Fragile X syndrome is caused by the loss of expression of the fragile X mental retardation protein, FMRP. FMRP is an RNA-binding protein that is highly expressed in neurons and undergoes multiple post-translational modifications including methylation on arginine. FMRP is methylated on the high-affinity RNA-binding motif, the RGG box, at positions 533, 538, 543 and 545 of murine FMRP. To identify the arginines important for FMRP function, we examined their role in polyribosome and mRNA association. We found that arginines 533 and 538 were required for normal FMRP polyribosome association whereas all four arginines played a role in RNA binding, depending on the identity of the RNA. The model G-quadruplex RNA sc1 required arginines 533 and 538 for normal association with FMRP, whereas AATYK mRNA did not. In vitro methylation of FMRP-bearing arginine substitutions inhibited sc1 binding but not AATYK binding. In addition, we found that PRMT1 co-immunoprecipitated with FMRP isolated from cells and that siRNAs directed against PRMT1 led to reduced FMRP methylation. Thus, two lines of experimentation demonstrate that PRMT1 acts on FMRP in cells. In summary, we provide evidence for the important role of the RGG box in polyribosome association. We also demonstrate for the first time that the different arginines of the RGG box are important for the binding of different RNAs. Finally, we show that PRMT1 methylates FMRP in cells, suggesting a model where methylation of the RGG box modulates either the quantity or the identity of the RNAs bound by FMRP.

Arginine methylation is a post-translational modification that regulates protein function. RNA-binding proteins are an important class of cell function mediators, some of which are methylated on arginine. Early studies of RNA-binding proteins and arginine methylation are briefly introduced, and the enzymes that mediate this post-translational modification are described. We review the most common RNA-binding domains and briefly discuss how they associate with RNAs. We address the following groups of RNA-binding proteins: hnRNP, Sm, Piwi, Vasa, FMRP, and HuD. hnRNPs were the first RNA-binding proteins found to be methylated on arginine. The Sm proteins function in RNA processing and germ cell specification. The Piwi proteins are largely germ cell specific that are also required for germ cell production, as is Vasa. FMRP participates in germ cell formation in Drosophila but is more widely known for its neuronal function. Similarly, HuD plays a role in nervous system development and function. We review the effects of arginine methylation on the function of each protein, then conclude by addressing remaining questions and future directions of arginine methylation as an important and emerging area of regulation.

The mating-specific G ␣ protein of Saccharomyces cerevisiae, Gpa1, stimulates adaptation to pheromone by a mechanism independent of G ␤␥ sequestration. Genetic evidence suggests that Gpa1 targets the Fus3 mitogenactivated protein kinase, and it has recently been shown that the two proteins interact in cells responding to pheromone. To test the possibility that Gpa1 downregulates the mating signal by affecting the localization of Fus3, we created a Fus3-green fluorescent protein (GFP) fusion protein. In vegetative cells, Fus3-GFP was found in both the cytoplasm and the nucleus. Pheromone stimulated a measurable increase in the ratio of nuclear to cytoplasmic Fus3-GFP. In contrast, the relative level of nuclear Fus3-GFP decreased as cells recovered from pheromone arrest and did not increase when cells adapted to chronic stimulus were challenged again. Accumulation of Fus3-GFP in the nuclei of stimulated cells was also inhibited by overexpression of either wild-type Gpa1, the E364K hyperadaptive mutant form of Gpa1, or the Msg5 dually specific phosphatase. The effects of Gpa1 and Msg5 on Fus3 are partially interdependent. In a genetic screen for adaptive defective mutants, a nonsense allele of the nucleocytoplasmic transport receptor, Kap104, was identified. Truncation of the Kap104 cargo-binding domain blocked the effect of both Gpa1E364K and Msg5 on Fus3-GFP localization. Based on these results, we propose that Gpa1 and Msg5 work in concert to downregulate the mating signal and that they do so by inhibiting the pheromone-induced increase of Fus3 in the nucleus. Kap104 is required for the G ␣ /phosphatase-mediated effect on Fus3 localization.

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.

Background: Like mammalian MAP kinases, the mating-specific Fus3 MAPK of yeast accumulates in the nuclei of stimulated cells. Because Fus3 does not appear to be subjected to active nucleocytoplasmic transport, it is not clear how its activation by mating pheromone effects the observed change in its localization. One possibility is that the activation of Fus3 changes its affinity for nuclear and cytoplasmic tethers.

SummaryFragile X mental retardation protein (FMRP) is required for normal cognition. FMRP has two autosomal paralogs, which although similar to FMRP, cannot compensate for the loss of FMRP expression in brain. The arginine-and glycine-rich region of FMRP (the RGG box) is unique; it is the high-affinity RNA-binding motif in FMRP and is encoded by exon 15. Alternative splicing occurs in the 59 end of exon 15, which is predicted to affect the structure of the distally encoded RGG box. Here, we provide evidence that isoform 3, which removes 25 amino acids from the 59 end of exon 15, has an altered conformation that reduces binding of a specific antibody and renders the RGG box unable to efficiently associate with polyribosomes. Isoform 3 is also compromised in its ability to form granules and to associate with a key messenger ribonucleoprotein Yb1 (also known as p50, NSEP1 and YBX1). Significantly, these functions are similarly compromised when the RGG box is absent from FMRP, suggesting an important regulatory role of the N-terminal region encoded by exon 15.