Fragile X protein family member FXR1P is regulated by microRNAs

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 39 untranslated region (39UTR) of FXR1 was significantly increased in the absence of miRNAs, confirming miRNA-mediated regulation of FXR1P, while a luciferase reporter bearing the FMR1 39UTR was not. We identified one of the regulatory regions in the 39UTR 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.

Emerging roles of non-coding RNAs in brain evolution, development, plasticity and disease

“…In fact, FXR1P is involved in the biogenesis of miR-9 and miR-124 (REF. 106) and, in turn, its expression is modulated by various miRNAs 107 . Ataxin 2 (ATX2) is another example of a protein involved in neurological disease that links RNA granules and miRNA pathways with synaptic functions.…”

Fragile hearts: New insights into translational control in cardiac muscle

“…While not much else is known about the regulation of FXR1 itself, Ceman and colleagues have recently revealed a mechanism whereby FXR1 (but not FMRP) expression is controlled directly by the miRNA pathway. Specifically, using a conditional Dicer knockdown cell line, FXR1 (but not FMRP) was shown to be regulated via the miR-25/32/92/363/367 seed sequence in its 3′ UTR (Cheever et al, 2010). These studies together with findings that FXR1 (but not FMRP) regulates miR-9 and miR-124 in the brain (Xu et al, 2011), suggesting that FXR1 may act as a critical node for controlling gene expression by as well as through the miRNA pathway.…”

Detection and Quantification of the Fragile X Mental Retardation Protein 1 (FMRP)

“…The reactivity of the above antibodies with multiple proteins was explained by the presence of several mRNA isoforms—produced by alternative splicing of FMR1 —found in human lymphoblastoid cells [14] and mouse tissues [19,20]. However, it has been reported that in FMR1 KO mouse brain MAB2160 [21,22] cross-reacts with FXR1, and mAb7G1-1 cross reacts with caprin 1 [21,22,23] and argonaute [24]. …”

“…The incongruence could (1) be caused the action of genetic and epigenetic factors responsible for the clinical phenotypes associated with PM; (2) stem from the way sample extraction [43] and Western blotting were performed; and (3) be caused by the lack of specificity of the anti-FMRP antibodies used. Background staining is obviously expected in immunoblots probed with mAb7G1-1, which is known to cross-react with two other proteins, caprin1 [21,22,23] and argonaute [24]; and with MAB2160—the antibody used in most Western blot experiments—which cross-reacts with FXR1 [22,45]. The MAB2160 epitope was mapped [35] to the sequence 34 NNWQPD 39 of human FMRP, which is conserved except for the change of D 39 to E in both mouse FMRP and in FXR1 (NNWQPE).…”