RNase L inhibitor pathway regulates both MyoD mRNA stability and muscle cell differentiation - PubMed (original) (raw)

The 2'-5' oligoadenylate/RNase L/RNase L inhibitor pathway regulates both MyoD mRNA stability and muscle cell differentiation

C Bisbal et al. Mol Cell Biol. 2000 Jul.

Abstract

The 2'-5' oligoadenylate (2-5A)/RNase L pathway is one of the enzymatic pathways induced by interferon. RNase L is a latent endoribonuclease which is activated by 2-5A and inhibited by a specific protein known as RLI (RNase L inhibitor). This system has an important role in regulating viral infection. Additionally, variations in RNase L activity have been observed during cell growth and differentiation but the significance of the 2-5A/RNase L/RLI pathway in these latter processes is not known. To determine the roles of RNase L and RLI in muscle differentiation, C2 mouse myoblasts were transfected with sense and antisense RLI cDNA constructs. Importantly, the overexpression of RLI in C2 cells was associated with diminished RNase L activity, an increased level of MyoD mRNA, and accelerated kinetics of muscle differentiation. Inversely, transfection of the RLI antisense construct was associated with increased RNase L activity, a diminished level of MyoD mRNA, and delayed differentiation. In agreement with these data, MyoD mRNA levels were also decreased in C2 cells transfected with an inducible RNase L construct. The effect of RNase L activity on MyoD mRNA levels was relatively specific because expression of several other mRNAs was not altered in C2 transfectants. Therefore, RNase L is directly involved in myoblast differentiation, probably through its role in regulating MyoD stability. This is the first identification of a potential mRNA target for RNase L.

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Figures

FIG. 1

Increases in the RLI and RNase L proteins during the differentiation of C2 myoblasts into myotubes. (A) C2 cells were grown in GM and then shifted to DM on day 1. At the indicated times, cells were harvested and lysed as described in Materials and Methods. Total protein samples (100 μg) were analyzed for RLI protein by Western blotting with a polyclonal RLI antiserum. (B) Densitometric analysis of the gel shown in panel A. A value of 100% corresponds to the amount of RLI protein in proliferating myoblasts at day 0. Error bars refer to the standard deviation obtained in three independent experiments. (C) C2 cells were grown in GM and then shifted to DM on day 1. At the indicated times, cells were harvested and lysed. Proteins (600 μg) were incubated with radiolabeled 2-5A4-3′-[32P]pCp (2-5ApCp) in a radiobinding assay; 100% corresponds to the amount of 2-5ApCp bound to RNase L in proliferating myoblasts. Error bars refer to the standard deviation obtained in three independent experiments.

FIG. 2

Increases in RLI and RNase L proteins during the differentiation of transfected C2 cells. (A) Pooled C2 cells stably transfected with an RLI cDNA sense construct (VS) and an RLI cDNA antisense construction (VAS) were grown in GM and then shifted to DM on day 1. At the indicated times, cells were lysed, and RLI expression was assessed in protein samples (100 μg) using an RLI-specific antiserum. (B) Densitometric analysis of the gel shown in panel A. A value of 100% corresponds to the amount of RLI protein at day 0 in proliferating C2 control myoblasts. Error bars refer to the standard deviation obtained in three independent experiments. Symbols: ◊, VS cells; ■, VAS cells. (C) The cells described in panel A were lysed, and proteins (600 μg) were incubated with radiolabeled 2-5A4-3′-[32P]pCp (2-5ApCp) in a radiobinding assay; 100% corresponds to the amount of 2-5ApCp bound to RNase L in proliferating C2 control myoblasts. Error bars refer to the standard deviation obtained in three independent experiments. Symbols: ◊, VS cells; ■, VAS cells.

FIG. 3

RLI mRNA levels increase during the differentiation of transfected C2 cells. Pooled stable control C2 cells transfected by an empty pcDNA3 vector (VV) (A), pooled stable C2 cells transfected with an RLI cDNA antisense construction (VAS) (B), and pooled stable C2 cells transfected with an RLI cDNA sense construction (VS) (C) were grown in GM and then shifted to DM on day 1. At the indicated times, cells were harvested, and mRNAs were extracted and analyzed by Northern blot using the murine RLI [32P]cDNA probe (VV, VAS, and VS [□]). The VS samples were also probed with a human RLI [32P]cDNA fragment (■) as indicated. Densitometric analysis of the gels are presented. A value of 100% corresponds to the amount of RLI mRNA in proliferating C2 control myoblasts at day 0. Error bars refer to standard deviation obtained in three independent experiments.

FIG. 4

Variations in RNase L activity following transfection with RLI sense and antisense constructs. Pooled stable control C2 cells transfected with an empty pcDNA3 vector (VV, panels A and B), the RLI antisense cDNA (VAS, panels C and D) and the RLI sense cDNA (VS, panels E and F) were seeded in GM and then shifted to DM (day 1). Cells were harvested at the indicated times. Proteins (100 μg) were incubated for 60 min at 30°C in the absence (A, C, and E) or presence (B, D, and F) of 2-5A4 (1 μM, final concentration). rRNAs were analyzed on 1.8% (wt/vol) agarose gels. Intact 28S and 18S rRNA migration is indicated at the left of each gel. Major rRNA degradation products are indicated by arrows.

FIG. 5

Kinetics of protein expression in differentiation C2 cells. Pooled stable control C2 cells transfected with an empty pcDNA3 vector (VV), the RLI sense cDNA (VS), and the RLI antisense cDNA (VAS) were seeded in GM and then shifted to DM (day 1). Cells were harvested at the indicated times. Proteins (100 μg) were analyzed by Western blotting with the different antisera as indicated.

FIG. 6

Decreased expression of MyoD in VAS cells and overexpression of MyoD in VS cells. Each polyclonal population of transfected C2 (VV, VAS, and VS) myoblasts grown in GM and myotubes differentiated in DM were fixed as described in Materials and Methods. Expression of MyoD was analyzed using the 5.8A mouse monoclonal antibody against MyoD. DNA was stained with Hoechst stain (0.1 mg/ml).

FIG. 7

MyoD mRNA stability is altered by the expression of RLI. Each polyclonal population of transfected C2 (VV, VAS, and VS) myoblasts grown in GM and myotubes grown in DM were treated with actinomycin D (5 μg/ml). At the indicated times, cells were harvested and the mRNAs were extracted and analyzed by Northern blotting with the different [32P]cDNA probes. mRNAs were visualized on a PhosphorImager 445-SI (Molecular Dynamics).

FIG. 8

The half-life of the MyoD mRNA is altered by the expression of RLI. Each polyclonal population of transfected C2 (VV, VAS, and VS) myoblasts (GM) and myotubes (DM) were treated with actinomycin D (5 μg/ml), and the mRNAs were analyzed as described in Fig. 7. After visualization on a PhosphorImager 445-SI (Molecular Dynamics), MyoD mRNAs were quantified by image analysis with the ImageQuant program (Molecular Dynamics). Each lane was normalized with the S26 probe. Densitometric analysis of the gel is presented. A value of 100% corresponds to the amount of MyoD mRNA at time zero in each polyclonal population of transfected C2 (VV, VAS, and VS) myoblasts (GM) (A) (□, control VV cells; ○, RLI sense VS cells) or myotubes (DM) (B) (■, control VV cells; ●, RLI sense VS cells; ⧫, RLI antisense cells). Error bars refer to the standard deviation obtained in three independent experiments.

FIG. 9

Nuclear run-on transcription analysis in RLI-transfected C2 cells. (A) Each polyclonal population of transfected C2 (VV, VAS, and VS) myoblasts grown in GM and myotubes grown in DM was harvested, and the nuclei were extracted as described in Materials and Methods. In vitro-elongated RNAs were hybridized to a blot of DNAs corresponding to MyoD, Myf5, myogenin, α-actin, and GAPDH. (B) After visualization on a PhosphorImager 445-SI (Molecular Dynamics), the mRNAs were quantified by image analysis with the ImageQuant program (Molecular Dynamics). A densitometric analysis of the autoradiography shown in panel A is presented. Error bars refer to the standard deviation obtained in three independent experiments.

FIG. 10

MyoD mRNA levels are decreased in a C2 clone overexpressing RNase L. (A) The RNase L3 clone was treated with increasing concentrations of IPTG during 8 h. Cells were then harvested and analyzed for RNase L 2-5A binding activity with the 2-5A radiocovalent binding assay. A densitometric analysis of the gel is shown. A value of 100% corresponds to the amount of 2-5A binding activity in proliferating RNase L3 clone myoblasts in the absence of IPTG. Error bars refer to the standard deviation obtained in three independent experiments. (B) RNase L rRNAs cleavage activity was assessed in the absence or presence of 2-5A (1 μM) as described in Fig. 4. (C) mRNAs were analyzed by Northern blotting using the indicated [32P]cDNA probes. Bands were visualized on a PhosphorImager 445-SI (Molecular Dynamics). A densitometric analysis of the gels is shown (◊, actin mRNA; □, Myf5 mRNA; ▵, S26 mRNA; ○, MyoD mRNA). A value of 100% corresponds to the amount of each mRNA in proliferating RNase L3 clone myoblasts in the absence of IPTG. Error bars refer to the standard deviation obtained in three independent experiments.

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The 2'-5' oligoadenylate/RNase L/RNase L inhibitor pathway regulates both MyoD mRNA stability and muscle cell differentiation - PubMed (original) (raw)