Alternative splicing modulates protein arginine methyltransferase-dependent methylation of fragile X syndrome mental retardation protein
ABSTRACT The fragile X mental retardation protein (FMRP) is an RNA binding protein that is methylated by an endogenous methyltransferase in rabbit reticulocyte lysates. We mapped the region of methylation to the C-terminal arginine-glycine-rich residues encoded by FMR1 exon 15. We additionally demonstrated that mutation of R(544) to K reduced the endogenous methylation by more than 80%, while a comparable mutant R(546)-K reduced the endogenous methylation by 20%. These mutations had no effect on the subcellular distribution of FMRP, recapitulating previous results using the methyltransferase inhibitor adenosine-2',3'-dialdehyde. Using purified recombinant protein arginine methyltransferases (PRMTs), we showed that the C-terminal domain could be methylated by PRMT1, PRMT3, and PRMT4 in vitro and that both the R(544)-K mutant and the R(546)-K mutant were refractory toward these enzymes. We also report that truncating the N-terminal 12 residues encoded by FMR1 exon 15, which occurs naturally via alternative splicing, had no effect on FMRP methylation, demonstrating conclusively that phosphorylation of serine residue 500 (S(500)), one of the 12 residues, was not required for methylation. Nevertheless, truncating 13 additional amino acids, as occurs in the smallest alternatively spliced variant of FMR1 exon 15, reduced methylation by more than 85%. This suggests that differential expression and methylation of the FMRP exon 15 variants may be an important means of regulating target mRNA translation, which is consonant with recently demonstrated functional effects mediated by inhibiting FMRP methylation in cultured cells.
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ABSTRACT: 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 and 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.Molecular Reproduction and Development 03/2012; 79(3):163-75. DOI:10.1002/mrd.22024 · 2.68 Impact Factor
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ABSTRACT: Exon 15 of the fragile X mental retardation protein gene (FMR1) is alternatively spliced into three variants. The amino acids encoded by the 5' end of the exon contain several regulatory determinants including phosphorylation sites and a potential conformational switch. Residues encoded by the 3' end of the exon specify FMRP's RGG box, an RNA binding domain that interacts with G-quartet motifs. Previous studies demonstrated that the exon 15-encoded N-terminal residues influence the extent of arginine methylation, independent of S 500 phosphorylation. In the present study we focus on the role the putative conformational switch plays in arginine methylation. Chemical and structural probing of Ex15 alternatively spliced variant proteins and several mutants leads to the following conclusions: Ex15c resides largely in a conformation that is refractory toward methylation; however, it can be methylated by supplementing extracts with recombinant PRMT1 or PRMT3. Protein modeling studies reveal that the RG-rich region is part of a three to four strand antiparallel beta-sheet, which in other RNA binding proteins functions as a platform for nucleic acid interactions. In the Ex15c variant the first strand of this sheet is truncated, and this significantly perturbs the side-chain conformations of the arginine residues in the RG-rich region. Mutating R 507 in the conformational switch to K also truncates the first strand of the beta-sheet, and corresponding decreases in in vitro methylation were found for this and R 507/R 544 and R 507/R 546 double mutants. These effects are not due to the loss of R 507 methylation as a conformational switch-containing peptide reacted under substrate excess and in methyl donor excess was not significantly methylated. Consistent with this, similar changes in beta-sheet structure and decreases in in vitro methylation were observed with a W 513-K mutant. These data support a novel model for FMRP arginine methylation and a role for conformational switch residues in arginine modification.Biochemistry 08/2008; 47(33):8491-503. DOI:10.1021/bi702298f · 3.19 Impact Factor
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ABSTRACT: Brain-derived neurotrophic factor (BDNF) has multiple alternative splicing variants and plays diverse biological functions in mammals, including neuronal survival, cholesterol metabolism, cell differentiation and tumor development. However, genomic structures of chicken BDNF (cBDNF) variants and its potential functions are still undefined. Here, we characterized two novel alternative splicing variants of cBDNF, cBDNF1 and cBDNF2, via combining comparative genomics methods and molecular techniques in inbred chicken line 6(3) and line 7(2), which have been developed to be resistant and susceptible, respectively, to Marek's disease tumor since 1939. Both cBDNFs consist of a bipartite transcript, with different 5' exons, exon I (298 bp) in cBDNF1 and exon II (286 bp) in cBDNF2, each of which is spliced to the common 3' exon IV. Exon I and IV are highly conserved between chicken and mammals, whereas exon II is unique for chicken. The amino acid sequence of cBDNF1 contains 8 additional amino acids in the N terminal compared to cBDNF2. cBDNF1 and cBDNF2 were only expressed in the hypothalamus among eight tissues, and cBDNF2 showed lower expression than that of cBDNF1 in both lines. The expression level of cBDNF1 was significantly higher in line 7(2) than in line 6(3) (P<0.01). Notably, the DNA methylation levels on the cis-regulatory region of cBDNF1 was negatively correlated with its expression level, which suggests that the mRNA expression level of cBDNF1 may be related to the DNA methylation status in the chickens. We also discussed a potential role of variant cBDNF1 in MD tumor resistance and susceptibility.Brain research 03/2009; 1269:1-10. DOI:10.1016/j.brainres.2009.01.071 · 2.83 Impact Factor