Muscle Specific Fragile X Related Protein 1 Isoforms are Sequestered in the Nucleus of Undifferentiated Myoblast
© Dubé et al; licensee BioMed Central Ltd. 2000
Received: 14 November 2000
Accepted: 7 December 2000
Published: 7 December 2000
The family of Fragile X Mental Retardation Proteins is composed of three members: Fragile Mental Retardation 1, Fragile X Related 1 and X Related 2 proteins. These proteins are associated with mRNPs within translating ribosomes and have the capacity to shuttle between the nucleus and the cytoplasm. Great attention has been given to FMRP due to its implication in human hereditary mental retardation while FXR1P and FXR2P have only recently been studied.
Using antibodies directed against several epitopes of FXR1P, we have detected protein isoforms generated by small peptides pocket inserts. Four isoforms of MW 70, 74, 78, 80 kDa are widely distributed in mouse organs, while in striated muscles these isoforms are replaced by proteins of 82 and 84 kDa containing an extra pocket of 27 aa. Expression of these muscle isoforms is an early event during in vitro differentiation of myoblasts into myotubes and correlates with the activation of muscle-specific genes. However, while FXR1P82,84 are associated with cytoplasmic mRNPs in myotubes, they are sequestered in the nuclei of undifferentiated myoblasts. These observations suggest that, in addition to a cytoplasmic function yet to be defined, FXR1P82,84 may play a nuclear role in pre-mRNA metabolism.
The pattern of subcellular partitioning of FXR1P isoforms during myogenesis is unique among the family of the FXR proteins. The model system described here should be considered as a powerful tool for ongoing attempts to unravel structure-function relationships of the different FMR family members since the potential role(s) of FXR1P as a compensatory factor in Fragile X syndrome is still elusive.
The Fragile X Mental Retardation (FMR) protein family is composed of three highly homologous members. The Fragile X Mental Retardation Protein (FMRP) is coded by the X-linked FMR1 gene and its absence is directly associated with human hereditary mental retardation [reviewed in 1,2]. Two other members of this family are the Fragile X Related 1 (FXR1P) and Fragile X Related 2 (FXR2P) proteins [3,4,5] that are coded by the FXR1 and FXR2 genes located at 3q28 and 17p13.1, respectively, in human. These genes are highly conserved in vertebrate evolution and contain two KH domains and a RGG box that are functional characteristic motifs in RNA-binding proteins [4,5,6,7]. In addition, they also contain a nuclear localization signal (NLS) as well as a nuclear export signal (NES) making them putative nucleocytoplasmic shuttling proteins [reviewed in 1,2]. Finally, FMRP as well as the other members of the family have been shown to be associated with messenger RiboNucleoParticles (mRNP) within actively translating ribosomes. This association suggests that their roles might be linked to RNA transport and/or translation [8,9,10,11,12].
Whereas absence of FMRP is the cause of Fragile X Mental Retardation in human, it is not known whether FXR1P and FXR2P are associated to any pathology or phenotype. Also it is not known whether these homologous proteins can compensate for the absence of FMRP in the case of the Fragile X syndrome. In vitro studies showed that all three members interact with themselves and with each other [5, 13, 14]. However, their distribution in certain mouse and human tissues showed individual pattern of expression [15, 16] indicating that each protein also may function autonomously .
FXR1P has been shown to have a complex expression pattern in different mammalian cell lines since six distinct isoforms were observed and their respective levels were shown to be cell type specific . In particular, it was observed that 4 distinct FXR1P isoforms of MW 70 and 74 kDa (previously referred to as short) and 78 and 80 kDa (long) are widely expressed in diverse cell lines as well as in different organs in mouse. However, in muscle, these isoforms are replaced by novel super long isoforms of MW 82 and 84 kDa. The replacement of the short and long isoforms by the super long isoforms is clearly apparent during myogenesis of myoblastic cell lines that can differentiate in vitro into myotubes. This model system which mimics, although imperfectly, muscle differentiation has permitted us to show in the present report that transition of the short and long isoforms to the super long is an early event that takes place concomitantly to the expression of muscle-specific genes. In addition, we also show that low levels of the super long isoforms are constitutively expressed in undifferentiated myoblasts and that they are sequestered in the nuclei, while in differentiated myotubes P82,84 are transferred to the cytoplasm where they are incorporated in mRNPs present in actively translating ribosomes.
Complex expression of FXR1P isoforms
Accumulation of FXR1P82,84 isoforms during in vitro myogenesis
Based on the observations reported above, we tested whether accumulation of P82,84 was regulated at the level of its mRNA. Total RNA was extracted from undifferentiated and from stimulated C2C4 myoblasts at different times and subjected to Northern blot analyses. Membranes were sequentially probed with 32P-labeled exon 15 and various cDNA inserts. The results presented in Figure 6 (lower panels) representative of three independent time-course analyses, showed that muscle specific exon 15 (81 nucleotides) hybridized to the two FXR1 transcripts of 3.2 and 2.4 kb [10,18]. In agreement with the results obtained at the protein level, these mRNA variants are present at low levels in non-differentiated myoblasts. Densitometric analysis showed a clear and significant increase (3.0 ± 0.5) of both transcripts at 15 h after serum deprivation, then after their steady state levels continued to augment to reach a 5 fold increase by 24 h. The beginning of this increase coincided in time with that of myogenin and cardiac actin mRNAs, which began to accumulate at 12-15 h. In contrast, myf 5 mRNA levels decreased gradually as expected . These results clearly indicate that accumulation of the FXR1 mRNA variants containing the muscle-specific exon 15 tightly correlates with the activation of muscle-specific genes.
FXR1P82,84 are absent from polyribosomes in undifferentiated myoblasts
Nuclear localization of FXR1P82,84 in undifferentiated myoblasts
Fragile X Related 1 protein belongs to the FMR family of RNA binding proteins which are specifically associated with mRNPs engaged in actively translating ribosomes. All members of this family share the same protein structure and contain key domains that are conserved in RNA binding proteins [reviewed in 27] as well as NL and NE signals which allow the proteins to shuttle between the cytoplasm and the nucleus [8,28,29,30]. Indeed, accumulation of the FMR proteins in the nucleus has been observed experimentaly with truncated variants of the proteins [28,30] as well as after overexpression experiments followed by treatment with Leptomycin B, an antibiotic that inhibits the nuclear export of NES-containing proteins [31,32]. Due to extensive alternative splicing of the FMRP primary transcript [33,34], it is believe that at least 20 different FMRP isoforms might exist. However, using the available antibodies, only 6 FMRP variants have been observed [35,36,37]. Similarly to FMR1, FXR1 primary transcript undergoes complex alternative splicing, however yielding in theory a much more modest number of protein isoforms. Recent studies  showed that 7 mRNA variants are detected, and our analyses in mouse organs revealed that unlike FMRP, the different FXR1P isoforms are tissue-specific. In general, P70,74,78,80 are widely expressed, albeit at different levels, and their respective ratio seem to be cell and tissue specific. In heart and skeletal muscle, these short and long protein isoforms are absent and are replaced by the P82,84 super long isoforms that are generated by a small peptide insert of 27 aa .
While FMRP is abundant in neurons, it is absent in muscle [12,35]. On the other hand, FXR1P82,84 specific to muscle has not been detected in neurons or in brain extracts [12 and this study]. This tissue specificity is not maintained in culture of undifferentiated myoblasts since both FMRP and P82,84 are detected in these cells . In addition, P70,74,78,80 isoforms which are absent in muscle are clearly present in myoblasts. These observations suggest that the individual FMR members may be interchangeable in certain physiological conditions such as cell deadaptation in culture  or under cellular reprogramming during wound healing . Using the model system of myoblast induced to differentiate into myotubes, we provide evidence that expression of the non-muscle to muscle-specific isoforms of FXR1P correlates with cellular differentiation. We observed that the levels of P82,84 and that of muscle mRNA variants begin to increase at an early time after stimulation concomitant with the expression of different markers of myogenesis. Accumulation of these super long isoforms is paralleled by the decrease of the short and long isoforms and finally at day 3 when approximately 80% of the cells underwent myotube differentiation, P82,84 accounted for the great majority of FXR1P. These results strongly suggest that the mechanisms by which FXR1P82,84 expression are controlled, reside at the level of splicing of the primary transcript. Further analyses are required to determine the factors associated with the expression of these muscle mRNA variants.
In undifferentiated myoblasts, P82,84 make exception in the FMR protein family since they are not associated with polysomal mRNPs and are sequestered in the nucleus. One possible explanation for this localization would have been that these isoforms contain, in addition to the canonical NLS found in all FMR protein members, a second nuclear localization signal. In fact, Tamanini et al.  recently reported that the 27 aa pocket insert contains a short arginine rich motif (QRRNRSRRR) identical to amino acids 35 to 44 present in a domain that confers nucleolar localization to the HIV Rev protein . Although a similar (PQRRNRSRRRRFRGQ) peptide present in the 27 aa pocket, when coupled to a dye, has been shown to penetrate the cell and target the nucleolus , transient expression of all FXR1P isoforms, with or without the 27 aa pocket, localize exclusively to the cytoplasm [39 and our unpublished results]. Thus, it is not established that localization of FXRP82,84 is under the control of this peptide sequence. On the other hand, proteins that have been considered to be strictly nucleoplasmic, such as the hnRNPs  or the Rev proteins , have subsequently been found to shuttle between the nucleus and the cytoplasm [42,43,44,45]. In addition, hnRNPs have been shown to participate in the processing of pre-mRNAs and in the transport of mRNAs to the cytoplasm, and have even been suggested to play a role in translational regulation [46,47,48]. We propose that FXR1P82,84 might be sequestered in nucleoplasmic complexes in undifferentiated myoblasts and once cells are committed to differentiate, P82,84 are transferred to the cytoplasm with mRNPs carrying the newly processed mRNAs specific to the differentiated stage of myotubes. According to our working hypothesis, a nuclear role, yet to be defined, for FXR1P82,84 in undifferentiated myoblasts, is conceivable.
Previous studies on the FMR proteins have shown that although the FXR1 protein is predominantly cytoplasmic, in rare occasions a nuclear localization has been observed in undifferentiated cells in several tissues of human foetuses and mouse embryo [15,49]. The model system of C2C4 myoblasts that can be manipulated in vitro to differentiate into myotubes provides strong evidence that specific isoforms of FXR1P are indeed sequestred in the nucleus in undifferentiated myoblasts. As a working hypothesis we propose that the pattern of nucleo-cytoplasmic partitioning of FXR1P isoforms is under the control of factors regulating cell differentiation. By extention, we also speculate that isoforms of FMRP, that for the moment have escaped detection due to the very restricted number of available antibodies, might play a nuclear role in mRNA maturation at specific stages of neuronal differentiation and plasticity. In conclusion, the model system described here should be considered as a powerful tool for ongoing attempts to unravel structure-function relationships of the different FMR family members since the potential role(s) of FXR1P and FXR2P as a compensatory factor(s) in Fragile X Mental Retardation is still elusive.
Material and Methods
The mouse myogenic cell line C2C4, a subclone of the mouse skeletal muscle cell line C2,  was obtained from Chistian Pinset, Institut Pasteur, Paris, France. Cultures were routinely maintained at low cell density in Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% fetal calf serum (FCS) plus antibiotics (100 units/ml penicillin, 50 μg/ml streptomycin). Cultures were usually initiated with 3 × 104 cells in 100-mm diameter petri dishes. After 4 days of culture, differentiation was induced by replacing the medium with DMEM containing 0.2% FCS.
Two synthetic polypeptides of 15 (DDSEKKPQRRNRSRR) and 17 (NRSRRRRFRGQAEDRQP) amino acids present in the muscle peptide insert of 27 aa  were synthesized on a branched lysine residue (MAP carrier core technique by Research Genetic, Huntsville, Alabama) and used for immunization of rabbits using standard protocols and two polyclonal sera #27-15 and #27-17 were obtained. The monoclonal antibody 3FX and the polyclonal #830 anti-serum that detect different epitopes, have been described previously .
Total proteins from cell cultures and from various mouse tissues were prepared as described . Immunodetection analyses were performed using the following antibodies: mAb3FX and #830 , #27-15 and #27-17 (this study) for FXR1P. Hybridoma supernatants for cardiac troponin T (CT3; developed by JJ-C Lin), myogenin (F5D; WE Wright), nestin (Rat-401; S Hockfield), and desmin (D3; DA Fischman) were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242 (http://www.uiowa.edu/~dshbwww/). Immunoreaction was detected using horse radish peroxidase-conjugated secondary antibodies followed by the ECL (Amersham Pharmacia Biotech) reaction and exposure to Kodak Biomax MR films. High resolution two-dimension gel electrophoresis was conducted essentially as described  with the following modifications. The first IEF dimension contained 9 M urea, 2% Ampholynes pH 3.5-10 (Amersham Pharmacia Biotech AB) and 2% of CHAPS (Sigma, St Louis, MO) instead of 2% NP-40, and electrophoresis was conducted for 14 000 V/h under a constant current of 300 V. The second SDS-PAGE dimension consisted of a 7.5% acrylamide gel. Distribution of the different FXR1P isoforms in nuclear, cytoplasmic and polyribosome fractions were conducted as described previously  in the presence of Complete™, Mini (Roche Diagnostics) protease inhibitors cocktail.
Total RNA was prepared using the Trizol reagent (GIBCO). Northern blot analyses were performed as described . RNA was estimated by optical density at 260 nm and corrected for even loading (5 μg) after dot blot hybridization with a 32P-labeled 18S rRNA probe followed by radioactivity determination by liquid scintillation counting. cDNA inserts for myogenin, myf5 , and cardiac actin  were 32P-labeled by random priming. To obtain a specific probe to FXR1 exon 15, the 81 bp was cloned in pCR®II plasmid and amplified by PCR using as primers CAGGAAACAGCTATGACC (forward) and ATACGACTCACTATAGGG (reverse). The short 297 bp fragment obtained was labeled by random priming. Densitometric analyses were performed on autoradiograms using the NIH Image 1.62f program.
Immunofluorescence microscopy and immunohistochemistry
Immunoreactions on cells grown on glass coverslips and on sections from mouse organs were performed as previously described . Fluorescent and light microscopy were viewed with a Nikon TE300 microscope connected to a CoolSnap camera (RS Photometrics) using a 100 x oil immersion objective. Confocal images were obtained with a Zeiss LSM-310 microscope and images were colored using the Adobe PhotoShop program.
We thank Sylvie Giroux and Michel Vincent for helpful discussions, Michael Rudnicky, and Christian Pinset for cDNA probes and cell lines, Sandra Tremblay for technical assistance and Yves Labelle for critical reading of the manuscript. This work was supported by the Natural Sciences and Engineering Research Council of Canada. M.D. was supported by a studentship from le Fonds pour la Formation des Chercheurs et l'Aide à la Recherche du Québec.
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