Limits...
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.


Comparison of IRES activities following DNA or RNA transfection.(A, C, E) C2C12 cells were transfected with bicistronic plasmids containing either the FGF1 IRES, or EMCV IRES or a hairpin (control without IRES) and with siRNA siM, sip54 or sic, and IRES activities measured as in Fig 4B, 4D and 4F C2C12 cells were transfected with siRNA siM, sip54 or sic and 24h later with bicistronic mRNAs containing either the FGF1 IRES, or the EMCV IRES, or a hairpin as above. mRNAs were transcribed in vitro, capped and polyadenylated, as described in Mat. & Meth. The LucF/LucR activity ratio was measured 12h post-transfection. Experiments were performed in biological triplicates and repeated three times. The Student test was used. A representative experiment is shown (mean +- standard deviation, *p<0.05, **p<0.01, ***<0.001). For A and B, the Luc R and Luc F values are shown in S4 File.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4558007&req=5

pone.0136466.g006: Comparison of IRES activities following DNA or RNA transfection.(A, C, E) C2C12 cells were transfected with bicistronic plasmids containing either the FGF1 IRES, or EMCV IRES or a hairpin (control without IRES) and with siRNA siM, sip54 or sic, and IRES activities measured as in Fig 4B, 4D and 4F C2C12 cells were transfected with siRNA siM, sip54 or sic and 24h later with bicistronic mRNAs containing either the FGF1 IRES, or the EMCV IRES, or a hairpin as above. mRNAs were transcribed in vitro, capped and polyadenylated, as described in Mat. & Meth. The LucF/LucR activity ratio was measured 12h post-transfection. Experiments were performed in biological triplicates and repeated three times. The Student test was used. A representative experiment is shown (mean +- standard deviation, *p<0.05, **p<0.01, ***<0.001). For A and B, the Luc R and Luc F values are shown in S4 File.

Mentions: To analyse the impact of the promoter on activation of IRES-dependent translation by hnRNPM and p54nrb, C2C12 cells were transfected with either bicistronic plasmid DNAs or in vitro transcribed capped and polyadenylated bicistronic mRNAs (Fig 6). DNA transfection resulted (as already shown in Fig 4) in a high FGF1 IRES activity, strongly induced by C2C12 differentiation, and downregulated by the knockdown of hnRNPM or p54nrb (Fig 6A and S4 File). In contrast, basal FGF1 IRES activity was 500 times lower following RNA transfection, and no induction by either hnRNPM or p54 was observed (Fig 6B and S4 File). These features were not observed with the EMCV IRES, whose activity was low after DNA or RNA transfection and was not affected by hnRNPM or p54 knockdown (Fig 6C and 6D). As a negative control, we used a construct containing a hairpin between the two cistrons instead of an IRES. The LucF/LucR ratios in the absence of an IRES were very low for both DNA and RNA transfections and were not affected by hnRNPM or p54 knockdown (Fig 6E and 6F). These results suggested that a nuclear step may be a prerequisite to IRES induction by the hnRNPM and p54nrb.


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)

Comparison of IRES activities following DNA or RNA transfection.(A, C, E) C2C12 cells were transfected with bicistronic plasmids containing either the FGF1 IRES, or EMCV IRES or a hairpin (control without IRES) and with siRNA siM, sip54 or sic, and IRES activities measured as in Fig 4B, 4D and 4F C2C12 cells were transfected with siRNA siM, sip54 or sic and 24h later with bicistronic mRNAs containing either the FGF1 IRES, or the EMCV IRES, or a hairpin as above. mRNAs were transcribed in vitro, capped and polyadenylated, as described in Mat. & Meth. The LucF/LucR activity ratio was measured 12h post-transfection. Experiments were performed in biological triplicates and repeated three times. The Student test was used. A representative experiment is shown (mean +- standard deviation, *p<0.05, **p<0.01, ***<0.001). For A and B, the Luc R and Luc F values are shown in S4 File.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0136466.g006: Comparison of IRES activities following DNA or RNA transfection.(A, C, E) C2C12 cells were transfected with bicistronic plasmids containing either the FGF1 IRES, or EMCV IRES or a hairpin (control without IRES) and with siRNA siM, sip54 or sic, and IRES activities measured as in Fig 4B, 4D and 4F C2C12 cells were transfected with siRNA siM, sip54 or sic and 24h later with bicistronic mRNAs containing either the FGF1 IRES, or the EMCV IRES, or a hairpin as above. mRNAs were transcribed in vitro, capped and polyadenylated, as described in Mat. & Meth. The LucF/LucR activity ratio was measured 12h post-transfection. Experiments were performed in biological triplicates and repeated three times. The Student test was used. A representative experiment is shown (mean +- standard deviation, *p<0.05, **p<0.01, ***<0.001). For A and B, the Luc R and Luc F values are shown in S4 File.
Mentions: To analyse the impact of the promoter on activation of IRES-dependent translation by hnRNPM and p54nrb, C2C12 cells were transfected with either bicistronic plasmid DNAs or in vitro transcribed capped and polyadenylated bicistronic mRNAs (Fig 6). DNA transfection resulted (as already shown in Fig 4) in a high FGF1 IRES activity, strongly induced by C2C12 differentiation, and downregulated by the knockdown of hnRNPM or p54nrb (Fig 6A and S4 File). In contrast, basal FGF1 IRES activity was 500 times lower following RNA transfection, and no induction by either hnRNPM or p54 was observed (Fig 6B and S4 File). These features were not observed with the EMCV IRES, whose activity was low after DNA or RNA transfection and was not affected by hnRNPM or p54 knockdown (Fig 6C and 6D). As a negative control, we used a construct containing a hairpin between the two cistrons instead of an IRES. The LucF/LucR ratios in the absence of an IRES were very low for both DNA and RNA transfections and were not affected by hnRNPM or p54 knockdown (Fig 6E and 6F). These results suggested that a nuclear step may be a prerequisite to IRES induction by the hnRNPM and p54nrb.

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.