Microrna-mediated regulation and the fragile X family of proteins

The 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 when FMRP is phosphorylated. We hypothesized that translational regulation by P-FMRP is accomplished through association with the miRNA pathway. Accordingly, we developed a phospho-specific antibody to P-FMRP and showed that P-FMRP associates with increased amounts of precursor miRNAs (pre-miRNA) compared to total FMRP. Furthermore, P-FMRP does not associate with Dicer or Dicer containing complexes in co-immunoprecipitation 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. 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 miRNAs and possibly a paucity of activating miRNAs. FMRP’s autosomal paralog, FXR1P, plays an important role in normal muscle development, has been implicated in fascioscapulohumeral muscular dystrophy (FSHD) and its absence or misregulation has been shown to cause cardiac abnormalities in 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, increases upon Dicer knockdown and consequent absence of miRNAs suggesting that FXR1P is regulated by miRNAs, while FMRP is not. Expression of a luciferase reporter bearing the FXR1 3’UTR was significantly increased in the absence of miRNAs, confirming miRNA-mediated regulation of FXR1P. We identified one of the regulatory regions by removing an 8-nucleotide miRNA seed sequence common to miRNAs 25, 32, 92, 363 and 367 in the 3’UTR of FXR1. Accordingly, over expression of a miRNA, miR-367, containing this common seed sequence decreased endogenous FXR1P expression in HEK-293T and HeLa cell lines. We report for the first time that FXR1P expression is regulated through miRNA binding to the miR-25/32/92/363/367 seed sequence binding site in the FXR1 3’UTR