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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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ALS-associated FUS mutants are deficient in alternative splicing and autoregulation.A) Confocal fluorescent microscopy showing the cellular localization of EGFP-FUS mutants in HEK293 cells. Magnification, 40×. Scale bar, 20 µm. Endogenous FUS protein was detected using anti-FUS antibody (Bethyl, BL1355). DNA in the nucleus was stained with DAPI or NucRed Dead 647. B) RT-PCR analysis of FUS exon 7 splice variants in pDUP-FUS-E7L coexpressed with either wildtype (wt) or mutant EGFP-FUS in HEK293 cells. Bar graphs represent mean ± SD (n = 3). EGFP-FUS mutants were compared with EGFP-FUS wildtype protein using student's t-tests. C) Western blot analysis of endogenous FUS and EGFP-FUS protein in HEK293 cells, using anti-FUS antibody (10F7). Bar graphs represent mean ± SEM (n = 3). EGFP, EGFP-FUS wildtype and mutants were compared with mock transfected cells using student's t-tests. In both panel B and C, * P≤0.05, ** P≤0.01. D) Confocal fluorescent microscopy showing the localization of endogenous FUS protein in the cytoplasmic aggregates of EGFP-FUS ΔE15 mutants expressed in HEK293 cells. Anti-FUS antibody (Bethyl, BL1355) recognizing C-terminus epitope detected only endogenous FUS protein but not ΔE15 mutants. Magnification, 40×. Scale bar, 20 µm.
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pgen-1003895-g006: ALS-associated FUS mutants are deficient in alternative splicing and autoregulation.A) Confocal fluorescent microscopy showing the cellular localization of EGFP-FUS mutants in HEK293 cells. Magnification, 40×. Scale bar, 20 µm. Endogenous FUS protein was detected using anti-FUS antibody (Bethyl, BL1355). DNA in the nucleus was stained with DAPI or NucRed Dead 647. B) RT-PCR analysis of FUS exon 7 splice variants in pDUP-FUS-E7L coexpressed with either wildtype (wt) or mutant EGFP-FUS in HEK293 cells. Bar graphs represent mean ± SD (n = 3). EGFP-FUS mutants were compared with EGFP-FUS wildtype protein using student's t-tests. C) Western blot analysis of endogenous FUS and EGFP-FUS protein in HEK293 cells, using anti-FUS antibody (10F7). Bar graphs represent mean ± SEM (n = 3). EGFP, EGFP-FUS wildtype and mutants were compared with mock transfected cells using student's t-tests. In both panel B and C, * P≤0.05, ** P≤0.01. D) Confocal fluorescent microscopy showing the localization of endogenous FUS protein in the cytoplasmic aggregates of EGFP-FUS ΔE15 mutants expressed in HEK293 cells. Anti-FUS antibody (Bethyl, BL1355) recognizing C-terminus epitope detected only endogenous FUS protein but not ΔE15 mutants. Magnification, 40×. Scale bar, 20 µm.

Mentions: Consistent with previous reports [6], [8], [9], [11], we observed predominant nuclear localization of wildtype FUS, minor cytoplasmic and mainly nuclear localization of R521G, moderate cytoplasmic accumulation and aggregation of R522G, and severe cytoplasmic aggregation with much less nuclear localization of the FUS ΔE15 mutant, following transient transfection in HEK293 cells (Figure 6A) and mouse motor neuron cells NSC-34 (Figure S8). The RRM mutant, like the wildtype FUS protein, was predominantly localized in the nucleus.


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)

ALS-associated FUS mutants are deficient in alternative splicing and autoregulation.A) Confocal fluorescent microscopy showing the cellular localization of EGFP-FUS mutants in HEK293 cells. Magnification, 40×. Scale bar, 20 µm. Endogenous FUS protein was detected using anti-FUS antibody (Bethyl, BL1355). DNA in the nucleus was stained with DAPI or NucRed Dead 647. B) RT-PCR analysis of FUS exon 7 splice variants in pDUP-FUS-E7L coexpressed with either wildtype (wt) or mutant EGFP-FUS in HEK293 cells. Bar graphs represent mean ± SD (n = 3). EGFP-FUS mutants were compared with EGFP-FUS wildtype protein using student's t-tests. C) Western blot analysis of endogenous FUS and EGFP-FUS protein in HEK293 cells, using anti-FUS antibody (10F7). Bar graphs represent mean ± SEM (n = 3). EGFP, EGFP-FUS wildtype and mutants were compared with mock transfected cells using student's t-tests. In both panel B and C, * P≤0.05, ** P≤0.01. D) Confocal fluorescent microscopy showing the localization of endogenous FUS protein in the cytoplasmic aggregates of EGFP-FUS ΔE15 mutants expressed in HEK293 cells. Anti-FUS antibody (Bethyl, BL1355) recognizing C-terminus epitope detected only endogenous FUS protein but not ΔE15 mutants. Magnification, 40×. Scale bar, 20 µm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814325&req=5

pgen-1003895-g006: ALS-associated FUS mutants are deficient in alternative splicing and autoregulation.A) Confocal fluorescent microscopy showing the cellular localization of EGFP-FUS mutants in HEK293 cells. Magnification, 40×. Scale bar, 20 µm. Endogenous FUS protein was detected using anti-FUS antibody (Bethyl, BL1355). DNA in the nucleus was stained with DAPI or NucRed Dead 647. B) RT-PCR analysis of FUS exon 7 splice variants in pDUP-FUS-E7L coexpressed with either wildtype (wt) or mutant EGFP-FUS in HEK293 cells. Bar graphs represent mean ± SD (n = 3). EGFP-FUS mutants were compared with EGFP-FUS wildtype protein using student's t-tests. C) Western blot analysis of endogenous FUS and EGFP-FUS protein in HEK293 cells, using anti-FUS antibody (10F7). Bar graphs represent mean ± SEM (n = 3). EGFP, EGFP-FUS wildtype and mutants were compared with mock transfected cells using student's t-tests. In both panel B and C, * P≤0.05, ** P≤0.01. D) Confocal fluorescent microscopy showing the localization of endogenous FUS protein in the cytoplasmic aggregates of EGFP-FUS ΔE15 mutants expressed in HEK293 cells. Anti-FUS antibody (Bethyl, BL1355) recognizing C-terminus epitope detected only endogenous FUS protein but not ΔE15 mutants. Magnification, 40×. Scale bar, 20 µm.
Mentions: Consistent with previous reports [6], [8], [9], [11], we observed predominant nuclear localization of wildtype FUS, minor cytoplasmic and mainly nuclear localization of R521G, moderate cytoplasmic accumulation and aggregation of R522G, and severe cytoplasmic aggregation with much less nuclear localization of the FUS ΔE15 mutant, following transient transfection in HEK293 cells (Figure 6A) and mouse motor neuron cells NSC-34 (Figure S8). The RRM mutant, like the wildtype FUS protein, was predominantly localized in the nucleus.

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