Fragile X syndrome results from the absence of the RNA binding FMR protein. Here, mRNA was coimmunoprecipitated with the FMRP ribonucleoprotein complex and used to interrogate microarrays. We identified 432 associated mRNAs from mouse brain. Quantitative RT-PCR confirmed some to be >60-fold enriched in the immunoprecipitant. In parallel studies, mRNAs from polyribosomes of fragile X cells were used to probe microarrays. Despite equivalent cytoplasmic abundance, 251 mRNAs had an abnormal polyribosome profile in the absence of FMRP. Although this represents <2% of the total messages, 50% of the coimmunoprecipitated mRNAs with expressed human orthologs were found in this group. Nearly 70% of those transcripts found in both studies contain a G quartet structure, demonstrated as an in vitro FMRP target. We conclude that translational dysregulation of mRNAs normally associated with FMRP may be the proximal cause of fragile X syndrome, and we identify candidate genes relevant to this phenotype.
Fragile X syndrome is caused by a loss of expression of the fragile X mental retardation protein (FMRP). FMRP is a selective RNA-binding protein which forms a messenger ribonucleoprotein (mRNP) complex that associates with polyribosomes. Recently, mRNA ligands associated with FMRP have been identified. However, the mechanism by which FMRP regulates the translation of its mRNA ligands remains unclear. MicroRNAs are small noncoding RNAs involved in translational control. Here we show that in vivo mammalian FMRP interacts with microRNAs and the components of the microRNA pathways including Dicer and the mammalian ortholog of Argonaute 1 (AGO1). Using two different Drosophila melanogaster models, we show that AGO1 is critical for FMRP function in neural development and synaptogenesis. Our results suggest that FMRP may regulate neuronal translation via microRNAs and links microRNAs with human disease.
Fragile X mental retardation protein, FMRP, is absent in patients with fragile X syndrome, a common form of mental retardation. FMRP is a nucleocytoplasmic RNA binding protein that is primarily associated with polyribosomes. FMRP is believed to be a translational repressor and may regulate the translation of certain mRNAs at the base of dendritic spines in neurons. However, little is known about the regulation of FMRP. Using mass spectrometry and site-directed mutagenesis, we show that FMRP is phosphorylated between residues 483 and 521, N-terminal to the RGG box, both in murine brain and in cultured cells. Primary phosphorylation occurs on the highly conserved serine 499, which triggers hierarchical phosphorylation of nearby serines. FMRP is phosphorylated within 2-4 h of synthesis, however, phosphorylation has no effect on the half-life of the protein. In contrast to the Drosophila ortholog dFxr, the phosphorylation status of mammalian FMRP does not influence its association with specific mRNAs in vivo. However, we find unphosphorylated FMRP associated with actively translating polyribosomes while a fraction of phosphorylated FMRP is associated with apparently stalled polyribosomes. Our data suggest that the phosphorylation may regulate FMRP and that the release of FMRP-induced translational suppression may involve a dephosphorylation signal.
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