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. 2012:2:603.
doi: 10.1038/srep00603. Epub 2012 Aug 28.

Widespread binding of FUS along nascent RNA regulates alternative splicing in the brain

Boris Rogelj  1 , Laura E Easton, Gireesh K Bogu, Lawrence W Stanton, Gregor Rot, Tomaž Curk, Blaž Zupan, Yoichiro Sugimoto, Miha Modic, Nejc Haberman, James Tollervey, Ritsuko Fujii, Toru Takumi, Christopher E Shaw, Jernej Ule
Affiliations

Widespread binding of FUS along nascent RNA regulates alternative splicing in the brain

Boris Rogelj et al. Sci Rep. 2012.

Abstract

Fused in sarcoma (FUS) and TAR DNA-binding protein 43 (TDP-43) are RNA-binding proteins pathogenetically linked to amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), but it is not known if they regulate the same transcripts. We addressed this question using crosslinking and immunoprecipitation (iCLIP) in mouse brain, which showed that FUS binds along the whole length of the nascent RNA with limited sequence specificity to GGU and related motifs. A saw-tooth binding pattern in long genes demonstrated that FUS remains bound to pre-mRNAs until splicing is completed. Analysis of FUS(-/-) brain demonstrated a role for FUS in alternative splicing, with increased crosslinking of FUS in introns around the repressed exons. We did not observe a significant overlap in the RNA binding sites or the exons regulated by FUS and TDP-43. Nevertheless, we found that both proteins regulate genes that function in neuronal development.

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Figures

Figure 1
Figure 1. FUS binds along the whole length of nascent RNAs.
(a) The proportion of cDNAs (out of all cDNAs that mapped to the mouse genome) from the FUS, TDP-43 and U2AF65 iCLIP experiments that mapped to different RNA regions. (UTR: untranslated region, ORF: open reading frame, ncRNA: non-coding RNA). (b) The map of FUS, TDP-43 and U2AF65 iCLIP crosslink sites at the 5′ regions of all protein-coding genes annotated by ENSEMBL 59, from 1 kb upstream of transcription start to 200 nts downstream. The data of each protein was first normalised to the average crosslinking on the sense strand within this region, and then to the crosslinking of U2AF65 within the interval 150-200 nts downstream of the transcription start site. Crosslinking distribution was then evaluated in 50 nt intervals. The solid lines show binding to antisense, and the dashed lines of the same colour show binding to the sense RNAs, relative to the orientation of the downstream gene. (c) The map of clustered crosslink sites (flank = 15) of FUS, TDP-43 and U2AF65 iCLIP identified at intron-exon junctions of all exons annotated by ENSEMBL 59. Crosslinking enrichment was determined by comparing to the average crosslinking of the same protein in the intronic region of 300–100 nts upstream of exons. (d) The average gene-normalized iCLIP cDNA density in 5 kb intervals of introns longer than 100 kb, at different distances relative to 3′ splice sites. (e) Gene-normalized cDNA density in 0.5 kb intervals within the Gabrb3 gene. The cDNA density in each interval was normalized to the average cDNA density within the whole gene. The maximum normalized cDNA density is shown at the right of each track.
Figure 2
Figure 2. Analysis of RNA sequence and structure specificity of FUS.
(a) Analysis of the probability of single-strandedness at positions −100 to +100 around the FUS, TDP-43 and U2AF65 crosslink sites. We calculated the probability of tetramers being located in single-stranded regions in 70-nucleotide sliding windows using RNAplfold, as described previously. (b–e) Comparison of pentamer enrichment at crosslink sites between replicate FUS experiments, or between FUS, TDP-43 and U2AF65 iCLIP. Pearson correlation coefficient (r) is shown. Pentamers centered at positions −4 to 0 relative to the crosslink site were evaluated and enrichment is determined by comparison to randomised positions in the same genomic regions. The sequences of the most enriched pentamers at TDP-43 (d) and U2AF65 (e) crosslink sites are shown, and two groups of pentamers circled in (e) are evaluated further in panels 2f and 2g. (f) Position of the average enrichment of the four listed pentamers (circled in red in Fig. 2e). The middle position of the pentamers is evaluated relative to the position of cross-link sites, and enrichment is determined by comparison to randomised positions in the same genomic regions. (g) Position of the average enrichment of the two listed pentamers (circled in purple in Fig. 2e). The middle position of the pentamers is evaluated relative to the position of cross-link sites, and enrichment is determined by comparison to randomised positions in the same genomic regions.
Figure 3
Figure 3. FUS, TDP-43 and U2AF65 crosslinking around two exons regulated by FUS.
(a) Crosslinking density within the D4Wsu53e gene. The exon repressed by FUS is marked by a blue arrow. The bar graph shows the cDNA count at individual crosslink sites in replicate FUS iCLIP experiments, and the grouped TDP-43 and U2AF65 iCLIP experiments. (b) Crosslinking density within the Ewsr1 gene. The exon enhanced by FUS is marked by a red arrow. The bar graph shows the cDNA count at individual crosslink sites in replicate FUS iCLIP experiments, and the grouped TDP-43 and U2AF65 iCLIP experiments.
Figure 4
Figure 4. Analysis of iCLIP crosslink clusters around the exons regulated by FUS.
The map of crosslink clusters at positions within 500 nt of alternative exons and flanking exons. Crosslink clusters were determined using flank = 200 and FDR<0.05. The exons are grouped by sequential analysis of crosslink cluster positions in three regions: group 1 is identified by clusters within the exon; group 2 by clusters downstream of the exon; and group 3 by clusters upstream of the exon. The cassette exons with ΔIrank > 1 (enhanced exons, red clusters) or ΔIrank < −1 (repressed exons, blue clusters) in analysis of FUS−/− brain RNA that contain at least one crosslink cluster in these regions are shown. The positions of FUS (a), TDP-43 (b) and U2AF65 (c) crosslink clusters are shown, and the gene symbol and exon number (according to genomic annotation) is shown on the left. Only the FUS-regulated exons that contain crosslink clusters for the corresponding RBP in the evaluated regions are shown.
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