2D exon 11 in TDP-43 knockdown SH-SY5Y cells (Fig. 4a). The additional binding sites in the 3′ UTR indicate that TDP-43 can remain associated with the MEF2D mRNA after splicing is finished and can thereby regulate additional aspects of RNA processing. Figure 4: TDP-43 regulates splicing of non-coding and protein-coding RNAs. Characterizing the RNA targets and position-dependent splici... http://www.nature.com/neuro/journal/v14/n4/full/nn.2778.html 8 of 17 03/06/2011 14:57 (a) TDP-43 cross-linking in MIAT ncRNA. Pink bars, UG repeat lengths; black bars, cross-link clusters. Analysis of MIAT exon 10 inclusion (gel electropherogram, left, and quantification, right) in control and TDP-43 knockdown SH-SY5Y cells is shown below. (b) TDP-43 cross-linking in MEF2D protein-coding transcript. Colours as in a. Analysis of MEF2D exon 11 inclusion (gel electropherogram, left, and quantification, right) in control and TDP-43 knockdown SH-SY5Y cells is shown below. (c) Analysis of BIM exon 3 splicing (gel electropherogram, left, and quantification, right) in control and TDP-43 knockdown SH-SY5Y cells, and in brain samples from subjects without (C23, C25, C30) and with FTLD-TDP (F19, F20, F21). Owing to the strong sequence-specificity in binding to long ncRNAs (Supplementary Fig. 4), we also speculated that TDP-43 might regulate processing of such ncRNAs. Using iCLIP, we identified an alternative exon in the long ncRNA myocardial infarction associated transcript (MIAT). Eleven cross-link clusters are dispersed over the 1-kb region of both introns that flank this exon (Fig. 4b). We identified a 43% decrease in inclusion of the MIAT exon 10 in TDP-43 knockdown SH-SY5Y cells (Fig. 4b and Supplementary Fig. 5b). We also identified a cross-link cluster in the CFTR pre-mRNA, which was the first identified RNA target of TDP-43 (ref. 3). The cross-link overlapped with the UG-rich element upstream of exon 9, which is required for TDP-43-dependent splicing regulation of this exon (Supplementary Fig. 6). Previous studies identified several candidate RNA targets of TDP-43, for which the direct in vivo interaction with TDP-43 has not been validated. Here, we identified TDP-43 binding sites in the FUS pre-mRNA16, hNFL 3′ UTR17, the CDK6 intron and 3′ UTR18, and the intronic region that overlaps with the mir558 gene19 (Supplementary Fig. 6). We also observed cross-link clusters in the mRNAs encoding HDAC6 and Casein kinase 1 and 2, which have been reported to regulate the phosphorylation and expression of TDP-43, respectively20, 21 (Supplementary Fig. 6). We performed GO term analysis of genes regulated by TDP-43 at the level of alternative splicing, as determined by analysis of SH-SY5Y knockdown cells. This identified several terms related to development, including neural tube formation, organ morphogenesis and chordate embryonic development (Table 1). Fifteen of the exons that are regulated by TDP-43 are within genes that encode proteins with well-characterized functions in neuronal survival or development (Table 2). TDP-43 promotes inclusion of alternative exon 3 in Bcl-2 interacting mediator of cell death (BIM, also called BCL2l11), and thereby suppresses production of the most cytotoxic BIM isoform (BIM )22. We have evaluated splicing of BIM in FTLD-TDP, and have found that the BIM isoform increases in tissue from subjects with FTLD-TDP to a similar extent as in TDP-43 knockdown cells (Fig. 4c). This result indicates that aberrant splicing might contribute to neurodegeneration in the human brain. Table 1: Significant GO terms of the exons regulated by TDP-43 S S Characterizing the RNA targets and position-dependent splici... http://www.nature.com/neuro/journal/v14/n4/full/nn.2778.html 9 of 17 03/06/2011 14:57 Table 2: Neuronal functions of proteins encoded by the alternative mRNA isoforms regulated by TDP-43 Discussion Our results show that TDP-43 interacts with a diverse spectrum of RNAs with important functions in the brain. We identified binding of TDP-43 to non-coding RNAs, introns and 3′ UTRs of mRNAs, indicating that TDP-43 has a role in coupling different aspects of gene expression. Even though only a minor portion of the protein was detected in the cytoplasm, we found a tenfold increase in TDP-43 binding to 3′ UTR elements when comparing RNAs bound to cytoplasmic and nuclear protein. We also identified changes in alternative splicing associated with decreased TDP-43 protein. Analysis of cross-link clusters in the regulated pre-mRNAs indicated that the splicing function of TDP-43 depends on the position where it interacts with pre-mRNAs. We found that TDP-43 preferentially bound to UG tandem repeats or long clusters of UG-rich motifs. These results agree with in vitro studies in which RNA binding of TDP-43 was shown to increase with the length of the UG repeat2, 3. iCLIP results also indicated that the increased affinity for long binding sites differentiates the RNA specificity of TDP-43 from CELF2, which recognizes shorter clusters of UG-rich motifs. As a single RRM domain generally recognizes four nucleotides23, this RNA specificity for long binding sites indicates that TDP-43 can cooperatively recognize multiple RNA binding sites and thereby achieve high-affinity RNA binding. The apparent RNA binding of TDP-43 in a homodimer state might contribute to its ability to recognize binding sites composed of multiple dispersed UG-rich motifs (Fig. 1a). TDP-43 cross-link clusters were located upstream from and within the silenced exons and immediately downstream of enhanced exons, which is also a characteristic of several other RNA-binding proteins, including the Nova proteins24. In addition, dispersed clusters of TDP-43 cross-linking further downstream of exons were associated with splicing silencing. A similar mode of splicing silencing, involving multiple binding sites flanking an alternative exon, has also been reported for Sex lethal (Sxl), heterogeneous nuclear ribonucleoprotein A1, C and L (hnRNP A1, L and C), and polypyrimidine tract binding protein (PTB)10, 25, 26. The glycine-rich C-terminal domains of Sxl and hnRNP A1 are required for the cooperative assembly of these proteins on multiple proximal binding sites and the associated splicing silencing25. TDP-43 also contains a glycine-rich C terminus. Mutations in the TARDBP gene that were identified in subjects with ALS or FTLD are concentrated in the region that encodes the glycine-rich C terminus5, indicating that the ability of TDP-43 to assemble cooperatively on long RNA binding sites is involved in the disease mechanisms. The tandem UG repeat sequences that are recognized by TDP-43 correspond to the most common microsatellite (AC) in the human genome. Variations in the lengths of microsatellites can introduce heritable phenotypic variation27 or lead to changes in transcription and alternative splicing28. Genes that are involved in organ morphogenesis and neurogenesis are enriched in the variable tandem repeats29. Similarly, the genes that contain the exons that are regulated by TDP-43 are enriched for neural tube formation and organ morphogenesis. This is consistent with the finding that a TDP-43 knockout is lethal for mice from embryonic day 3.5 (ref. 30). It could be speculated that the ability of TDP-43 to regulate splicing by binding the UG repeats in the human genome might contribute to variation in the splicing regulation of genes that are involved in development. TDP-43 regulates the splicing of several transcripts that encode proteins involved in neuronal survival, as well as seven mRNAs that encode proteins that are relevant for neurodegenerative diseases (Table 2). Deltacatenin (CTNND1) is involved in the development and maintenance of dendritic spines31, 32. Furthermore, CTNND1 interacts with presenilin-1 to inhibit production of β-amyloid peptide, the main constituent of amyloid Characterizing the RNA targets and position-dependent splici... http://www.nature.com/neuro/journal/v14/n4/full/nn.2778.html 10 of 17 03/06/2011 14:57 plaques in the brains of individuals with Alzheimer's disease33. MEF2D is a transcription factor that has been reported to contribute to neuronal survival in Parkinson's disease34. BIM is a core factor in the neuron-specific JNK-mediated apoptotic pathway, and loss of BIM protects mice from ischemia-induced damage35, 36. The BIM splice isoforms that are regulated by TDP-43 have been functionally characterized; BIM antagonizes the pro-survival Bcl-2 family members more effectively than BIM , whereas BIM is the least potent22. Production of the most cytotoxic BIM isoform (BIM ) was increased in TDP-43 knockdown cells and in tissue from subjects with FTLD-TDP. However, analysis of other exons regulated by TDP-43 did not indicate a significant correlation with the splicing changes present in samples from subjects with FTLD-TDP (unpublished observation). We also found that TDP-43 bound to long ncRNAs in highly sequence-specific manner in tissue from subjects with or without FTLD-TDP. The greatest increase in TDP-43 binding in samples from subjects with FTLD-TDP is seen in the NEAT1 ncRNA. Expression of NEAT1 and MALAT1 is markedly increased in FTLD-TDP, which might be the primary cause of the increased association of TDP-43 with these RNAs. NEAT1 functions in paraspeckle assembly11 and the MALAT1 ncRNA recruits splicing factors to nuclear speckles and affects phosphorylation of serine/arginine-rich splicing factor proteins37. In conclusion, we have characterized the splicing function of TDP-43 and quantified the changes in its RNA binding in FTLD-TDP. Analysis of these RNAs in model organisms and in neurons containing TDP-43 inclusions will be required for full understanding of the mechanisms of loss or gain of TDP-43 function in the neurodegenerative process. Methods iCLIP analysis. The iCLIP protocol was performed as described10, with the following modifications. SH-SY5Y neuroblastoma or H9 human embryonic stem cells were irradiated once with 150 mJ cm−2 in a Stratlinker 2400 at 254 nm, brain tissue was dissociated in cold PBS and the suspension was cross-linked four times with 100 mJ cm−2. TDP-43 was immunoprecipitated with protein A Dynabeads (Invitrogen) conjugated to rabbit-anti TDP-43 (Proteintech, 10782-2-AP). For iCLIP of CELF2, protein G Dynabeads conjugated to mouse anti-CELF2 (Sigma, C9367) were used. In both cases, the region corresponding to 55–100-kDa complexes was excised from the membrane to isolate the RNA. High-throughput sequencing using Illumina GA2 was done using 54 or 72 cycles (Supplementary Table 1). The barcode sequences corresponding to the individual experiment were as described (Supplementary Table 1). The random barcodes were registered and the barcodes were removed before mapping the sequences to the human genome sequence (version GRCh37/hg19) allowing one mismatch using Bowtie version 0.10.1 (command line: -a -m 1 -v 1). Nucleocytoplasmic fractionation. We resuspended 50 mg of cross-linked SH-SY5Y cells or brain tissue in 1 ml of cold cytoplasmic lysis buffer (50 mM Tris-HCl, pH 7.4, 10 mM NaCl, 0.5% NP-40 (vol/vol), 0.25% Triton X-100 (vol/vol), 1 mM EDTA, 0.5% RNAsin (vol/vol)). After rotation at 4 °C for 5 min, homogeniser was used to complete the lysis. After centrifugation at 4 °C, 3,000 g for 5 min, supernatant was collected and centrifugation was repeated at 4 °C, 10,000 g for 10 min. Supernatant was collected and 200 μl of 0.5% SDS (vol/vol), 0.25% sodium deoxycholate (vol/vol), 0.5 M NaCl was added; this was used as cytoplasmic fraction for CLIP or western blot analyses. The pellet (after 3,000 g spin) was resuspended in 1 ml of CLIP lysis buffer10, sonicated and used as nuclear fraction for CLIP or western blot analyses. The radioactive CLIP protein–RNA complexes on nitrocellulose membrane were quantified using phosphoimager. Sequence analyses. Analysis of reproducibility of cross-link sites was done as described10. Identification of significant iCLIP cross-link clusters and z-score analysis of enriched pentamers was done as described38. For analysis of S