Mental retardation linked to mutations in the HSD17B10 gene interfering with neurosteroid and isoleucine metabolism.

Mutations in the HSD17B10 gene were identified in two previously described mentally retarded males. A point mutation c.776G>C was found from a survivor (SV), whereas a potent mutation, c.419C>T, was identified in another deceased case (SF) with undetectable hydroxysteroid (17beta) dehydrogenase 10 (HSD10) activity. Protein levels of mutant HSD10(R130C) in patient SF and HSD10(E249Q) in patient SV were about half that of HSD10 in normal controls. The E249Q mutation appears to affect HSD10 subunit interactions, resulting in an allosteric regulatory enzyme. For catalyzing the oxidation of allopregnanolone by NAD+ the Hill coefficient of the mutant enzyme is approximately 1.3. HSD10(E249Q) was unable to catalyze the dehydrogenation of 2-methyl-3-hydroxybutyryl-CoA and the oxidation of allopregnanolone, a positive modulator of the gamma-aminobutyric acid type A receptor, at low substrate concentrations. Neurosteroid homeostasis is critical for normal cognitive development, and there is increasing evidence that a blockade of isoleucine catabolism alone does not commonly cause developmental disabilities. The results support the theory that an imbalance in neurosteroid metabolism could be a major cause of the neurological handicap associated with hydroxysteroid (17beta) dehydrogenase 10 deficiency.

On BC1 RNA and the fragile X mental retardation protein.

The fragile X mental retardation protein (FMRP), the functional absence of which causes fragile X syndrome, is an RNA-binding protein that has been implicated in the regulation of local protein synthesis at the synapse. The mechanism of FMRP's interaction with its target mRNAs, however, has remained controversial. In one model, it has been proposed that BC1 RNA, a small non-protein-coding RNA that localizes to synaptodendritic domains, operates as a requisite adaptor by specifically binding to both FMRP and, via direct base-pairing, to FMRP target mRNAs. Other models posit that FMRP interacts with its target mRNAs directly, i.e., in a BC1-independent manner. Here five laboratories independently set out to test the BC1-FMRP model. We report that specific BC1-FMRP interactions could be documented neither in vitro nor in vivo. Interactions between BC1 RNA and FMRP target mRNAs were determined to be of a nonspecific nature. Significantly, the association of FMRP with bona fide target mRNAs was independent of the presence of BC1 RNA in vivo. The combined experimental evidence is discordant with a proposed scenario in which BC1 RNA acts as a bridge between FMRP and its target mRNAs and rather supports a model in which BC1 RNA and FMRP are translational repressors that operate independently.

Dendritic BC1 RNA in translational control mechanisms.

Translational control at the synapse is thought to be a key determinant of neuronal plasticity. How is such control implemented? We report that small untranslated BC1 RNA is a specific effector of translational control both in vitro and in vivo. BC1 RNA, expressed in neurons and germ cells, inhibits a rate-limiting step in the assembly of translation initiation complexes. A translational repression element is contained within the unique 3' domain of BC1 RNA. Interactions of this domain with eukaryotic initiation factor 4A and poly(A) binding protein mediate repression, indicating that the 3' BC1 domain targets a functional interaction between these factors. In contrast, interactions of BC1 RNA with the fragile X mental retardation protein could not be documented. Thus, BC1 RNA modulates translation-dependent processes in neurons and germs cells by directly interacting with translation initiation factors.

Tissue and developmental regulation of fragile X mental retardation 1 exon 12 and 15 isoforms.

The pre-mRNA of the fragile X mental retardation 1 gene (FMR1) is subject to exon skipping and alternative splice site selection, which can generate up to 12 isoforms. The expression and function of these variants in vivo has not yet been fully explored. In the present study, we investigated the distribution of Fmr1 exon 12 and exon 15 isoforms. Exon 12 encodes an extension of KH(2) domain, one of the RNA binding domains in the FMR1 gene product (FMRP) and we show that exon 12 variant proteins differentially interact with kissing complex RNA. Alternative splicing at exon 15 produces FMRPs differing in RNA binding ability and each is distinguished by unique post-translational modifications. Using semiquantitative RT-PCR and Northern blotting, we found that particular Fmr1 exon 12 and exon 15 isoforms change during neuronal differentiation. Interestingly, Fmr1 exon 12 variants display tissue-specific and developmental differences, while exon 15-containing transcripts vary less. Altogether, the spatio-temporal plasticity of FMR1 mRNA is consistent with complex RNA processing that is mis-regulated in fragile X syndrome.

Chemical and structural probing of the N-terminal residues encoded by FMR1 exon 15 and their effect on downstream arginine methylation.

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.

Translational regulation by non-protein-coding RNAs: different targets, common themes.

There are several classes of small non-protein-coding RNA (npcRNA) that play important roles in cellular metabolism including mRNA decoding, RNA processing and mRNA stability. Indeed, altered expression of some of these npcRNAs has been associated with cancer, neurodegenerative diseases such as Alzheimer's disease, as well as various types of mental retardation and psychiatric disorders. The basis of this correlation is currently not understood. However, recent studies have begun to shed light on one of the mechanism(s) by which these RNAs exert their effects, namely, translational control. These data provide hope that rational treatments for these varied disorders may be in sight. Here, we review this new body of work.

Protein methyltransferase activities in commercial in vitro translation systems.

Protein arginine methylation is a well-known post-translational modification that has been shown to occur in rabbit reticulocyte in vitro translation lysates (RRL); however, it is not known whether this is a general feature of in vitro-produced proteins from other eukaryotic cell-free translation systems, particularly insect-derived lysates (ICL). Because methylation can affect protein localization, RNA binding and protein-protein interactions this may be of great importance as in vitro-produced proteins are often used in assays of protein function. Here, I report the presence of base-stable and base-labile methyltransferase activities in RRL, ICL and wheat germ in vitro extracts (WGE). Indeed, the presence of CARM1 in RRL and ICL and a class II protein arginine methyltransferase activity (PRMT5/7) is documented in all three systems. Additionally, the lysine methyltransferase that modifies eukaryotic elongation factor 1A (eEF-1A) was detected in ICL and WGE. Importantly, using a defined set of substrates under identical conditions I show that all three in vitro systems contain different complements of the various methyltransferases. These data suggest that three systems can be used in a complementary fashion to investigate the effect(s) of post-translational modification on protein function.

Generation of polyclonal antiserum for the detection of methylarginine proteins.

This report describes an approach for the study of the biology of methylarginine proteins based on the generation of immunological reagents capable of recognizing the methylarginine status of cellular proteins. Two forms of an immunizing peptide were prepared based upon an amino acid sequence motif found most prevalently among verified dimethylarginine-containing proteins. One form of the peptide was constructed with 7 arginine residues alternating with 8 glycine residues. None of the arginines used in the synthesis were methylated. The alternative form of the peptide was synthesized with the identical repeating GRG sequence, but with asymmetrical dimethylarginine at each arginine residue. A methylarginine-specific antiserum was generated using the latter peptide. ELISA and western blotting of glycine arginine-rich peptides, each synthesized with or without asymmetric dimethylarginine, demonstrate the methyl specificity of the antiserum. The methylarginine-specific antibody co-localizes with the highly methylated native nucleolin protein conspicuously concentrated in the nucleolus. The methylarginine-specific antiserum recognizes a GRG peptide and bacterially expressed RBP16 only after incubation of the peptide or RBP16 with recombinant protein arginine methyltransferase 1, or cell extracts, respectively. Proteins isolated from cells in different developmental states exhibit different patterns of reactivity observed by western blots. Finally, the methylarginine-specific reagent interacts specifically with the methylarginine of cellular hnRNPA1 and human fragile X mental retardation protein expressed in cultured PC12 cells. An immunological reagent capable of detecting the methylarginine status of cellular methylproteins will facilitate the cellular and molecular analysis of protein arginine methylation in a wide variety of research and biomedical applications.

Oxidative stress reveals heterogeneity of FMRP granules in PC12 cell neurites.

PC12 cells are a well-known model of parasympathetic neurons. They have also been used to study the dynamics of heterologously expressed fragile X mental retardation (FMRP) granule trafficking down neurites. Here, we demonstrate that undifferentiated and differentiated PC12 cells harbor endogenous FMRP-containing granules. These granules are not stress granules because they do not associate with an authentic stress granule marker protein T-cell internal antigen 1 (TIA-1). Treatment with sodium arsenite induces stress granule formation in undifferentiated and differentiated PC12 cells. In NGF-treated cells, FMRP-containing stress granules are observed in the soma, neurites and growth cones by co-immunostaining with anti-TIA-1 antibody. These data demonstrate that all three microdomains respond similarly to oxidative stress. Nevertheless, we find significantly less co-localization of FMRP and TIA-1 and FMRP and its homologs in the neurites of differentiated PC12 cells treated with sodium arsenite than in the soma or growth cones. The heterogeneity of these granules suggests that FMRP has multiple roles in neurites.

GABAAergic stimulation modulates intracellular protein arginine methylation.

Changes in cytoplasmic pH are known to regulate diverse cellular processes and influence neuronal activities. In neurons, the intracellular alkalization is shown to occur after stimulating several channels and receptors. For example, it has previously demonstrated in P19 neurons that a sustained intracellular alkalinization can be mediated by the Na(+)/H(+) antiporter. In addition, the benzodiazepine binding subtypes of the γ-amino butyric acid type A (GABAA) receptor mediate a transient intracellular alkalinization when they are stimulated. Because the activities of many enzymes are sensitive to pH shift, here we investigate the effects of intracellular pH modulation resulted from stimulating GABAA receptor on the protein arginine methyltransferases (PRMT) activities. We show that the major benzodiazepine subtype (2α1, 2ß2, 1γ2) is constitutively expressed in both undifferentiated P19 cells and retinoic acid (RA) differentiated P19 neurons. Furthermore stimulation with diazepam and, diazepam plus muscimol produce an intracellular alkalinization that can be detected ex vivo with the fluorescence dye. The alkalinization results in significant perturbation in protein arginine methylation activity as measured in methylation assays with specific protein substrates. Altered protein arginine methylation is also observed when cells are treated with the GABAA agonist muscimol but not an antagonist, bicuculline. These data suggest that pH-dependent and pH-independent methylation pathways can be activated by GABAAergic stimulation, which we verified using hippocampal slice preparations from a mouse model of fragile X syndrome.

Introduction: reminiscing on models and modeling.

This chapter answers three basic questions, which are: (1) Why build models, (2) why build models of fragile X syndrome, and (3) what has been learned from the models of fragile X syndrome that have been made? The first question is used to frame the other two questions, providing the appropriate context by which the rest of the book should be examined. Of necessity the last two questions are only addressed briefly, and from one man's point of view, as they contain the subject matter of the entirety of the book. Thus, the reader is introduced to the various topics under review and urged to read for him/herself their contents, drawing such conclusions as he/she thinks are warranted.

Vignettes: models in absentia.

In this chapter, I will concisely summarize the salient features of all of the fragile X models (ex vivo, non-mouse, mouse, novel mouse, and human) that were not able to be described by their creators in separate chapters. By doing so, it is hoped that this book will become more of an encyclopedic compendium.

Conformational-dependent and independent RNA binding to the fragile x mental retardation protein.

The interaction between the fragile X mental retardation protein (FMRP) and BC1 RNA has been the subject of controversy. We probed the parameters of RNA binding to FMRP in several ways. Nondenaturing agarose gel analysis showed that BC1 RNA transcripts produced by in vitro transcription contain a population of conformers, which can be modulated by preannealing. Accordingly, FMRP differentially binds to the annealed and unannealed conformer populations. Using partial RNase digestion, we demonstrate that annealed BC1 RNA contains a unique conformer that FMRP likely binds. We further demonstrate that this interaction is 100-fold weaker than that the binding of eEF-1A mRNA and FMRP, and that preannealing is not a general requirement for FMRP's interaction with RNA. In addition, binding does not require the N-terminal 204 amino acids of FMRP, methylated arginine residues and can be recapitulated by both fragile X paralogs. Altogether, our data continue to support a model in which BC1 RNA functions independently of FMRP.

Protein methylation and stress granules: posttranslational remodeler or innocent bystander?

Stress granules contain a large number of post-translationally modified proteins, and studies have shown that these modifications serve as recruitment tags for specific proteins and even control the assembly and disassembly of the granules themselves. Work originating from our laboratory has focused on the role protein methylation plays in stress granule composition and function. We have demonstrated that both asymmetrically and symmetrically dimethylated proteins are core constituents of stress granules, and we have endeavored to understand when and how this occurs. Here we seek to integrate this data into a framework consisting of the currently known post-translational modifications affecting stress granules to produce a model of stress granule dynamics that, in turn, may serve as a benchmark for understanding and predicting how post-translational modifications regulate other granule types.

mRNPs take shape by CLIPPING and PAIRING.

The interaction of RNA-binding proteins (RBPs) with RNA is a crucial aspect of normal cellular metabolism. Yet, the diverse number of RBPs and RNA motifs to which they bind, the wide range of interaction strengths and the fact that RBPs associate in dynamic complexes have made it challenging to determine whether a particular RNA-binding protein binds a particular RNA. Recent work by three different laboratories has led to the development of new tools to query such interactions in the more physiological environs of cultured cells. The use of these methods has led to insights into (1) the networks of RNAs regulated by a particular protein, (2) the identification of new protein partners within messenger ribonucleoprotein particles and (3) the flux of RNA-binding proteins on an mRNA throughout its lifecycle. Here, I examine these new methods and discuss their relative strengths and current limitations.

Alternative splicing modulates protein arginine methyltransferase-dependent methylation of fragile X syndrome mental retardation protein.

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.

Methylation regulates the intracellular protein-protein and protein-RNA interactions of FMRP.

FMRP, the fragile X mental retardation protein, is an RNA-binding protein that interacts with approximately 4% of fetal brain mRNA. We have recently shown that a methyltransferase (MT) co-translationally methylates FMRP in vitro and that methylation modulates the ability of FMRP to bind mRNA. Here, we recapitulate these in vitro data in vivo, demonstrating that methylation of FMRP affects its ability to bind to FXR1P and regulate the translation of FMRP target mRNAs. Additionally, using double-label fluorescence confocal microscopy, we identified a subpopulation of FMRP-containing small cytoplasmic granules that are distinguishable from larger stress granules. Using the oxidative-stress induced accumulation of abortive pre-initiation complexes as a measure of the association of FMRP with translational components, we have demonstrated that FMRP associates with ribosomes during initiation and, more importantly, that methylation regulates this process by influencing the ratio of FMRP-homodimer-containing mRNPs to FMRP-FXR1P-heterodimer-containing mRNPs. These data suggest a vital role for methylation in normal FMRP functioning.

PAD: the smoking gun behind arginine methylation signaling?

Post-translational modifications (PTM) supply the proteome with functional and regulatory diversity. Modifications including phosphorylation, acetylation and methylation have been identified in eucaryotic proteins. For all but the last, corresponding "de-modifying" enzymes exist to remove the PTM tag returning the protein to its basal state. Recently, a novel mechanism in which peptidylarginine deiminase (PAD4) converts histone H3 and H4 methyl arginine residues into citrulline was proposed to regulate estrogen-responsive gene transcription.1,2 These data, the first to provide a mechanistic basis for the dynamic changes observed in a subset of protein arginine methylated substrates,3 lead to a host of questions concerning the generality of this mechanism for non-histone targets of the protein arginine methyltransferases (PRMTs).

Déjà vu all over again: FMRP binds U-rich target mRNAs.

The fragile X mental retardation protein (FMRP) contains three RNA binding domains, two of which the KH2 domain and the C-terminal arginine-glycine-rich (RG-rich) region participate in RNA binding. Because fragile X syndrome is the leading cause of inherited mental retardation, there has been an intensive search for the messenger RNA (mRNA) targets that interact with FMRP in vivo. Initial work led to the conclusion that FMRP binds to a nucleic acid tertiary structure element called a G-quartet. Recent studies have shown that FMRP also binds mRNAs containing U-pentameric sequences. Interestingly, both motifs are mimicked by homoribopolymers (poly (rG) and poly (rU)) that were first used to determine that FMRP functioned as an RNA binding protein. The consequences of these discoveries and future areas of investigation are discussed.