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ALS-associated FUS mutations result in compromised FUS alternative splicing and autoregulation.

Zhou Y, Liu S, Liu G, Oztürk A, Hicks GG - PLoS Genet. (2013)

Bottom Line: Overexpression of FUS led to the repression of exon 7 splicing and a reduction of endogenous FUS protein.Conversely, the repression of exon 7 was reduced by knockdown of FUS protein, and moreover, it was rescued by expression of EGFP-FUS.Taken together, FUS autoregulation by alternative splicing provides insight into a molecular mechanism by which FUS-regulated pre-mRNA processing can impact a significant number of targets important to neurodegeneration.

View Article: PubMed Central - PubMed

Affiliation: Manitoba Institute of Cell Biology, University of Manitoba, Winnipeg, Manitoba, Canada ; Department of Biochemistry & Medical Genetics, University of Manitoba, Winnipeg, Manitoba, Canada ; Regenerative Medicine Program, University of Manitoba, Winnipeg, Manitoba, Canada.

ABSTRACT
The gene encoding a DNA/RNA binding protein FUS/TLS is frequently mutated in amyotrophic lateral sclerosis (ALS). Mutations commonly affect its carboxy-terminal nuclear localization signal, resulting in varying deficiencies of FUS nuclear localization and abnormal cytoplasmic accumulation. Increasing evidence suggests deficiencies in FUS nuclear function may contribute to neuron degeneration. Here we report a novel FUS autoregulatory mechanism and its deficiency in ALS-associated mutants. Using FUS CLIP-seq, we identified significant FUS binding to a highly conserved region of exon 7 and the flanking introns of its own pre-mRNAs. We demonstrated that FUS is a repressor of exon 7 splicing and that the exon 7-skipped splice variant is subject to nonsense-mediated decay (NMD). Overexpression of FUS led to the repression of exon 7 splicing and a reduction of endogenous FUS protein. Conversely, the repression of exon 7 was reduced by knockdown of FUS protein, and moreover, it was rescued by expression of EGFP-FUS. This dynamic regulation of alternative splicing describes a novel mechanism of FUS autoregulation. Given that ALS-associated FUS mutants are deficient in nuclear localization, we examined whether cells expressing these mutants would be deficient in repressing exon 7 splicing. We showed that FUS harbouring R521G, R522G or ΔExon15 mutation (minor, moderate or severe cytoplasmic localization, respectively) directly correlated with respectively increasing deficiencies in both exon 7 repression and autoregulation of its own protein levels. These data suggest that compromised FUS autoregulation can directly exacerbate the pathogenic accumulation of cytoplasmic FUS protein in ALS. We showed that exon 7 skipping can be induced by antisense oligonucleotides targeting its flanking splice sites, indicating the potential to alleviate abnormal cytoplasmic FUS accumulation in ALS. Taken together, FUS autoregulation by alternative splicing provides insight into a molecular mechanism by which FUS-regulated pre-mRNA processing can impact a significant number of targets important to neurodegeneration.

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FUS binds to exon 7 and flanking introns of its own pre-mRNA in vivo.A) The enrichment of FUS CLIP tags in exon 7 (E7) and the flanking introns of FUS own pre-mRNA, as determined by a peak finding algorithm CisGenome. B) Cross-species conservation of FUS gene. The conservation track of UCSC genome browser (http://genome.ucsc.edu/) was used to display the PhastCons conservation score of 46 vertebrate species. C) FUS RNA-IP followed by RT-PCR of FUS exon 7. RT-PCR of FUS constitutive exon 5 is a control. Medium RNase concentration (M; 0.1 µg/ml) or high RNase concentration (H; 1 µg/ml) was used to treat cell lysates before immunoprecipitation. D) FUS exon 7-skipped splice variant is subject to nonsense mediated decay (NMD). Cycloheximide (CHX) was used to treat cells for 6 h to inhibit NMD. FUS exon 7 splice variants were detected by [γ-32P] ATP labeled PCR. The exon skipping ratio is equal to the intensity of the exon 7-skipped band divided by the intensity sum of both splice variants. Bar graphs represent mean ± SEM (n = 5 or 6). For all the quantification, student's t-tests were performed. * P≤0.05, ** P≤0.01.
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pgen-1003895-g002: FUS binds to exon 7 and flanking introns of its own pre-mRNA in vivo.A) The enrichment of FUS CLIP tags in exon 7 (E7) and the flanking introns of FUS own pre-mRNA, as determined by a peak finding algorithm CisGenome. B) Cross-species conservation of FUS gene. The conservation track of UCSC genome browser (http://genome.ucsc.edu/) was used to display the PhastCons conservation score of 46 vertebrate species. C) FUS RNA-IP followed by RT-PCR of FUS exon 7. RT-PCR of FUS constitutive exon 5 is a control. Medium RNase concentration (M; 0.1 µg/ml) or high RNase concentration (H; 1 µg/ml) was used to treat cell lysates before immunoprecipitation. D) FUS exon 7-skipped splice variant is subject to nonsense mediated decay (NMD). Cycloheximide (CHX) was used to treat cells for 6 h to inhibit NMD. FUS exon 7 splice variants were detected by [γ-32P] ATP labeled PCR. The exon skipping ratio is equal to the intensity of the exon 7-skipped band divided by the intensity sum of both splice variants. Bar graphs represent mean ± SEM (n = 5 or 6). For all the quantification, student's t-tests were performed. * P≤0.05, ** P≤0.01.

Mentions: Out of the 87 cassette exons enriched with FUS CLIP clusters, the top candidate was exon 7 and flanking introns 6 and 7 of the pre-mRNAs of FUS itself (Table S3). This CLIP cluster was also in the top 10 of all 1928 FUS CLIP clusters identified, as ranked by fold enrichment. The number of FUS CLIP tags within exon 7 and its flanking introns were 8.1 fold higher than the control mouse IgG CLIP tags (FDR = 0.043), as determined by the peak finding algorithm CisGenome [33] (Figure 2A). The region encompassing FUS intron6-exon7-intron7 is ∼3 kb and highly conserved in 38 vertebrate species (Figure 2B). Human and mouse DNA sequences share 77% identity within this region, while the average similarity throughout other introns of FUS is 40%. Sub-regions with highly enriched CLIP tags (over 100 overlapping CLIP tags in the center) were used for de novo consensus RNA motif analysis. Analysis of all the CLIP tags within these selected regions using the Homer algorithm revealed that GU or GGU containing sequences are statistically enriched over background (all pre-mRNA sequences) (Figure S5). This is consistent with previous reports that GGUG, GUGGU, or GGU containing RNA sequences are potential FUS binding motifs [29], [30], [40].


ALS-associated FUS mutations result in compromised FUS alternative splicing and autoregulation.

Zhou Y, Liu S, Liu G, Oztürk A, Hicks GG - PLoS Genet. (2013)

FUS binds to exon 7 and flanking introns of its own pre-mRNA in vivo.A) The enrichment of FUS CLIP tags in exon 7 (E7) and the flanking introns of FUS own pre-mRNA, as determined by a peak finding algorithm CisGenome. B) Cross-species conservation of FUS gene. The conservation track of UCSC genome browser (http://genome.ucsc.edu/) was used to display the PhastCons conservation score of 46 vertebrate species. C) FUS RNA-IP followed by RT-PCR of FUS exon 7. RT-PCR of FUS constitutive exon 5 is a control. Medium RNase concentration (M; 0.1 µg/ml) or high RNase concentration (H; 1 µg/ml) was used to treat cell lysates before immunoprecipitation. D) FUS exon 7-skipped splice variant is subject to nonsense mediated decay (NMD). Cycloheximide (CHX) was used to treat cells for 6 h to inhibit NMD. FUS exon 7 splice variants were detected by [γ-32P] ATP labeled PCR. The exon skipping ratio is equal to the intensity of the exon 7-skipped band divided by the intensity sum of both splice variants. Bar graphs represent mean ± SEM (n = 5 or 6). For all the quantification, student's t-tests were performed. * P≤0.05, ** P≤0.01.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3814325&req=5

pgen-1003895-g002: FUS binds to exon 7 and flanking introns of its own pre-mRNA in vivo.A) The enrichment of FUS CLIP tags in exon 7 (E7) and the flanking introns of FUS own pre-mRNA, as determined by a peak finding algorithm CisGenome. B) Cross-species conservation of FUS gene. The conservation track of UCSC genome browser (http://genome.ucsc.edu/) was used to display the PhastCons conservation score of 46 vertebrate species. C) FUS RNA-IP followed by RT-PCR of FUS exon 7. RT-PCR of FUS constitutive exon 5 is a control. Medium RNase concentration (M; 0.1 µg/ml) or high RNase concentration (H; 1 µg/ml) was used to treat cell lysates before immunoprecipitation. D) FUS exon 7-skipped splice variant is subject to nonsense mediated decay (NMD). Cycloheximide (CHX) was used to treat cells for 6 h to inhibit NMD. FUS exon 7 splice variants were detected by [γ-32P] ATP labeled PCR. The exon skipping ratio is equal to the intensity of the exon 7-skipped band divided by the intensity sum of both splice variants. Bar graphs represent mean ± SEM (n = 5 or 6). For all the quantification, student's t-tests were performed. * P≤0.05, ** P≤0.01.
Mentions: Out of the 87 cassette exons enriched with FUS CLIP clusters, the top candidate was exon 7 and flanking introns 6 and 7 of the pre-mRNAs of FUS itself (Table S3). This CLIP cluster was also in the top 10 of all 1928 FUS CLIP clusters identified, as ranked by fold enrichment. The number of FUS CLIP tags within exon 7 and its flanking introns were 8.1 fold higher than the control mouse IgG CLIP tags (FDR = 0.043), as determined by the peak finding algorithm CisGenome [33] (Figure 2A). The region encompassing FUS intron6-exon7-intron7 is ∼3 kb and highly conserved in 38 vertebrate species (Figure 2B). Human and mouse DNA sequences share 77% identity within this region, while the average similarity throughout other introns of FUS is 40%. Sub-regions with highly enriched CLIP tags (over 100 overlapping CLIP tags in the center) were used for de novo consensus RNA motif analysis. Analysis of all the CLIP tags within these selected regions using the Homer algorithm revealed that GU or GGU containing sequences are statistically enriched over background (all pre-mRNA sequences) (Figure S5). This is consistent with previous reports that GGUG, GUGGU, or GGU containing RNA sequences are potential FUS binding motifs [29], [30], [40].

Bottom Line: Overexpression of FUS led to the repression of exon 7 splicing and a reduction of endogenous FUS protein.Conversely, the repression of exon 7 was reduced by knockdown of FUS protein, and moreover, it was rescued by expression of EGFP-FUS.Taken together, FUS autoregulation by alternative splicing provides insight into a molecular mechanism by which FUS-regulated pre-mRNA processing can impact a significant number of targets important to neurodegeneration.

View Article: PubMed Central - PubMed

Affiliation: Manitoba Institute of Cell Biology, University of Manitoba, Winnipeg, Manitoba, Canada ; Department of Biochemistry & Medical Genetics, University of Manitoba, Winnipeg, Manitoba, Canada ; Regenerative Medicine Program, University of Manitoba, Winnipeg, Manitoba, Canada.

ABSTRACT
The gene encoding a DNA/RNA binding protein FUS/TLS is frequently mutated in amyotrophic lateral sclerosis (ALS). Mutations commonly affect its carboxy-terminal nuclear localization signal, resulting in varying deficiencies of FUS nuclear localization and abnormal cytoplasmic accumulation. Increasing evidence suggests deficiencies in FUS nuclear function may contribute to neuron degeneration. Here we report a novel FUS autoregulatory mechanism and its deficiency in ALS-associated mutants. Using FUS CLIP-seq, we identified significant FUS binding to a highly conserved region of exon 7 and the flanking introns of its own pre-mRNAs. We demonstrated that FUS is a repressor of exon 7 splicing and that the exon 7-skipped splice variant is subject to nonsense-mediated decay (NMD). Overexpression of FUS led to the repression of exon 7 splicing and a reduction of endogenous FUS protein. Conversely, the repression of exon 7 was reduced by knockdown of FUS protein, and moreover, it was rescued by expression of EGFP-FUS. This dynamic regulation of alternative splicing describes a novel mechanism of FUS autoregulation. Given that ALS-associated FUS mutants are deficient in nuclear localization, we examined whether cells expressing these mutants would be deficient in repressing exon 7 splicing. We showed that FUS harbouring R521G, R522G or ΔExon15 mutation (minor, moderate or severe cytoplasmic localization, respectively) directly correlated with respectively increasing deficiencies in both exon 7 repression and autoregulation of its own protein levels. These data suggest that compromised FUS autoregulation can directly exacerbate the pathogenic accumulation of cytoplasmic FUS protein in ALS. We showed that exon 7 skipping can be induced by antisense oligonucleotides targeting its flanking splice sites, indicating the potential to alleviate abnormal cytoplasmic FUS accumulation in ALS. Taken together, FUS autoregulation by alternative splicing provides insight into a molecular mechanism by which FUS-regulated pre-mRNA processing can impact a significant number of targets important to neurodegeneration.

Show MeSH
Related in: MedlinePlus