[Show abstract][Hide abstract] ABSTRACT: main purpose of this work is to understand how two members of the fragile X family of RNA binding proteins, fragile X mental retardation protein (FMRP) and FXR1P, are regulated by post-translational modifications and microRNAs (miRNAs), respectively. Both proteins play key roles in normal development and function. The absence of FMRP leads to the cognitive defects seen in Fragile X syndrome, the leading cause of hereditary mental retardation, while loss of FXR1P expression in mice is fatal after birth, likely due to cardiac and muscle abnormalities. Small, genomically encoded miRNAs are involved in almost every biological process, specifically in the regulation of mRNA translation. Although their biogenesis is relatively well defined, it is still unclear how they are recruited to their mRNA targets. FMRP and its autosomal paralogs, FXR1P and FXR2P, in addition to the single Drosophila ortholog, dFmrp, associate physically and functionally with the miRNA pathway. Constitutively phosphorylated FMRP (P-FMRP) is found associated with stalled untranslating polyribosomes and translation of at least one mRNA is downregulated
[Show abstract][Hide abstract] ABSTRACT: FXR1P is one of two autosomal paralogs of the fragile X mental retardation protein FMRP. The absence of FMRP causes fragile X syndrome, the leading cause of hereditary mental retardation. FXR1P plays an important role in normal muscle development and has been implicated in facioscapulohumeral muscular dystrophy (FSHD). Its absence also causes cardiac abnormalities in both mice and zebrafish. To examine miRNA-mediated regulation of FMRP and FXR1P, we studied their expression in a conditional Dicer knockdown cell line, DT40. We found that FXR1P, but not FMRP, is significantly increased upon Dicer knockdown and the consequent reduction of miRNAs, suggesting that FXR1P is regulated by miRNAs while FMRP is not in DT40 cells. Expression of a luciferase reporter bearing the 3' untranslated region (3'UTR) of FXR1 was significantly increased in the absence of miRNAs, confirming miRNA-mediated regulation of FXR1P, while a luciferase reporter bearing the FMR1 3'UTR was not. We identified one of the regulatory regions in the 3'UTR of FXR1 by removing a conserved, 8-nucleotide miRNA seed sequence common to miRNAs 25, 32, 92, 363, and 367 and demonstrated loss of miRNA-mediated suppression. Treatment with specific miRNA hairpin inhibitors to each of the miRNAs in the seed sequence showed that miRs 92b, 363, and 367 regulated FXR1P expression. Accordingly, overexpression of the miRNA 367 mimic significantly decreased endogenous FXR1P expression in human cell lines HEK-293T and HeLa. We report for the first time that FXR1P is regulated through miRNA binding, with one site being the miR-25/32/92/363/367 seed sequence.
[Show abstract][Hide abstract] ABSTRACT: Small, genomically-encoded microRNAs are important factors in the regulation of mRNA translation. Although their biogenesis is relatively well-defined, it is still unclear how they are recruited to their mRNA targets. The fragile X mental retardation protein family members, FMRP, FXR1P and FXR2P are RNA binding proteins that regulate translation of their cargo mRNAs. All three proteins, in addition to the single Drosophila ortholog, dFmrp, associate physically and functionally with the microRNA pathway. In this review, we summarize what is known about the role of the fragile X family members in translation regulation, highlighting evidence for their association with the microRNA pathway. In addition, we present a new model for the effect of phosphorylation on FMRP function, where phosphorylation of FMRP inhibits Dicer binding, leading to the accumulation of precursor microRNAs and possibly a paucity of activating microRNAs.
[Show abstract][Hide abstract] ABSTRACT: Fragile X syndrome is caused by an absence of the protein product of the fragile X mental retardation gene (FMR1). The fragile X mental retardation protein (FMRP) is an RNA-binding protein that regulates translation of associated mRNAs; however, the mechanism for this regulation remains unknown. Constitutively, phosphorylated FMRP (P-FMRP) is found associated with stalled untranslating polyribosomes, and translation of at least one mRNA is down-regulated when FMRP is phosphorylated. Based on our hypothesis that translational regulation by P-FMRP is accomplished through association with the microRNA (miRNA) pathway, we developed a phospho-specific antibody to P-FMRP and showed that P-FMRP associates with increased amounts of precursor miRNAs (pre-miRNA) compared with total FMRP. Furthermore, P-FMRP does not associate with Dicer or Dicer-containing complexes in coimmunoprecipitation experiments or in an in vitro capture assay using a P-FMRP peptide sequence bound to agarose beads. These data show that Dicer-containing complexes bind FMRP at amino acids 496-503 and that phosphorylation disrupts this association with a consequent increase in association with pre-miRNAs. In sum, we propose that in addition to regulating translation, phosphorylation of FMRP regulates its association with the miRNA pathway by modulating association with Dicer.
[Show abstract][Hide abstract] ABSTRACT: Fragile X syndrome is the most common form of inherited mental retardation and is caused by the absence of expression of the FMR1 gene. The protein encoded by this gene, Fmrp, is an RNA-binding protein that binds a subset of mRNAs and regulates their translation, leading to normal cognitive function. Although the association with RNAs is well established, it is still unknown how Fmrp finds and assembles with its RNA cargoes and how these activities are regulated. We show here that Fmrp is post-translationally methylated, primarily on its arginine-glycine-glycine box. We identify the four arginines that are methylated and show that cellular Fmrp is monomethylated and asymmetrically dimethylated. We also show that the autosomal paralog Fxr1 and the Drosophila ortholog dFmr1 are methylated post-translationally. Recombinant protein arginine methyl transferase 1 (PRMT1) methylates Fmrp on the same arginines in vitro as in cells. In vitro methylation of Fmrp results in reduced binding to the minimal RNA sequence sc1, which encodes a stem loop G-quartet structure. Our data identify an additional mechanism, arginine methylation, for modifying Fmrp function and suggest that methylation occurs to limit or modulate RNA binding by Fmrp.
Human Molecular Genetics 02/2006; 15(1):87-96. DOI:10.1093/hmg/ddi429 · 6.39 Impact Factor