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. 1997 Mar 1;17(5):1539-47.
doi: 10.1523/JNEUROSCI.17-05-01539.1997.

Fragile X mental retardation protein: nucleocytoplasmic shuttling and association with somatodendritic ribosomes

Y Feng  1 , C A Gutekunst, D E Eberhart, H Yi, S T Warren, S M Hersch
Affiliations

Fragile X mental retardation protein: nucleocytoplasmic shuttling and association with somatodendritic ribosomes

Y Feng et al. J Neurosci. .

Abstract

Fragile X syndrome, a leading cause of inherited mental retardation, is attributable to the unstable expansion of a CGG-repeat within the FMR1 gene that results in the absence of the encoded protein. The fragile X mental retardation protein (FMRP) is a ribosome-associated RNA-binding protein of uncertain function that contains nuclear localization and export signals. We show here detailed cellular localization studies using both biochemical and immunocytochemical approaches. FMRP was highly expressed in neurons but not glia throughout the rat brain, as detected by light microscopy. Although certain structures, such as hippocampus, revealed a strong signal, the regional variation in staining intensity appeared to be related to neuron size and density. In human cell lines and mouse brain, FMRP co-fractionated primarily with polysomes and rough endoplasmic reticulum. Ultrastructural studies in rat brain revealed high levels of FMRP immunoreactivity in neuronal perikarya, where it is concentrated in regions rich in ribosomes, particularly near or between rough endoplasmic reticulum cisternae. Immunogold studies also provided evidence of nucleocytoplasmic shuttling of FMRP, which was localized in neuronal nucleoplasm and within nuclear pores. Moreover, labeling was observed in large- and small-caliber dendrites, in dendritic branch points, at the origins of spine necks, and in spine heads, all known locations of neuronal polysomes. Dendritic localization, which was confirmed by co-fractionation of FMRP with synaptosomal ribosomes, suggests a possible role of FMRP in the translation of proteins involved in dendritic structure or function and relevant for the mental retardation occurring in fragile X syndrome.

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Figures

Fig. 1.
Fig. 1.
FMRP distribution in subcellular fractions of human lymphoblastoid cells. A is a schematic illustration of subcellular fractionation of EBV-transformed human lymphoblastoid cells. The descriptor under key fractions refers to the panels below(B–D), with INT as interface. A detailed description and protocol is provided in Materials and Methods. InB, the left panel shows SDS-PAGE immunoblot analysis of FMRP and P0 in crude subcellular fractions. Total protein (3 μg) from each fraction of B1–B5 was loaded. Densitometric analysis of immunoblot signals was used to calculate the total yield of FMRP in each corresponding fraction, based on the total fraction volume. The relative percentage of FMRP in each fraction was plotted as shown in the right panel. Cshows the SDS-PAGE immunoblot analysis of FMRP and P0 in separated postnuclear supernatant fraction. C1–C4 in sucrose gradient fractionation 1 represent cytosol; low-density membranes (plasma membrane, Golgi, and smooth ER); high-density membranes (RER); and free polysome pellet, respectively. Total protein (1.5 μg) from each fraction was loaded. D shows the SDS-PAGE immunoblot analysis of FMRP and P0 in various ER components.D1–D3 in sucrose gradient fractionation 2 represent smooth ER, light RER, and heavy RER. Total protein (3 μg) from each fraction was loaded.
Fig. 2.
Fig. 2.
FMRP immunocytochemistry in the rat brain.A is a coronal section demonstrating widespread FMRP labeling. The most intense labeling is in the cellular layers of the hippocampus (h) and pyriform cortex (p), which are regions with extremely high neuronal densities. The deeper layers of the cerebral cortex (c) are also well labeled. B demonstrates FMRP immunoreactivity in the frontal cortex at higher magnification. It appears that most neurons in each cortical layer are FMRP-positive.C illustrates the cellular pattern of FMRP immunoreactivity in layer V pyramidal cells from frontal cortex. Staining is very dense in perikarya and proximal dendrites (triangles). In contrast, nuclear staining (arrows) is uncertain. D is an electron micrograph of the soma of a cerebral cortical pyramidal cell. With immunoperoxidase, dense cytoplasmic staining is evident. Although the nucleus is somewhat dark, FMRP immunoreactivity is not clearly present. Scale bars: A, 50 mm; B, 100 μm;C, 50 μm; D, 1 μm.
Fig. 3.
Fig. 3.
A, Electron micrograph of the soma of a labeled cerebral cortical pyramidal neuron. Immunogold particles are present both in the nucleus (n) and perikaryon. Little of the cytoplasmic label is associated with the plasma membrane, mitochondria, or Golgi apparatus (arrow).B, C, Few particles were found in the nuclei or cytoplasm of astrocytes (a) or oligodendrocytes (o). D, Tangential section through the nuclear envelope of a pyramidal cell showing three nuclear pores (arrows), one of which (longer arrow) contains an immunogold particle. E, Cross-section through the nuclear envelope showing immunogold particles within nuclear pores (arrows).F–H, In perikarya, most immunogold particles are clustered between the cisternae of RER. These regions are especially rich in free ribosomes, visible here as fine electron-dense particles (asterisks). A few immunogold particles are also seen in direct contact with the cisternae (arrows). Scale bars: A–C, 1 μm;D–H, 500 μm.
Fig. 4.
Fig. 4.
Electron micrographs demonstrating FMRP localization in cellular processes in cerebral cortex.A–C, Dendrites (d) in cross-section and longitudinal section showing that immunogold particles are either free in the cytoplasm or clustered around cisternae of smooth ER (arrows) or at the origins of dendritic spines (triangles).D–F, Dendritic spines (s) containing immunogold particles, which are either free in the cytoplasm or associated with the spine apparatus (arrows).G, Rare axon terminals (a) contain immunogold particles that are cytoplasmic in location.H, FMRP-immunoreactive axon terminals (a) are more easily identified using immunoperoxidase. Scale bars:A–H, 500 nm.
Fig. 5.
Fig. 5.
Association of FMRP with translating ribosomes in rat cortex. A, Subcellular fractionation of rat cortex by velocity centrifugation. The fractionation procedures are illustrated on the left panel, with Sindicating supernatant and P indicating pellet. A detailed description and protocol are provided in Materials and Methods. The right panel shows SDS-PAGE immunoblot analysis of FMRP and other marker proteins in the corresponding fractions as indicated. Based on Bradford assay, 20 μg of total protein from each fraction was used in this blot. B, Association of FMRP with polysomes in synaptosomal lysate. SDS-PAGE immunoblot analysis was performed using linear sucrose gradient fractions containing synaptosomal lysates with the presence of Mg2+ or EDTA, as described in Materials and Methods. The signals of FMRP and P0 protein are indicated on theright. The sedimentation of ribosomal components in human lymphoblasts monitored at OD254 in a parallel gradient are indicated on top of the corresponding fractions.
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