Muscle specific fragile X related protein 1 isoforms are sequestered in the nucleus of undifferentiated myoblast

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

Figure 1

Schematic representation of the FXR1 protein structure. a) Localization of the RNA-binding domains (KH and RGG; yellow) and the nuclear localization (NL) and export (NE) signals (green). b) Structure of the different FXR1P isoforms at the C-termini generated by the four small peptide inserts (in blue) deduced from the sequence of individual mRNA variants according to Kirkpatrick's et al. [18] numbering and after compilation of GenPept access No AF124386.1 to 124394.1. The boxes (exon 12, 13, 15 and 16, in blue) correspond to the peptide inserts present or absent in the different protein isoforms. The red zones indicate the regions recognized by the different antibodies.

Figure 2

Distribution of FXR1P isoforms in extracts from different tissues and organs in adult mouse. Equal amounts of proteins (60 μg) were resolved by SDS-PAGE (7.5% acrylamide) and immunoblotted with three antibodies detecting different epitopes in FXR1P (see Results section). Note the specificity of #27-15 antiserum to the FXR1P82,84 isoforms and the cross-reaction of mAb3FX with FXR2P (94 kDa).

Figure 3

Two-dimensional immunoblot analysis of proteins extracted from heart and limb muscle from adult mouse. Hundred and fifty μg of protein extracts were resolved by IEF in the first dimension and by 7.5% SDS-PAGE in the second dimension. Arrows indicate the positions of the major P82,84 modified variants at pH 6.0, 5.7 and 5.4.

Figure 4

Comparative immunostaining of FXR1P82,84 in mouse heart and limb muscle. The red staining deposits indicate the localization of P82,84 in striated muscle as dot-like structures reminiscent of costameres associated with Z bands. Nuclei were counterstained with hematoxylin. Scale bars: = 10 μm.

Figure 5

Time course analyses of FXR1P isoforms levels in C2C4 cells induced to differentiate. Equal amounts of proteins (approximately 40 μg) from unstimulated myoblast (C: control) and stimulated cells 1 to 3 days after serum deprivation were separated by SDS-PAGE and processed for immunoblot analyses. All FXR1P isoforms were revealed with mAb3FX while P82,84 were detected with #27-15. dps: days post-stimulation.

Figure 6

Accumulation of FXR1P82,84 is an early event and coincides with expression of different myogenetic markers. Protein (40 μg) and RNA (5 μg) were prepared at the indicated times and subjected to immunoblot (Western) and Northern analyses, respectively. Exposure times for Western analyses were between 15 and 30 sec. Northern blots were exposed for 36 h while the 81 bp insert required prolonged exposure up to 72 h due to the short probe used. hps: hours post-stimulation after serum deprivation.

Figure 7

FXR1P isoforms are associated with mRNPs present in polyribosomes. Aliquots containing ~12 ^A260 units of post-nuclear supernatants from non-induced myoblasts and from differentiated myotubes at day 3 were analyzed by sedimentation velocity through sucrose density gradients. Each collected fraction from gradients containing MgCl₂ or EDTA (lower panels) was analyzed for the presence of all FXR1P isoforms using three different antibodies. Note that none of the antibodies used detect P82,84 in cytoplasmic extracts from myoblasts.

Figure 8

Low levels of FXR1P82,84 are present in nuclear preparation of undifferentiated myoblasts and differentiated myotubes, while in differentiated myotubes, accumulation of P82,84 is restricted to the cytoplasmic fraction. N: nuclear, and C: cytoplasmic fractions, respectively.

Figure 9

Intracellular localization of the short, long and super long FXR1P isoforms by indirect immunofluorescence in myoblasts and myotubes as detected with the different antibodies to FXR1P. Confocal (upper panels) and light (lower panels) microscopy analyses after reaction with the different anti-FXR1P antibodies. Nuclei were counterstained with DAPI. Scale bars: = 5 μm.