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The RNA-binding protein Staufen1 impairs myogenic differentiation via a c-myc-dependent mechanism.

Ravel-Chapuis A, Crawford TE, Blais-Crépeau ML, Bélanger G, Richer CT, Jasmin BJ - Mol. Biol. Cell (2014)

Bottom Line: Cells overexpressing Staufen1 differentiated poorly, as evidenced by reductions in the differentiation and fusion indices and decreases in MyoD, myogenin, MEF2A, and MEF2C, independently of Staufen-mediated mRNA decay.By contrast, the knockdown of Staufen1 decreased c-myc levels in myoblasts.Collectively our results show that Staufen1 is highly expressed during early stages of differentiation/development and that it can impair differentiation by regulating c-myc, thereby highlighting the multifunctional role of Staufen1 in skeletal muscle cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, ON K1H 8M5, Canada.

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MyoD does not rescue the differentiation defect. (A) Representative Western blots showing MyoD protein level during myogenic differentiation of Staufen1-HA–stable cell lines. β-Actin was used as a loading control. (B) Relative quantification of MyoD protein levels normalized to β-actin (n = 4). (C) Relative quantification of MyoD mRNA levels as determined by qRT-PCR. Levels were normalized to cyclophylin-B (n = 4). (D) Stable cell lines were transfected with control or MyoD expression vectors. At 24 h after transfection, cells were allowed to differentiate for the indicated time period. Western blots showing MyoD (lower band, endogenous MyoD; upper band, ectopic MyoD-Flag-myc protein) and myogenin expression levels. β-Actin was used to show equal loading. (E) Immunofluorescence using a pan-MyHC antibody. (F, G) Fusion and differentiation indices of C2C12 cells differentiated for 72 h. Scale bars, 100 μm. Asterisks indicate significance (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).
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Figure 6: MyoD does not rescue the differentiation defect. (A) Representative Western blots showing MyoD protein level during myogenic differentiation of Staufen1-HA–stable cell lines. β-Actin was used as a loading control. (B) Relative quantification of MyoD protein levels normalized to β-actin (n = 4). (C) Relative quantification of MyoD mRNA levels as determined by qRT-PCR. Levels were normalized to cyclophylin-B (n = 4). (D) Stable cell lines were transfected with control or MyoD expression vectors. At 24 h after transfection, cells were allowed to differentiate for the indicated time period. Western blots showing MyoD (lower band, endogenous MyoD; upper band, ectopic MyoD-Flag-myc protein) and myogenin expression levels. β-Actin was used to show equal loading. (E) Immunofluorescence using a pan-MyHC antibody. (F, G) Fusion and differentiation indices of C2C12 cells differentiated for 72 h. Scale bars, 100 μm. Asterisks indicate significance (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).

Mentions: We then sought to investigate whether Staufen1 regulates expression of the myogenic regulatory factor MyoD, a crucial regulator of myogenesis (Tapscott et al., 1988; Berkes and Tapscott, 2005; Buckingham and Vincent, 2009; Bismuth and Relaix, 2010; Bentzinger et al., 2012). We found that during proliferation and differentiation, expression of MyoD was significantly reduced (p < 0.05) in both Staufen1-HA–overexpressing clones compared with control cells (Figure 6, A–C). To assess whether the addition of exogenous MyoD could rescue the differentiation defect, we performed MyoD add-back experiments. Briefly, stable cell lines were transfected with MyoD-Flag-myc or control vectors. After transfection, cells were allowed to differentiate for 3 d. First, the increase in MyoD expression was confirmed by Western blot using MyoD antibodies. In these blots, the lower band corresponds to endogenous MyoD and the upper band to ectopic MyoD-Flag-myc (Figure 6D). Note that we were able to restore normal MyoD levels in Staufen1-HA stable cell lines (compare CTL+pcDNA3 and #25+MyoD). Then we assessed the efficiency of differentiation by measuring expression of myogenin and by immunofluorescence using a pan-MyHC antibody. As illustrated in Figure 6, E–G, no rescue of differentiation was observed in the Staufen1-HA stable cell line upon addition of MyoD-Flag-myc (compare both Staufen1-HA cells transfected with MyoD-Flag-myc to control cells transfected with MyoD-Flag-myc and to Staufen1-HA cells without MyoD-Flag-myc). Taken together, these findings show that the inhibition of differentiation induced by Staufen1 is MyoD independent.


The RNA-binding protein Staufen1 impairs myogenic differentiation via a c-myc-dependent mechanism.

Ravel-Chapuis A, Crawford TE, Blais-Crépeau ML, Bélanger G, Richer CT, Jasmin BJ - Mol. Biol. Cell (2014)

MyoD does not rescue the differentiation defect. (A) Representative Western blots showing MyoD protein level during myogenic differentiation of Staufen1-HA–stable cell lines. β-Actin was used as a loading control. (B) Relative quantification of MyoD protein levels normalized to β-actin (n = 4). (C) Relative quantification of MyoD mRNA levels as determined by qRT-PCR. Levels were normalized to cyclophylin-B (n = 4). (D) Stable cell lines were transfected with control or MyoD expression vectors. At 24 h after transfection, cells were allowed to differentiate for the indicated time period. Western blots showing MyoD (lower band, endogenous MyoD; upper band, ectopic MyoD-Flag-myc protein) and myogenin expression levels. β-Actin was used to show equal loading. (E) Immunofluorescence using a pan-MyHC antibody. (F, G) Fusion and differentiation indices of C2C12 cells differentiated for 72 h. Scale bars, 100 μm. Asterisks indicate significance (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).
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Figure 6: MyoD does not rescue the differentiation defect. (A) Representative Western blots showing MyoD protein level during myogenic differentiation of Staufen1-HA–stable cell lines. β-Actin was used as a loading control. (B) Relative quantification of MyoD protein levels normalized to β-actin (n = 4). (C) Relative quantification of MyoD mRNA levels as determined by qRT-PCR. Levels were normalized to cyclophylin-B (n = 4). (D) Stable cell lines were transfected with control or MyoD expression vectors. At 24 h after transfection, cells were allowed to differentiate for the indicated time period. Western blots showing MyoD (lower band, endogenous MyoD; upper band, ectopic MyoD-Flag-myc protein) and myogenin expression levels. β-Actin was used to show equal loading. (E) Immunofluorescence using a pan-MyHC antibody. (F, G) Fusion and differentiation indices of C2C12 cells differentiated for 72 h. Scale bars, 100 μm. Asterisks indicate significance (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).
Mentions: We then sought to investigate whether Staufen1 regulates expression of the myogenic regulatory factor MyoD, a crucial regulator of myogenesis (Tapscott et al., 1988; Berkes and Tapscott, 2005; Buckingham and Vincent, 2009; Bismuth and Relaix, 2010; Bentzinger et al., 2012). We found that during proliferation and differentiation, expression of MyoD was significantly reduced (p < 0.05) in both Staufen1-HA–overexpressing clones compared with control cells (Figure 6, A–C). To assess whether the addition of exogenous MyoD could rescue the differentiation defect, we performed MyoD add-back experiments. Briefly, stable cell lines were transfected with MyoD-Flag-myc or control vectors. After transfection, cells were allowed to differentiate for 3 d. First, the increase in MyoD expression was confirmed by Western blot using MyoD antibodies. In these blots, the lower band corresponds to endogenous MyoD and the upper band to ectopic MyoD-Flag-myc (Figure 6D). Note that we were able to restore normal MyoD levels in Staufen1-HA stable cell lines (compare CTL+pcDNA3 and #25+MyoD). Then we assessed the efficiency of differentiation by measuring expression of myogenin and by immunofluorescence using a pan-MyHC antibody. As illustrated in Figure 6, E–G, no rescue of differentiation was observed in the Staufen1-HA stable cell line upon addition of MyoD-Flag-myc (compare both Staufen1-HA cells transfected with MyoD-Flag-myc to control cells transfected with MyoD-Flag-myc and to Staufen1-HA cells without MyoD-Flag-myc). Taken together, these findings show that the inhibition of differentiation induced by Staufen1 is MyoD independent.

Bottom Line: Cells overexpressing Staufen1 differentiated poorly, as evidenced by reductions in the differentiation and fusion indices and decreases in MyoD, myogenin, MEF2A, and MEF2C, independently of Staufen-mediated mRNA decay.By contrast, the knockdown of Staufen1 decreased c-myc levels in myoblasts.Collectively our results show that Staufen1 is highly expressed during early stages of differentiation/development and that it can impair differentiation by regulating c-myc, thereby highlighting the multifunctional role of Staufen1 in skeletal muscle cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, ON K1H 8M5, Canada.

Show MeSH
Related in: MedlinePlus