The HMG (high-mobility group)-box-containing chromatin-remodelling factor SRY (sex-determining region on the Y chromosome) plays a key role in sex determination. Its role in the nucleus is critically dependent on two NLSs (nuclear localization signals) that flank its HMG domain: the C-terminally located ‘β-NLS’ that mediates nuclear transport through Impβ1 (importin β1) and the N-terminally located ‘CaM-NLS’ which is known to recognize the calcium-binding protein CaM (calmodulin). In the present study, we examined a number of missense mutations in the SRY CaM-NLS from human XY sex-reversed females for the first time, showing that they result in significantly reduced nuclear localization of GFP (green fluorescent protein)–SRY fusion proteins in transfected cells compared with wild-type. The CaM antagonist CDZ (calmidazolium chloride) was found to significantly reduce wild-type SRY nuclear accumulation, indicating dependence of SRY nuclear import on CaM. Intriguingly, the CaM-NLS mutants were all resistant to CDZ's effects, implying a loss of interaction with CaM, which was confirmed by direct binding experiments. CaM-binding/resultant nuclear accumulation was the only property of SRY found to be impaired by two of the CaM-NLS mutations, implying that inhibition of CaM-dependent nuclear import is the basis of sex reversal in these cases. Importantly, the CaM-NLS is conserved in other HMG-box-domain-containing proteins such as SOX-2, -9, -10 and HMGN1, all of which were found for the first time to rely on CaM for optimal nuclear localization. CaM-dependent nuclear translocation is thus a common mechanism for this family of important transcription factors.

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.

Background: Electrotransfer of plasmid DNA into skeletal muscle is a promising strategy for the delivery of therapeutic molecules targeting various muscular diseases, cancer and lower-limb ischemia. Internal Ribosome Entry Sites (IRESs) allow co-expression of proteins of interest from a single transcriptional unit. IRESs are RNA elements that have been found in viral RNAs as well as a variety of cellular mRNAs with long 5' untranslated regions. While the encephalomyocarditis virus (EMCV) IRES is often used in expression vectors, we have shown that the FGF-1 IRES is equally active to drive short term transgene expression in mouse muscle. To compare the ability of the FGF-1 IRES to drive long term expression against the EMCV and FGF-2 IRESs, we performed analyses of expression kinetics using bicistronic vectors that express the bioluminescent renilla and firefly luciferase reporter genes. Long term expression of bicistronic vectors was also compared to that of monocistronic vectors. Bioluminescence was quantified ex vivo using a luminometer and in vivo using a CCD camera that monitors luminescence within live animals.