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A novel myogenic function residing in the 5' non-coding region of Insulin receptor substrate-1 (Irs-1) transcript.

Nagano H, Yamagishi N, Tomida C, Yano C, Aibara K, Kohno S, Abe T, Ohno A, Hirasaka K, Okumura Y, Mills EM, Nikawa T, Teshima-Kondo S - BMC Cell Biol. (2015)

Bottom Line: The overexpression of the 5'UTR markedly reduced Rb mRNA expression, and this reduction was fully dependent on the complementary element and was not compensated by IRS-1 protein.Conversely, knockdown of FL-Irs-1 mRNA increased Rb mRNA expression and enhanced myoblast differentiation into myotubes.Our findings suggest that the FL-Irs-1 transcript regulates myogenic differentiation as a regulatory RNA in myoblasts.

View Article: PubMed Central - PubMed

Affiliation: Department of Nutritional Physiology, Institute of Health Biosciences, Tokushima University Graduate School, Tokushima, 770-8503, Japan. h_nagano1231@yahoo.co.jp.

ABSTRACT

Background: There is evidence that several messenger RNAs (mRNAs) are bifunctional RNAs, i.e. RNA transcript carrying both protein-coding capacity and activity as functional non-coding RNA via 5' and 3' untranslated regions (UTRs).

Results: In this study, we identified a novel bifunctional RNA that is transcribed from insulin receptor substrate-1 (Irs-1) gene with full-length 5'UTR sequence (FL-Irs-1 mRNA). FL-Irs-1 mRNA was highly expressed only in skeletal muscle tissue. In cultured skeletal muscle C2C12 cells, the FL-Irs-1 transcript functioned as a bifunctional mRNA. The FL-Irs-1 transcript produced IRS-1 protein during differentiation of myoblasts into myotubes; however, this transcript functioned as a regulatory RNA in proliferating myoblasts. The FL-Irs-1 5'UTR contains a partial complementary sequence to Rb mRNA, which is a critical factor for myogenic differentiation. The overexpression of the 5'UTR markedly reduced Rb mRNA expression, and this reduction was fully dependent on the complementary element and was not compensated by IRS-1 protein. Conversely, knockdown of FL-Irs-1 mRNA increased Rb mRNA expression and enhanced myoblast differentiation into myotubes.

Conclusions: Our findings suggest that the FL-Irs-1 transcript regulates myogenic differentiation as a regulatory RNA in myoblasts.

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FL-Irs-1transcript regulates C2C12 differentiation. (A,B) Effect of knockdown of FL-Irs-1 mRNA on differentiation of C2C12 myoblast. Fusion index (A) and a mean diameter of myotubes (B) are shown. Mean ± SD, n = 50, *p < 0.01 versus no treatment (none). (C) Effect of knockdown of FL- Irs-1 mRNA on expression levels of myosin heavy chain in differentiating C2C12 cells (DM2, 4 and 6).
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Fig2: FL-Irs-1transcript regulates C2C12 differentiation. (A,B) Effect of knockdown of FL-Irs-1 mRNA on differentiation of C2C12 myoblast. Fusion index (A) and a mean diameter of myotubes (B) are shown. Mean ± SD, n = 50, *p < 0.01 versus no treatment (none). (C) Effect of knockdown of FL- Irs-1 mRNA on expression levels of myosin heavy chain in differentiating C2C12 cells (DM2, 4 and 6).

Mentions: In myoblasts, the FL-Irs-1 transcript was highly expressed and was not involved in IRS-1 protein production. Therefore, we hypothesized that FL-Irs-1 mRNA functions as an ncRNA with activity independent of its protein-coding function. To address this hypothesis, we examined the effect of knockdown of FL-Irs-1 transcript on the C2C12 cell phenotype. The knockdown of FL- Irs-1 mRNA markedly enhanced differentiation into myotubes (Figure 2). The results of fusion index show that the number of differentiated myotubes was increased more than 3-fold by knockdown of FL- Irs-1 mRNA compared with untransfected and sh-control-transfected cells (Figure 2A). The mean diameter of myotubes was also increased by approximately 130% after knockdown of FL-Irs-1 mRNA (Figure 2B). The expression of myosin heavy chain, which is a myogenic differentiation marker, was also enhanced by knockdown of FL-Irs-1 mRNA (Figure 2C). In contrast, knockdown of s5′-Irs-1 mRNA did not affect C2C12 cell differentiation (Additional file 2: Figure S2). These data indicate that accelerated differentiation by knockdown of FL-Irs-1 transcript was not caused by a reduction of IRS-1 protein, but instead, by a blockade of FL-Irs-1 transcript function.Figure 2


A novel myogenic function residing in the 5' non-coding region of Insulin receptor substrate-1 (Irs-1) transcript.

Nagano H, Yamagishi N, Tomida C, Yano C, Aibara K, Kohno S, Abe T, Ohno A, Hirasaka K, Okumura Y, Mills EM, Nikawa T, Teshima-Kondo S - BMC Cell Biol. (2015)

FL-Irs-1transcript regulates C2C12 differentiation. (A,B) Effect of knockdown of FL-Irs-1 mRNA on differentiation of C2C12 myoblast. Fusion index (A) and a mean diameter of myotubes (B) are shown. Mean ± SD, n = 50, *p < 0.01 versus no treatment (none). (C) Effect of knockdown of FL- Irs-1 mRNA on expression levels of myosin heavy chain in differentiating C2C12 cells (DM2, 4 and 6).
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Related In: Results  -  Collection

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Fig2: FL-Irs-1transcript regulates C2C12 differentiation. (A,B) Effect of knockdown of FL-Irs-1 mRNA on differentiation of C2C12 myoblast. Fusion index (A) and a mean diameter of myotubes (B) are shown. Mean ± SD, n = 50, *p < 0.01 versus no treatment (none). (C) Effect of knockdown of FL- Irs-1 mRNA on expression levels of myosin heavy chain in differentiating C2C12 cells (DM2, 4 and 6).
Mentions: In myoblasts, the FL-Irs-1 transcript was highly expressed and was not involved in IRS-1 protein production. Therefore, we hypothesized that FL-Irs-1 mRNA functions as an ncRNA with activity independent of its protein-coding function. To address this hypothesis, we examined the effect of knockdown of FL-Irs-1 transcript on the C2C12 cell phenotype. The knockdown of FL- Irs-1 mRNA markedly enhanced differentiation into myotubes (Figure 2). The results of fusion index show that the number of differentiated myotubes was increased more than 3-fold by knockdown of FL- Irs-1 mRNA compared with untransfected and sh-control-transfected cells (Figure 2A). The mean diameter of myotubes was also increased by approximately 130% after knockdown of FL-Irs-1 mRNA (Figure 2B). The expression of myosin heavy chain, which is a myogenic differentiation marker, was also enhanced by knockdown of FL-Irs-1 mRNA (Figure 2C). In contrast, knockdown of s5′-Irs-1 mRNA did not affect C2C12 cell differentiation (Additional file 2: Figure S2). These data indicate that accelerated differentiation by knockdown of FL-Irs-1 transcript was not caused by a reduction of IRS-1 protein, but instead, by a blockade of FL-Irs-1 transcript function.Figure 2

Bottom Line: The overexpression of the 5'UTR markedly reduced Rb mRNA expression, and this reduction was fully dependent on the complementary element and was not compensated by IRS-1 protein.Conversely, knockdown of FL-Irs-1 mRNA increased Rb mRNA expression and enhanced myoblast differentiation into myotubes.Our findings suggest that the FL-Irs-1 transcript regulates myogenic differentiation as a regulatory RNA in myoblasts.

View Article: PubMed Central - PubMed

Affiliation: Department of Nutritional Physiology, Institute of Health Biosciences, Tokushima University Graduate School, Tokushima, 770-8503, Japan. h_nagano1231@yahoo.co.jp.

ABSTRACT

Background: There is evidence that several messenger RNAs (mRNAs) are bifunctional RNAs, i.e. RNA transcript carrying both protein-coding capacity and activity as functional non-coding RNA via 5' and 3' untranslated regions (UTRs).

Results: In this study, we identified a novel bifunctional RNA that is transcribed from insulin receptor substrate-1 (Irs-1) gene with full-length 5'UTR sequence (FL-Irs-1 mRNA). FL-Irs-1 mRNA was highly expressed only in skeletal muscle tissue. In cultured skeletal muscle C2C12 cells, the FL-Irs-1 transcript functioned as a bifunctional mRNA. The FL-Irs-1 transcript produced IRS-1 protein during differentiation of myoblasts into myotubes; however, this transcript functioned as a regulatory RNA in proliferating myoblasts. The FL-Irs-1 5'UTR contains a partial complementary sequence to Rb mRNA, which is a critical factor for myogenic differentiation. The overexpression of the 5'UTR markedly reduced Rb mRNA expression, and this reduction was fully dependent on the complementary element and was not compensated by IRS-1 protein. Conversely, knockdown of FL-Irs-1 mRNA increased Rb mRNA expression and enhanced myoblast differentiation into myotubes.

Conclusions: Our findings suggest that the FL-Irs-1 transcript regulates myogenic differentiation as a regulatory RNA in myoblasts.

Show MeSH