Fragile X mental retardation protein is associated with translating polyribosomes in neuronal cells

Abstract

Fragile X mental retardation protein (FMRP) is an RNA binding protein encoded by the gene FMR1, whose expression is impaired in patients with fragile X mental retardation. The association of FMRP with polyribosomes in non-neural cell lines has previously suggested that FMRP is involved in translational regulation. However, the relevance of these studies to neuronal function has been questioned by the finding that FMRP in brain is not associated with polyribosomes, but is part of small ribonucleo-protein complexes that do not appear to include ribosomes. Here we optimize methods to analyze brain polyribosomes, allowing us to definitively demonstrate that FMRP forms complexes with cortical brain polyribosomes. Moreover, we demonstrate in neuroblastoma cells that the FMRP-polyribosome complexes are sensitive to puromycin, a drug that targets actively translating ribosomes. These data indicate that FMRP associates with functional polyribosomes in neurons.

Figure 1.

Association of FMRP with polyribosomes in extracts from brain cortex of mice at different ages. Cytoplasmic extracts were prepared from brain cortices of 9-d-old (A), 5-month-old (B), and 53-d-old (C) mice and centrifuged on a 20-50% w/w linear sucrose gradient. Fractions were collected and analyzed by Western blot with antibodies against FMRP and ribosomal S6 protein (rS6). Extracts treated with 30 mm EDTA or RNase (C) were analyzed in parallel. Fractions from the top to the bottom of the gradient are shown from left to right. The positions of the 80 S ribosome monomer and 60 S subunit are indicated.

Figure 2.

Effects of treatment with different detergents on the association of FMRP with polyribosomes. A, Cortical extracts were prepared from 8-d-old mice in complete absence of detergent and analyzed as in Figure 1. B, Treatment with deoxycholic acid disrupts the interaction of FMRP with polyribosomes. Cytoplasmic extracts from mouse brain cortex were treated with 1% NP-40 (left panel) or 1% NP-40 and 0.5% deoxycholic acid (right panel). Subsequently, the extracts were fractionated on a 20-50% w/w sucrose linear density gradient. Fractions were collected and analyzed by Western blot using anti-FMRP (IC3) and anti-L7 antibodies.

Figure 3.

Association of FMRP with translating polyribosomes. Cytoplasmic extracts were prepared from Neuro-2a cells. Cells were treated with 0.35 mm cycloheximide for 10 min or with 1 mm puromycin for 3 hr in the culture medium before lysis. The extracts were analyzed on sucrose density gradients as in Figure 1.