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Promoter-Dependent Translation Controlled by p54nrb and hnRNPM during Myoblast Differentiation.

Ainaoui N, Hantelys F, Renaud-Gabardos E, Bunel M, Lopez F, Pujol F, Planes R, Bahraoui E, Pichereaux C, Burlet-Schiltz O, Parini A, Garmy-Susini B, Prats AC - PLoS ONE (2015)

Bottom Line: Importantly, mRNA transfection and promoter deletion experiments clearly demonstrate the impact of the FGF1 promoter on the activation of IRES-dependent translation via p54nrb and hnRNPM.Accordingly, knockdown of either p54 or hnRNPM also blocks endogenous FGF1 induction and myotube formation, demonstrating the physiological relevance of this mechanism and the role of these two proteins in myogenesis.Our study demonstrates the cooperative function of hnRNPM and p54nrb as regulators of IRES-dependent translation and indicates the involvement of a promoter-dependent mechanism.

View Article: PubMed Central - PubMed

Affiliation: TRADGENE, UPS (EA4554), Toulouse, France.

ABSTRACT
Fibroblast growth factor 1 (FGF1) is induced during myoblast differentiation at both transcriptional and translational levels. Here, we identify hnRNPM and p54nrb/NONO present in protein complexes bound to the FGF1 promoter and to the mRNA internal ribosome entry site (IRES). Knockdown or overexpression of these proteins indicate that they cooperate in activating IRES-dependent translation during myoblast differentiation, in a promoter-dependent manner. Importantly, mRNA transfection and promoter deletion experiments clearly demonstrate the impact of the FGF1 promoter on the activation of IRES-dependent translation via p54nrb and hnRNPM. Accordingly, knockdown of either p54 or hnRNPM also blocks endogenous FGF1 induction and myotube formation, demonstrating the physiological relevance of this mechanism and the role of these two proteins in myogenesis. Our study demonstrates the cooperative function of hnRNPM and p54nrb as regulators of IRES-dependent translation and indicates the involvement of a promoter-dependent mechanism.

No MeSH data available.


Influence of transcription level on the activity of the FGF1 IRES A.C2C12 cells were transfected with bicistronic plasmids containing either the complete promoter 1A or a deleted promoter lacking nucleotides 1 to 391. Transfected cells were treated 24h later with siRNA siM, sip54 or sic, and luciferase activities were measured as in Fig 6. (A) mRNA expression in the presence of promoter 1A and 1AΔ1–391 reflected by the LucR activities. (B) Activity of FGF1 IRES A in the presence of promoter 1A and 1AΔ1–391, reflected by the Luc F/ LucR ratio as above. Experiments were performed in biological triplicates and repeated three times. The statistical test used is the Student test. (mean +- standard deviation, *p<0.05, **p<0.01, ***<0.001).
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pone.0136466.g007: Influence of transcription level on the activity of the FGF1 IRES A.C2C12 cells were transfected with bicistronic plasmids containing either the complete promoter 1A or a deleted promoter lacking nucleotides 1 to 391. Transfected cells were treated 24h later with siRNA siM, sip54 or sic, and luciferase activities were measured as in Fig 6. (A) mRNA expression in the presence of promoter 1A and 1AΔ1–391 reflected by the LucR activities. (B) Activity of FGF1 IRES A in the presence of promoter 1A and 1AΔ1–391, reflected by the Luc F/ LucR ratio as above. Experiments were performed in biological triplicates and repeated three times. The statistical test used is the Student test. (mean +- standard deviation, *p<0.05, **p<0.01, ***<0.001).

Mentions: To go further with this hypothesis, we transfected C2C12 with a construct containing a deleted form of promoter F1A, P1AΔ1–391 (Fig 7). Measurements of LucR activity showed that this promoter is about ten times less efficient than the wild type promoter, whereas it was still sensitive to hnRNPM and p54 knockdown (Fig 7A). In contrast, in the presence of the deleted form of the promoter, IRES activity was no longer induced and was very mildly altered by the knockdown of hnRNPM or p54nrb (Fig 7B).


Promoter-Dependent Translation Controlled by p54nrb and hnRNPM during Myoblast Differentiation.

Ainaoui N, Hantelys F, Renaud-Gabardos E, Bunel M, Lopez F, Pujol F, Planes R, Bahraoui E, Pichereaux C, Burlet-Schiltz O, Parini A, Garmy-Susini B, Prats AC - PLoS ONE (2015)

Influence of transcription level on the activity of the FGF1 IRES A.C2C12 cells were transfected with bicistronic plasmids containing either the complete promoter 1A or a deleted promoter lacking nucleotides 1 to 391. Transfected cells were treated 24h later with siRNA siM, sip54 or sic, and luciferase activities were measured as in Fig 6. (A) mRNA expression in the presence of promoter 1A and 1AΔ1–391 reflected by the LucR activities. (B) Activity of FGF1 IRES A in the presence of promoter 1A and 1AΔ1–391, reflected by the Luc F/ LucR ratio as above. Experiments were performed in biological triplicates and repeated three times. The statistical test used is the Student test. (mean +- standard deviation, *p<0.05, **p<0.01, ***<0.001).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4558007&req=5

pone.0136466.g007: Influence of transcription level on the activity of the FGF1 IRES A.C2C12 cells were transfected with bicistronic plasmids containing either the complete promoter 1A or a deleted promoter lacking nucleotides 1 to 391. Transfected cells were treated 24h later with siRNA siM, sip54 or sic, and luciferase activities were measured as in Fig 6. (A) mRNA expression in the presence of promoter 1A and 1AΔ1–391 reflected by the LucR activities. (B) Activity of FGF1 IRES A in the presence of promoter 1A and 1AΔ1–391, reflected by the Luc F/ LucR ratio as above. Experiments were performed in biological triplicates and repeated three times. The statistical test used is the Student test. (mean +- standard deviation, *p<0.05, **p<0.01, ***<0.001).
Mentions: To go further with this hypothesis, we transfected C2C12 with a construct containing a deleted form of promoter F1A, P1AΔ1–391 (Fig 7). Measurements of LucR activity showed that this promoter is about ten times less efficient than the wild type promoter, whereas it was still sensitive to hnRNPM and p54 knockdown (Fig 7A). In contrast, in the presence of the deleted form of the promoter, IRES activity was no longer induced and was very mildly altered by the knockdown of hnRNPM or p54nrb (Fig 7B).

Bottom Line: Importantly, mRNA transfection and promoter deletion experiments clearly demonstrate the impact of the FGF1 promoter on the activation of IRES-dependent translation via p54nrb and hnRNPM.Accordingly, knockdown of either p54 or hnRNPM also blocks endogenous FGF1 induction and myotube formation, demonstrating the physiological relevance of this mechanism and the role of these two proteins in myogenesis.Our study demonstrates the cooperative function of hnRNPM and p54nrb as regulators of IRES-dependent translation and indicates the involvement of a promoter-dependent mechanism.

View Article: PubMed Central - PubMed

Affiliation: TRADGENE, UPS (EA4554), Toulouse, France.

ABSTRACT
Fibroblast growth factor 1 (FGF1) is induced during myoblast differentiation at both transcriptional and translational levels. Here, we identify hnRNPM and p54nrb/NONO present in protein complexes bound to the FGF1 promoter and to the mRNA internal ribosome entry site (IRES). Knockdown or overexpression of these proteins indicate that they cooperate in activating IRES-dependent translation during myoblast differentiation, in a promoter-dependent manner. Importantly, mRNA transfection and promoter deletion experiments clearly demonstrate the impact of the FGF1 promoter on the activation of IRES-dependent translation via p54nrb and hnRNPM. Accordingly, knockdown of either p54 or hnRNPM also blocks endogenous FGF1 induction and myotube formation, demonstrating the physiological relevance of this mechanism and the role of these two proteins in myogenesis. Our study demonstrates the cooperative function of hnRNPM and p54nrb as regulators of IRES-dependent translation and indicates the involvement of a promoter-dependent mechanism.

No MeSH data available.