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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 overexpression increases c-myc protein expression levels and cell proliferation. (A) Representative Western blot showing c-myc protein levels during myogenic differentiation of stable C2C12 cells. β-Actin was used to show equal loading. (B) Relative quantification of c-myc protein levels normalized to β-actin (n = 3). (C) Relative quantification of c-myc mRNA level in stable cell lines as determined by qRT-PCR. Levels were normalized to cyclophylin-B (n = 3). (D) C2C12 cells were transfected with sh-Staufen1 or control vectors. Western blots showing decreased Staufen1 and c-myc protein levels. β-Actin was used to show equal loading. (E) HSSM primary cells were transduced with sh-Staufen1 or control lentiviruses. Western blots were performed as in D. The arrow shows the specific c-myc band, as determined in separate experiments by knocking down c-myc expression (unpublished data). (F) 293T cells were cotransfected with luciferase vectors containing the c-myc 5′UTR and with Staufen1 or control plasmids. After transfection, the relative activity of luciferase was determined along with luciferase mRNA levels by qRT-PCR. Data are also normalized to luciferase levels in absence of Staufen1 and to an empty vector (n = 3). (G) Polysome profiling of proliferating stable C2C12 cells. Top, polysome profile obtained by continuous reading of absorbance at 254 nm. Bottom, levels of c-myc mRNAs were measured by qRT-PCR from 10 1-ml sucrose gradient fractions. (H) Cell proliferation assays performed with stable C2C12 cell lines (n = 3). Asterisks indicate significance (*p ≤ 0.05, **p ≤ 0.01).
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Figure 8: Staufen1 overexpression increases c-myc protein expression levels and cell proliferation. (A) Representative Western blot showing c-myc protein levels during myogenic differentiation of stable C2C12 cells. β-Actin was used to show equal loading. (B) Relative quantification of c-myc protein levels normalized to β-actin (n = 3). (C) Relative quantification of c-myc mRNA level in stable cell lines as determined by qRT-PCR. Levels were normalized to cyclophylin-B (n = 3). (D) C2C12 cells were transfected with sh-Staufen1 or control vectors. Western blots showing decreased Staufen1 and c-myc protein levels. β-Actin was used to show equal loading. (E) HSSM primary cells were transduced with sh-Staufen1 or control lentiviruses. Western blots were performed as in D. The arrow shows the specific c-myc band, as determined in separate experiments by knocking down c-myc expression (unpublished data). (F) 293T cells were cotransfected with luciferase vectors containing the c-myc 5′UTR and with Staufen1 or control plasmids. After transfection, the relative activity of luciferase was determined along with luciferase mRNA levels by qRT-PCR. Data are also normalized to luciferase levels in absence of Staufen1 and to an empty vector (n = 3). (G) Polysome profiling of proliferating stable C2C12 cells. Top, polysome profile obtained by continuous reading of absorbance at 254 nm. Bottom, levels of c-myc mRNAs were measured by qRT-PCR from 10 1-ml sucrose gradient fractions. (H) Cell proliferation assays performed with stable C2C12 cell lines (n = 3). Asterisks indicate significance (*p ≤ 0.05, **p ≤ 0.01).

Mentions: In parallel to the characterization of myogenic regulatory factors involved in muscle differentiation, we also performed a targeted approach to identify mRNAs regulated by Staufen1 and responsible for the impaired myogenesis. Although we examined the potential role of several candidates, including p21, RalGDS, cdk5, p35, junD, and c-jun, we were particularly interested in the transcription factor c-myc because it plays a role in the balance between proliferation/differentiation and is a known potent myogenic inhibitor (Miner and Wold, 1991). Thus we analyzed the levels of c-myc protein and mRNA by Western blot and qRT-PCR, respectively, in control versus Staufen1-HA–stable cell lines. In these experiments, we observed a significant increase (p < 0.05) in c-myc protein levels in Staufen1-overexpressing cells without a parallel increase in mRNA expression (Figure 8, A–C). In addition, we next determined whether knocking down Staufen1 expression had a reverse effect on c-myc protein levels in muscle cells. Thus C2C12 cells were transiently transfected with a short hairpin RNA (shRNA) targeting Staufen1 or a control. Western blot analyses showed a slight knockdown of Staufen1 in these experiments, together with a modest decrease in c-myc protein expression (Figure 8D). To circumvent the limitations linked to the relatively low transfection efficiency of C2C12 cells and improve the extent of the Staufen1 knockdown, we also transduced human primary HSMM cells with lentiviruses expressing one of two different shRNAs targeting Staufen1 or a control lentivirus. Western blots showed that both shRNAs induced a marked reduction in Staufen1 expression. As expected on the basis of our overexpression studies, down-regulation of Staufen1 was accompanied by a decrease in c-myc protein levels (Figure 8E). Taken together, data obtained with these varied experimental systems show that Staufen1 clearly regulates c-myc protein levels in skeletal muscle cells. Finally, to complement this work, we also assessed the levels of c-myc during muscle regeneration. After an initial decrease in c-myc levels at 2 d after cardiotoxin injection, the level of c-myc increased and essentially mirrored that of Staufen1 during early to mid phases of muscle regeneration, as expected based on our work with cells in culture (Figure 2, A and D).


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 overexpression increases c-myc protein expression levels and cell proliferation. (A) Representative Western blot showing c-myc protein levels during myogenic differentiation of stable C2C12 cells. β-Actin was used to show equal loading. (B) Relative quantification of c-myc protein levels normalized to β-actin (n = 3). (C) Relative quantification of c-myc mRNA level in stable cell lines as determined by qRT-PCR. Levels were normalized to cyclophylin-B (n = 3). (D) C2C12 cells were transfected with sh-Staufen1 or control vectors. Western blots showing decreased Staufen1 and c-myc protein levels. β-Actin was used to show equal loading. (E) HSSM primary cells were transduced with sh-Staufen1 or control lentiviruses. Western blots were performed as in D. The arrow shows the specific c-myc band, as determined in separate experiments by knocking down c-myc expression (unpublished data). (F) 293T cells were cotransfected with luciferase vectors containing the c-myc 5′UTR and with Staufen1 or control plasmids. After transfection, the relative activity of luciferase was determined along with luciferase mRNA levels by qRT-PCR. Data are also normalized to luciferase levels in absence of Staufen1 and to an empty vector (n = 3). (G) Polysome profiling of proliferating stable C2C12 cells. Top, polysome profile obtained by continuous reading of absorbance at 254 nm. Bottom, levels of c-myc mRNAs were measured by qRT-PCR from 10 1-ml sucrose gradient fractions. (H) Cell proliferation assays performed with stable C2C12 cell lines (n = 3). Asterisks indicate significance (*p ≤ 0.05, **p ≤ 0.01).
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Figure 8: Staufen1 overexpression increases c-myc protein expression levels and cell proliferation. (A) Representative Western blot showing c-myc protein levels during myogenic differentiation of stable C2C12 cells. β-Actin was used to show equal loading. (B) Relative quantification of c-myc protein levels normalized to β-actin (n = 3). (C) Relative quantification of c-myc mRNA level in stable cell lines as determined by qRT-PCR. Levels were normalized to cyclophylin-B (n = 3). (D) C2C12 cells were transfected with sh-Staufen1 or control vectors. Western blots showing decreased Staufen1 and c-myc protein levels. β-Actin was used to show equal loading. (E) HSSM primary cells were transduced with sh-Staufen1 or control lentiviruses. Western blots were performed as in D. The arrow shows the specific c-myc band, as determined in separate experiments by knocking down c-myc expression (unpublished data). (F) 293T cells were cotransfected with luciferase vectors containing the c-myc 5′UTR and with Staufen1 or control plasmids. After transfection, the relative activity of luciferase was determined along with luciferase mRNA levels by qRT-PCR. Data are also normalized to luciferase levels in absence of Staufen1 and to an empty vector (n = 3). (G) Polysome profiling of proliferating stable C2C12 cells. Top, polysome profile obtained by continuous reading of absorbance at 254 nm. Bottom, levels of c-myc mRNAs were measured by qRT-PCR from 10 1-ml sucrose gradient fractions. (H) Cell proliferation assays performed with stable C2C12 cell lines (n = 3). Asterisks indicate significance (*p ≤ 0.05, **p ≤ 0.01).
Mentions: In parallel to the characterization of myogenic regulatory factors involved in muscle differentiation, we also performed a targeted approach to identify mRNAs regulated by Staufen1 and responsible for the impaired myogenesis. Although we examined the potential role of several candidates, including p21, RalGDS, cdk5, p35, junD, and c-jun, we were particularly interested in the transcription factor c-myc because it plays a role in the balance between proliferation/differentiation and is a known potent myogenic inhibitor (Miner and Wold, 1991). Thus we analyzed the levels of c-myc protein and mRNA by Western blot and qRT-PCR, respectively, in control versus Staufen1-HA–stable cell lines. In these experiments, we observed a significant increase (p < 0.05) in c-myc protein levels in Staufen1-overexpressing cells without a parallel increase in mRNA expression (Figure 8, A–C). In addition, we next determined whether knocking down Staufen1 expression had a reverse effect on c-myc protein levels in muscle cells. Thus C2C12 cells were transiently transfected with a short hairpin RNA (shRNA) targeting Staufen1 or a control. Western blot analyses showed a slight knockdown of Staufen1 in these experiments, together with a modest decrease in c-myc protein expression (Figure 8D). To circumvent the limitations linked to the relatively low transfection efficiency of C2C12 cells and improve the extent of the Staufen1 knockdown, we also transduced human primary HSMM cells with lentiviruses expressing one of two different shRNAs targeting Staufen1 or a control lentivirus. Western blots showed that both shRNAs induced a marked reduction in Staufen1 expression. As expected on the basis of our overexpression studies, down-regulation of Staufen1 was accompanied by a decrease in c-myc protein levels (Figure 8E). Taken together, data obtained with these varied experimental systems show that Staufen1 clearly regulates c-myc protein levels in skeletal muscle cells. Finally, to complement this work, we also assessed the levels of c-myc during muscle regeneration. After an initial decrease in c-myc levels at 2 d after cardiotoxin injection, the level of c-myc increased and essentially mirrored that of Staufen1 during early to mid phases of muscle regeneration, as expected based on our work with cells in culture (Figure 2, A and D).

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