Fibroblast growth factor 1 induced during myogenesis by a transcription–translation coupling mechanism

Fibroblast growth factor 1 (FGF1) is involved in muscle development and regeneration. The FGF1 gene contains four tissue-specific promoters allowing synthesis of four transcripts with distinct leader regions. Two of these transcripts contain internal ribosome entry sites (IRESs), which are RNA elements allowing mRNA translation to occur in conditions of blockade of the classical cap-dependent mechanism. Here, we investigated the function and the regulation of FGF1 during muscle differentiation and regeneration. Our data show that FGF1 protein expression is induced in differentiating myoblasts and regenerating mouse muscle, whereas siRNA knock-down demonstrated FGF1 requirement for myoblast differentiation. FGF1 induction occurred at both transcriptional and translational levels, involving specific activation of both promoter A and IRES A, whereas global cap-dependent translation was inhibited. Furthermore, we identified, in the FGF1 promoter A distal region, a cis-acting element able to activate the IRES A-driven translation. These data revealed a mechanism of molecular coupling of mRNA transcription and translation, involving a unique process of IRES activation by a promoter element. The crucial role of FGF1 in myoblast differentiation provides physiological relevance to this novel mechanism. This finding also provides a new insight into the molecular mechanisms linking different levels of gene expression regulation

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

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

Influence of transcription level on the activity of the FGF1 IRES A.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136466#pone.0136466.g006" target="_blank">Fig 6</a>. (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).</p

Effect of hnRNPM and/or p54^nrb knockdown on the regulation of FGF1 IRES A activity during myoblast differentiation.

<p>C2C12 cells were first transfected with bicistronic plasmids and 48h later with siRNAs targeting hnRNPM (siM), p54^nrb (sip54) or control (sic), (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136466#pone.0136466.g003" target="_blank">Fig 3</a>). Luciferase activities were measured as described in Mat. & Meth two days after siRNA transfection, from C2C12 myoblasts maintained in proliferation (grey histogram) or serum-starved to induce differentiation (day 2, black histogram). The bicistronic cassette contains LucR and LucF reporter genes, separated by different IRESs (FGF1, FGF2 or EMCV). LucR and LucF activities reflect the cap-dependent and IRES dependent translation, respectively [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136466#pone.0136466.ref004" target="_blank">4</a>]. The knockdown efficiency was checked by Western blot (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136466#pone.0136466.s002" target="_blank">S2 File</a>). (A, B) FGF1 IRES activities were measured in C2C12 myoblasts in proliferation (grey histogram) and differentiation two days after serum-starvation treatment (black histogram). The bicistronic constructs are schematized. The bicistronic cassette is under the control of either the CMV promoter (A) or the FGF1 promoter A (B). Single and double knockdowns are shown in left and right panels, respectively. IRES activities are represented as LucF/LucR ratios. (C, D). Absence of effect of hnRNPM and/or p54^nrb knockdown on the regulation of EMCV and FGF2 IRESs during myoblast differentiation. Activities of EMCV (C) and FGF2 (D) IRESs were measured as above. For all panels, experiments were performed in biological triplicates and repeated at least three times. A representative experiment is shown (mean +- standard deviation).</p

Cooperative effect of p54^nrb and hnRNPM on FGF1 IRES activation.

<p>(A) Schema of the constructs used for C2C12 cell co-transfection. The target plasmid is the bicistronic dual luciferase vector with the FGF1 promoter and IRES. The effector plasmids express p54^nrb (p54) or hnRNPM (HM), or co-express p54^nrb and hnRNPM. The latter plasmid is a bicistronic contruct containing the FGF1 IRES. (B) Western blots of transfected proliferating C2C12 cell extracts using antibodies against p54(αHM), hnRNPM (αHM) of GAPDH as a control (αGAPDH). (C-E) Luciferase activities measurement of co-transfected proliferating or differentiating C2C12 cell extracts. LucR activity reflects the FGF1 mRNA promoter activity (as cap-dependent translation does not significantly vary, as shown by RNA transfection, see Table B in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136466#pone.0136466.s004" target="_blank">S4 File</a>) (C). LucF reflects IRES-dependent translation but is also dependent on mRNA amount (D). LucF/LucR ratio reflects the IRES activity normalized to mRNA amount, expressed relatively to the control (co-transfection with empty vector) (E). 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).</p