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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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Staufen1 expression is modulated during skeletal muscle degeneration/regeneration. (A) Western blots showing Staufen1, c-myc, and myogenin protein levels in regenerating TA muscles. TA muscles were injected with CTX to induce muscle degeneration and harvested at 2, 4, 7, and 14 d postinjection. Saline-injected muscles were used as controls. GAPDH and Ponceau staining were used to show equal loading. (B–D) Relative quantification of myogenin, Staufen1, and c-myc expression levels, respectively (n = 3). (E, F) Immunofluorescence on cryostat cross sections of control and CTX-injected TA muscles. Sections were stained with a Staufen1 (red) and laminin (green) antibodies and nuclei with DAPI (blue). Arrows point to marked staining of Staufen1. Same exposure parameters were used to allow the comparison of signal intensity. Using these parameters, a no-primary-antibody control shows no signal. Scale bars, 20 μm. Asterisks indicate significance (*p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001).
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Figure 2: Staufen1 expression is modulated during skeletal muscle degeneration/regeneration. (A) Western blots showing Staufen1, c-myc, and myogenin protein levels in regenerating TA muscles. TA muscles were injected with CTX to induce muscle degeneration and harvested at 2, 4, 7, and 14 d postinjection. Saline-injected muscles were used as controls. GAPDH and Ponceau staining were used to show equal loading. (B–D) Relative quantification of myogenin, Staufen1, and c-myc expression levels, respectively (n = 3). (E, F) Immunofluorescence on cryostat cross sections of control and CTX-injected TA muscles. Sections were stained with a Staufen1 (red) and laminin (green) antibodies and nuclei with DAPI (blue). Arrows point to marked staining of Staufen1. Same exposure parameters were used to allow the comparison of signal intensity. Using these parameters, a no-primary-antibody control shows no signal. Scale bars, 20 μm. Asterisks indicate significance (*p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001).

Mentions: Western blots were performed using TA protein extracts obtained 2, 4, 7, and 14 d after cardiotoxin injection. Myogenin protein expression was initially analyzed. Note that in contrast to myotubes, adult muscles express low levels of myogenic regulatory factors, including myogenin, because their expression is repressed by electrical activity generated by motor neurons (Eftimie et al., 1991). Myogenin showed an increase (p < 0.001) in expression immediately after injury, thereby confirming the induction of muscle regeneration. This was followed by a steady decrease (p < 0.001 and p < 0.05) in myogenin expression levels as the regeneration process advanced to completion 14 days after cardiotoxin injection (Figure 2, A and B). As a control, GAPDH expression was measured and showed a slight decrease in protein levels at days 2 and 4 postinjury, as previously described (Orengo et al., 2011). In addition, Ponceau staining was used to confirm relatively even loading. Next we measured Staufen1 protein expression and observed that Staufen1 gradually increased (p < 0.001) from 2 to 7 d after cardiotoxin treatment (Figure 2, A and C). Staufen1 expression then returned to control levels 14 d postinjury when muscle fibers are fully regenerated. Variations of Staufen1 levels within different time points reflect interindividual variability of protein expression, which is commonly observed when using animal tissues. This induction of Staufen1 after cardiotoxin injection follows a pattern similar to the one observed with CUGBP1, which is also involved in the regulation of myogenic differentiation (Orengo et al., 2011).


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)

Staufen1 expression is modulated during skeletal muscle degeneration/regeneration. (A) Western blots showing Staufen1, c-myc, and myogenin protein levels in regenerating TA muscles. TA muscles were injected with CTX to induce muscle degeneration and harvested at 2, 4, 7, and 14 d postinjection. Saline-injected muscles were used as controls. GAPDH and Ponceau staining were used to show equal loading. (B–D) Relative quantification of myogenin, Staufen1, and c-myc expression levels, respectively (n = 3). (E, F) Immunofluorescence on cryostat cross sections of control and CTX-injected TA muscles. Sections were stained with a Staufen1 (red) and laminin (green) antibodies and nuclei with DAPI (blue). Arrows point to marked staining of Staufen1. Same exposure parameters were used to allow the comparison of signal intensity. Using these parameters, a no-primary-antibody control shows no signal. Scale bars, 20 μm. Asterisks indicate significance (*p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001).
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Figure 2: Staufen1 expression is modulated during skeletal muscle degeneration/regeneration. (A) Western blots showing Staufen1, c-myc, and myogenin protein levels in regenerating TA muscles. TA muscles were injected with CTX to induce muscle degeneration and harvested at 2, 4, 7, and 14 d postinjection. Saline-injected muscles were used as controls. GAPDH and Ponceau staining were used to show equal loading. (B–D) Relative quantification of myogenin, Staufen1, and c-myc expression levels, respectively (n = 3). (E, F) Immunofluorescence on cryostat cross sections of control and CTX-injected TA muscles. Sections were stained with a Staufen1 (red) and laminin (green) antibodies and nuclei with DAPI (blue). Arrows point to marked staining of Staufen1. Same exposure parameters were used to allow the comparison of signal intensity. Using these parameters, a no-primary-antibody control shows no signal. Scale bars, 20 μm. Asterisks indicate significance (*p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001).
Mentions: Western blots were performed using TA protein extracts obtained 2, 4, 7, and 14 d after cardiotoxin injection. Myogenin protein expression was initially analyzed. Note that in contrast to myotubes, adult muscles express low levels of myogenic regulatory factors, including myogenin, because their expression is repressed by electrical activity generated by motor neurons (Eftimie et al., 1991). Myogenin showed an increase (p < 0.001) in expression immediately after injury, thereby confirming the induction of muscle regeneration. This was followed by a steady decrease (p < 0.001 and p < 0.05) in myogenin expression levels as the regeneration process advanced to completion 14 days after cardiotoxin injection (Figure 2, A and B). As a control, GAPDH expression was measured and showed a slight decrease in protein levels at days 2 and 4 postinjury, as previously described (Orengo et al., 2011). In addition, Ponceau staining was used to confirm relatively even loading. Next we measured Staufen1 protein expression and observed that Staufen1 gradually increased (p < 0.001) from 2 to 7 d after cardiotoxin treatment (Figure 2, A and C). Staufen1 expression then returned to control levels 14 d postinjury when muscle fibers are fully regenerated. Variations of Staufen1 levels within different time points reflect interindividual variability of protein expression, which is commonly observed when using animal tissues. This induction of Staufen1 after cardiotoxin injection follows a pattern similar to the one observed with CUGBP1, which is also involved in the regulation of myogenic differentiation (Orengo et al., 2011).

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