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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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Proposed model of FUS autoregulation and its contribution to ALS pathogenesis.A) A model illustrates FUS autoregulation as a feedback loop to control the homeostasis of FUS protein levels. When FUS protein levels are high, FUS downregulates its own protein by repressing exon 7 inclusion to produce exon 7-skipped transcripts for NMD. When FUS protein levels are low, the repression of exon 7 is reduced and more splice variants with exon 7-included are produced for translation. B) The deficient FUS autoregulation in ALS-associated FUS mutants may contribute to its abnormal cytoplasmic accumulation. Mutations within the nuclear localization signal result in cytoplasmic retention of FUS mutants and reduction of nuclear FUS. The reduction of nuclear FUS leads to the reduction of FUS exon 7 repression, which would in turn produce more exon 7-included transcripts for translation, thereby driving elevated FUS protein synthesis. This compromised FUS autoregulation forms a feed-forward loop, potentially exacerbating the abnormal cytoplasmic accumulation of FUS mutants. FUS mutations and FUS mutants are indicated with a red asterisk.
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pgen-1003895-g008: Proposed model of FUS autoregulation and its contribution to ALS pathogenesis.A) A model illustrates FUS autoregulation as a feedback loop to control the homeostasis of FUS protein levels. When FUS protein levels are high, FUS downregulates its own protein by repressing exon 7 inclusion to produce exon 7-skipped transcripts for NMD. When FUS protein levels are low, the repression of exon 7 is reduced and more splice variants with exon 7-included are produced for translation. B) The deficient FUS autoregulation in ALS-associated FUS mutants may contribute to its abnormal cytoplasmic accumulation. Mutations within the nuclear localization signal result in cytoplasmic retention of FUS mutants and reduction of nuclear FUS. The reduction of nuclear FUS leads to the reduction of FUS exon 7 repression, which would in turn produce more exon 7-included transcripts for translation, thereby driving elevated FUS protein synthesis. This compromised FUS autoregulation forms a feed-forward loop, potentially exacerbating the abnormal cytoplasmic accumulation of FUS mutants. FUS mutations and FUS mutants are indicated with a red asterisk.

Mentions: Here we report a novel autoregulatory mechanism of FUS by alternative splicing and NMD. The model shown in Figure 8A illustrates FUS autoregulation as a feedback loop to control the homeostasis of FUS protein levels. High levels of FUS protein lead to increased FUS binding to exon 7 and its flanking introns, promoting exon 7 skipping and NMD to reduce excessive FUS protein. Low levels of FUS protein would favor exon 7 inclusion, resulting in increased FUS protein production. Alternative splicing-mediated NMD and highly conserved intronic sequences represent an emerging common mechanism utilized by RNA binding proteins (RBPs) to maintain their homeostasis [43], [50], [51]. FUS now joins this increasing list of autoregulated RBPs, including PTB, hnRNP L, Nova and TDP-43 [43], [50]–[52]. FUS regulates many aspects of gene expression including transcription, alternative splicing and RNA transportation [23]–[25]. Dynamic regulation and conserved targets suggest it is important to keep these functional activities of FUS in tight control, and that FUS likely has a co-factor role in coordinating them. For example, loss of FUS can cause genomic instability and developmental defects in mouse, Drosophila and Zebrafish [18], [19], [53]. Conversely, high levels of FUS are associated with cancer and ALS, and moreover, are known genetic determinants of these diseases. Overexpression of FUS is observed in liposarcoma and leukemia with FUS translocations [54], [55]. Aberrant accumulation of FUS mutant protein is a characteristic pathology of FUS-associated ALS [8], [9]. Depletion of FUS in the mouse nervous system affects the abundance or the splicing of about 1000 mRNAs [30], suggesting that maintaining equilibrated FUS protein levels is critical for RNA processing.


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)

Proposed model of FUS autoregulation and its contribution to ALS pathogenesis.A) A model illustrates FUS autoregulation as a feedback loop to control the homeostasis of FUS protein levels. When FUS protein levels are high, FUS downregulates its own protein by repressing exon 7 inclusion to produce exon 7-skipped transcripts for NMD. When FUS protein levels are low, the repression of exon 7 is reduced and more splice variants with exon 7-included are produced for translation. B) The deficient FUS autoregulation in ALS-associated FUS mutants may contribute to its abnormal cytoplasmic accumulation. Mutations within the nuclear localization signal result in cytoplasmic retention of FUS mutants and reduction of nuclear FUS. The reduction of nuclear FUS leads to the reduction of FUS exon 7 repression, which would in turn produce more exon 7-included transcripts for translation, thereby driving elevated FUS protein synthesis. This compromised FUS autoregulation forms a feed-forward loop, potentially exacerbating the abnormal cytoplasmic accumulation of FUS mutants. FUS mutations and FUS mutants are indicated with a red asterisk.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1003895-g008: Proposed model of FUS autoregulation and its contribution to ALS pathogenesis.A) A model illustrates FUS autoregulation as a feedback loop to control the homeostasis of FUS protein levels. When FUS protein levels are high, FUS downregulates its own protein by repressing exon 7 inclusion to produce exon 7-skipped transcripts for NMD. When FUS protein levels are low, the repression of exon 7 is reduced and more splice variants with exon 7-included are produced for translation. B) The deficient FUS autoregulation in ALS-associated FUS mutants may contribute to its abnormal cytoplasmic accumulation. Mutations within the nuclear localization signal result in cytoplasmic retention of FUS mutants and reduction of nuclear FUS. The reduction of nuclear FUS leads to the reduction of FUS exon 7 repression, which would in turn produce more exon 7-included transcripts for translation, thereby driving elevated FUS protein synthesis. This compromised FUS autoregulation forms a feed-forward loop, potentially exacerbating the abnormal cytoplasmic accumulation of FUS mutants. FUS mutations and FUS mutants are indicated with a red asterisk.
Mentions: Here we report a novel autoregulatory mechanism of FUS by alternative splicing and NMD. The model shown in Figure 8A illustrates FUS autoregulation as a feedback loop to control the homeostasis of FUS protein levels. High levels of FUS protein lead to increased FUS binding to exon 7 and its flanking introns, promoting exon 7 skipping and NMD to reduce excessive FUS protein. Low levels of FUS protein would favor exon 7 inclusion, resulting in increased FUS protein production. Alternative splicing-mediated NMD and highly conserved intronic sequences represent an emerging common mechanism utilized by RNA binding proteins (RBPs) to maintain their homeostasis [43], [50], [51]. FUS now joins this increasing list of autoregulated RBPs, including PTB, hnRNP L, Nova and TDP-43 [43], [50]–[52]. FUS regulates many aspects of gene expression including transcription, alternative splicing and RNA transportation [23]–[25]. Dynamic regulation and conserved targets suggest it is important to keep these functional activities of FUS in tight control, and that FUS likely has a co-factor role in coordinating them. For example, loss of FUS can cause genomic instability and developmental defects in mouse, Drosophila and Zebrafish [18], [19], [53]. Conversely, high levels of FUS are associated with cancer and ALS, and moreover, are known genetic determinants of these diseases. Overexpression of FUS is observed in liposarcoma and leukemia with FUS translocations [54], [55]. Aberrant accumulation of FUS mutant protein is a characteristic pathology of FUS-associated ALS [8], [9]. Depletion of FUS in the mouse nervous system affects the abundance or the splicing of about 1000 mRNAs [30], suggesting that maintaining equilibrated FUS protein levels is critical for RNA processing.

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