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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 reduces Rb mRNA levels. (A) Expression levels of Rb mRNA in C2C12 myoblasts (GM) and myotubes (DM4). (B) Partial sequence complementarity between the 5′UTR of FL-Irs-1 transcript (nt 373 – nt 405) and Rb mRNA (nt 131 – nt 165). (C) Schematic representation of position of complementary element in the 5′UTR of FL-Irs-1 transcript. A position of an LNA-based antisense oligonucleotide (FL-5′-AS-ODN) is shown. (D) Effect of FL-Irs-1 5′UTR on Rb mRNA expression levels. C2C12 myoblasts were untransfected (−) or transfected with an Rb mRNA-expressing plasmid in combination with the indicated effector plasmid (pFL-5′UTR, pFL-5′UTR-del). Rb mRNA levels were measured by qRT-PCR. Mean ± SD, n = 4-5, *p < 0.01 versus control transfectant (pRB + pLuc). (E) Effect of FL-5′-AS-ODN on Rb mRNA reduction by FL-Irs-1 5′UTR. C2C12 myoblasts were co-transfected with plasmids expressing Rb mRNA and the FL-Irs-1 5′UTR RNA in combination with the indicated AS-ODN. Mean ± SD, n = 4-5, *p < 0.01 versus control transfectant (pRB + pLuc). (F) Effect of knockdown of endogenous FL-Irs-1 transcript on endogenous Rb mRNA levels. C2C12 myoblasts were transfected with the indicated shRNA or siRNA. Mean ± SD, n = 4, *p < 0.01 versus no treatment (none). (G) Effect of supplementary expression of IRS-1 protein on Rb mRNA levels under FL-Irs-1 transcript knockdown conditions. C2C12 myoblasts were transfected with the sh-FL-Irs-1 plasmid in combination with a plasmid encoding IRS-1 protein fused to V5 tag (pIRS-1-V5). Expression levels of exogenous IRS-1 protein were determined by immunoblot analysis using anti-V5 antibody. Mean ± SD, n = 3, *p < 0.01 versus control transfectant (sh-Control).
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Fig3: FL-Irs-1transcript reduces Rb mRNA levels. (A) Expression levels of Rb mRNA in C2C12 myoblasts (GM) and myotubes (DM4). (B) Partial sequence complementarity between the 5′UTR of FL-Irs-1 transcript (nt 373 – nt 405) and Rb mRNA (nt 131 – nt 165). (C) Schematic representation of position of complementary element in the 5′UTR of FL-Irs-1 transcript. A position of an LNA-based antisense oligonucleotide (FL-5′-AS-ODN) is shown. (D) Effect of FL-Irs-1 5′UTR on Rb mRNA expression levels. C2C12 myoblasts were untransfected (−) or transfected with an Rb mRNA-expressing plasmid in combination with the indicated effector plasmid (pFL-5′UTR, pFL-5′UTR-del). Rb mRNA levels were measured by qRT-PCR. Mean ± SD, n = 4-5, *p < 0.01 versus control transfectant (pRB + pLuc). (E) Effect of FL-5′-AS-ODN on Rb mRNA reduction by FL-Irs-1 5′UTR. C2C12 myoblasts were co-transfected with plasmids expressing Rb mRNA and the FL-Irs-1 5′UTR RNA in combination with the indicated AS-ODN. Mean ± SD, n = 4-5, *p < 0.01 versus control transfectant (pRB + pLuc). (F) Effect of knockdown of endogenous FL-Irs-1 transcript on endogenous Rb mRNA levels. C2C12 myoblasts were transfected with the indicated shRNA or siRNA. Mean ± SD, n = 4, *p < 0.01 versus no treatment (none). (G) Effect of supplementary expression of IRS-1 protein on Rb mRNA levels under FL-Irs-1 transcript knockdown conditions. C2C12 myoblasts were transfected with the sh-FL-Irs-1 plasmid in combination with a plasmid encoding IRS-1 protein fused to V5 tag (pIRS-1-V5). Expression levels of exogenous IRS-1 protein were determined by immunoblot analysis using anti-V5 antibody. Mean ± SD, n = 3, *p < 0.01 versus control transfectant (sh-Control).

Mentions: We examined the expression levels of Rb mRNA in myoblasts and myotubes. Myoblasts expressed low levels of Rb mRNA and myotubes expressed higher levels (Figure 3A). The Rb mRNA expression profile had an inverse relationship with FL-Irs-1 transcript levels (Figure 1C, C2C12 cells).Figure 3


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 reduces Rb mRNA levels. (A) Expression levels of Rb mRNA in C2C12 myoblasts (GM) and myotubes (DM4). (B) Partial sequence complementarity between the 5′UTR of FL-Irs-1 transcript (nt 373 – nt 405) and Rb mRNA (nt 131 – nt 165). (C) Schematic representation of position of complementary element in the 5′UTR of FL-Irs-1 transcript. A position of an LNA-based antisense oligonucleotide (FL-5′-AS-ODN) is shown. (D) Effect of FL-Irs-1 5′UTR on Rb mRNA expression levels. C2C12 myoblasts were untransfected (−) or transfected with an Rb mRNA-expressing plasmid in combination with the indicated effector plasmid (pFL-5′UTR, pFL-5′UTR-del). Rb mRNA levels were measured by qRT-PCR. Mean ± SD, n = 4-5, *p < 0.01 versus control transfectant (pRB + pLuc). (E) Effect of FL-5′-AS-ODN on Rb mRNA reduction by FL-Irs-1 5′UTR. C2C12 myoblasts were co-transfected with plasmids expressing Rb mRNA and the FL-Irs-1 5′UTR RNA in combination with the indicated AS-ODN. Mean ± SD, n = 4-5, *p < 0.01 versus control transfectant (pRB + pLuc). (F) Effect of knockdown of endogenous FL-Irs-1 transcript on endogenous Rb mRNA levels. C2C12 myoblasts were transfected with the indicated shRNA or siRNA. Mean ± SD, n = 4, *p < 0.01 versus no treatment (none). (G) Effect of supplementary expression of IRS-1 protein on Rb mRNA levels under FL-Irs-1 transcript knockdown conditions. C2C12 myoblasts were transfected with the sh-FL-Irs-1 plasmid in combination with a plasmid encoding IRS-1 protein fused to V5 tag (pIRS-1-V5). Expression levels of exogenous IRS-1 protein were determined by immunoblot analysis using anti-V5 antibody. Mean ± SD, n = 3, *p < 0.01 versus control transfectant (sh-Control).
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Fig3: FL-Irs-1transcript reduces Rb mRNA levels. (A) Expression levels of Rb mRNA in C2C12 myoblasts (GM) and myotubes (DM4). (B) Partial sequence complementarity between the 5′UTR of FL-Irs-1 transcript (nt 373 – nt 405) and Rb mRNA (nt 131 – nt 165). (C) Schematic representation of position of complementary element in the 5′UTR of FL-Irs-1 transcript. A position of an LNA-based antisense oligonucleotide (FL-5′-AS-ODN) is shown. (D) Effect of FL-Irs-1 5′UTR on Rb mRNA expression levels. C2C12 myoblasts were untransfected (−) or transfected with an Rb mRNA-expressing plasmid in combination with the indicated effector plasmid (pFL-5′UTR, pFL-5′UTR-del). Rb mRNA levels were measured by qRT-PCR. Mean ± SD, n = 4-5, *p < 0.01 versus control transfectant (pRB + pLuc). (E) Effect of FL-5′-AS-ODN on Rb mRNA reduction by FL-Irs-1 5′UTR. C2C12 myoblasts were co-transfected with plasmids expressing Rb mRNA and the FL-Irs-1 5′UTR RNA in combination with the indicated AS-ODN. Mean ± SD, n = 4-5, *p < 0.01 versus control transfectant (pRB + pLuc). (F) Effect of knockdown of endogenous FL-Irs-1 transcript on endogenous Rb mRNA levels. C2C12 myoblasts were transfected with the indicated shRNA or siRNA. Mean ± SD, n = 4, *p < 0.01 versus no treatment (none). (G) Effect of supplementary expression of IRS-1 protein on Rb mRNA levels under FL-Irs-1 transcript knockdown conditions. C2C12 myoblasts were transfected with the sh-FL-Irs-1 plasmid in combination with a plasmid encoding IRS-1 protein fused to V5 tag (pIRS-1-V5). Expression levels of exogenous IRS-1 protein were determined by immunoblot analysis using anti-V5 antibody. Mean ± SD, n = 3, *p < 0.01 versus control transfectant (sh-Control).
Mentions: We examined the expression levels of Rb mRNA in myoblasts and myotubes. Myoblasts expressed low levels of Rb mRNA and myotubes expressed higher levels (Figure 3A). The Rb mRNA expression profile had an inverse relationship with FL-Irs-1 transcript levels (Figure 1C, C2C12 cells).Figure 3

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