Hervé Moine

The fragile X mental retardation protein binds specifically to its mRNA via a purine quartet motif

Fragile X syndrome is caused by the absence of protein FMRP, the function of which is still poorly understood. Previous studies have suggested that FMRP may be involved in various aspects of mRNA metabolism, including transport, stability and/or translatability. FMRP was shown to interact with a subset of brain mRNAs as well as with its own mRNA; however, no specific RNA‐binding site could be identified precisely. Here, we report the identification and characterization of a specific and high affinity binding site for FMRP in the RGG‐coding region of its own mRNA. This site contains a purine quartet motif that is essential for FMRP binding and can be substituted by a heterologous quartet‐forming motif. The specific binding of FMRP to its target site was confirmed further in a reticulocyte lysate through its ability to repress translation of a reporter gene harboring the RNA target site in the 5′‐untranslated region. Our data address interesting questions concerning the role of FMRP in the post‐transcriptional control of its own gene and possibly other target genes.

G‐quadruplexes in RNA biology

G‐quadruplexes are noncanonical structures formed by G‐rich DNA and RNA sequences that fold into a four‐stranded conformation. Experimental studies and computational predictions show that RNA G‐quadruplexes are present in transcripts associated with telomeres, in noncoding sequences of primary transcripts and within mature transcripts. RNA G‐quadruplexes at these specific locations play important roles in key cellular functions, including telomere homeostasis and gene expression. Indeed, RNA G‐quadruplexes appear as important regulators of pre‐mRNA processing (splicing and polyadenylation), RNA turnover, mRNA targeting and translation. The regulatory mechanisms controlled by RNA G‐quadruplexes involve the binding of protein factors that modulate G‐quadruplex conformation and/or serve as a bridge to recruit additional protein regulators. In this review, we summarize the current knowledge on the role of G‐quadruplexes in RNA biology with particular emphasis on the molecular mechanisms underlying their specific function in RNA metabolism occurring in physiological or pathological conditions. WIREs RNA 2012, 3:495–507. doi: 10.1002/wrna.1113

A Single Internal Ribosome Entry Site Containing a G Quartet RNA Structure Drives Fibroblast Growth Factor 2 Gene Expression at Four Alternative Translation Initiation Codons

The 484-nucleotide (nt) alternatively translated region (ATR) of the human fibroblast growth factor 2 (FGF-2) mRNA contains four CUG and one AUG translation initiation codons. Although the 5′-end proximal CUG codon is initiated by a cap-dependent translation process, the other four initiation codons are initiated by a mechanism of internal entry of ribosomes. We undertook here a detailed analysis of the cis-acting elements defining the FGF-2 internal ribosome entry site (IRES). A thorough deletion analysis study within the 5′-ATR led us to define a 176-nt region as being necessary and sufficient for IRES function at four codons present in a downstream 308-nt RNA segment. Unexpectedly, a single IRES module is therefore responsible for translation initiation at four distantly localized codons. The determination of the FGF-2 5′-ATR RNA secondary structure by enzymatic and chemical probing experiments showed that the FGF-2 IRES contained two stem-loop regions and a G quartet motif that constitute novel structural determinants of IRES function.

Sequestration of DROSHA and DGCR8 by expanded CGG RNA Repeats Alters microRNA processing in fragile X-associated tremor/ataxia syndrome

Fragile X-associated tremor/ataxia syndrome (FXTAS) is an inherited neurodegenerative disorder caused by the expansion of 55-200 CGG repeats in the 5 UTR of FMR1. These expanded CGG repeats are transcribed and accumulate in nuclear RNA aggregates that sequester one or more RNA-binding proteins, thus impairing their functions. Here, we have identified that the double-stranded RNA-binding protein DGCR8 binds to expanded CGG repeats, resulting in the partial sequestration of DGCR8 and its partner, DROSHA, within CGG RNA aggregates. Consequently, the processing of microRNAs (miRNAs) is reduced, resulting in decreased levels of mature miRNAs in neuronal cells expressing expanded CGG repeats and in brain tissue from patients with FXTAS. Finally, overexpression of DGCR8 rescues the neuronal cell death induced by expression of expanded CGG repeats. These results support a model in which a human neurodegenerative disease originates from the alteration, in trans, of the miRNA-processing machinery.

G-quadruplex RNA structure as a signal for neurite mRNA targeting.

Targeting of messenger RNAs (mRNAs) in neuron processes relies on cis‐acting regulatory elements, the nature of which is poorly understood. Here, we report that approximately 30% of the best‐known dendritic mRNAs contain a guanine (G)–quadruplex consensus in their 3′‐untranslated region. Among these mRNAs, we show by using RNA structure probing that a G–quadruplex is present in the mRNAs of two key postsynaptic proteins: PSD‐95 and CaMKIIa. The G–quadruplex structure is necessary and sufficient for the potent and fast localization of mRNAs in cortical neurites and this occurs in a metabotropic glutamate receptor‐responsive manner. Thus, G–quadruplex seems to be a common neurite localization signal.

In Vitro and in Cellulo Evidences for Association of the Survival of Motor Neuron Complex with the Fragile X Mental Retardation Protein

Spinal muscular atrophy (SMA) is caused by reduced levels of the survival of motor neuron (SMN) protein. Although the SMN complex is essential for assembly of spliceosomal U small nuclear RNPs, it is still not understood why reduced levels of the SMN protein specifically cause motor neuron degeneration. SMN was recently proposed to have specific functions in mRNA transport and translation regulation in neuronal processes. The defective protein in Fragile X mental retardation syndrome (FMRP) also plays a role in transport of mRNPs and in their translation. Therefore, we examined possible relationships of SMN with FMRP. We observed granules containing both transiently expressed red fluorescent protein(RFP)-tagged SMN and green fluorescent protein(GFP)-tagged FMRP in cell bodies and processes of rat primary neurons of hypothalamus in culture. By immunoprecipitation experiments, we detected an association of FMRP with the SMN complex in human neuroblastoma SH-SY5Y cells and in murine motor neuron MN-1 cells. Then, by in vitro experiments, we demonstrated that the SMN protein is essential for this association. We showed that the COOH-terminal region of FMRP, as well as the conserved YG box and the region encoded by exon 7 of SMN, are required for the interaction. Our findings suggest a link between the SMN complex and FMRP in neuronal cells.

Internal Ribosome Entry Site Structural Motifs Conserved among Mammalian Fibroblast Growth Factor 1 Alternatively Spliced mRNAs

ABSTRACT Fibroblast growth factor 1 (FGF-1) is a powerful angiogenic factor whose gene structure contains four promoters, giving rise to a process of alternative splicing resulting in four mRNAs with alternative 5′ untranslated regions (5′ UTRs). Here we have identified, by using double luciferase bicistronic vectors, the presence of internal ribosome entry sites (IRESs) in the human FGF-1 5′ UTRs, particularly in leaders A and C, with distinct activities in mammalian cells. DNA electrotransfer in mouse muscle revealed that the IRES present in the FGF-1 leader A has a high activity in vivo. We have developed a new regulatable TET OFF bicistronic system, which allowed us to rule out the possibility of any cryptic promoter in the FGF-1 leaders. FGF-1 IRESs A and C, which were mapped in fragments of 118 and 103 nucleotides, respectively, are flexible in regard to the position of the initiation codon, making them interesting from a biotechnological point of view. Furthermore, we show that FGF-1 IRESs A of murine and human origins show similar IRES activity profiles. Enzymatic and chemical probing of the FGF-1 IRES A RNA revealed a structural domain conserved among mammals at both the nucleotide sequence and RNA structure levels. The functional role of this structural motif has been demonstrated by point mutagenesis, including compensatory mutations. These data favor an important role of IRESs in the control of FGF-1 expression and provide a new IRES structural motif that could help IRES prediction in 5′ UTR databases.

Escherichia coli threonyl-tRNA synthetase and tRNAThr modulate the binding of the ribosome to the translational initiation site of the ThrS mRNA

Escherichia coli threonyl-tRNA synthetase binds to the leader region of its own mRNA at two major sites: the first shares some analogy with the anticodon arm of several tRNA(Thr) isoacceptors and the second corresponds to a stable stem-loop structure upstream from the first one. The binding of the enzyme to its mRNA target site represses its translation by preventing the ribosome from binding to its attachment site. The enzyme is still able to bind to derepressed mRNA mutants resulting from single substitutions in the anticodon-like arm. This binding is restricted to the stem-loop structure of the second site. However, the interaction of the enzyme with this site fails to occlude ribosome binding. tRNA(Thr) is able to displace the wild-type mRNA from the enzyme at both sites and suppresses the inhibitory effect of the synthetase on the formation of the translational initiation complex. Our results show that tRNA(Thr) acts as an antirepressor on the synthesis of its cognate aminoacyl-tRNA synthetase. This repression/derepression double control allows precise adjustment of the rate of synthesis of threonyl-tRNA synthetase to the tRNA level in the cell.

The expression of E.coli threonyl-tRNA synthetase is regulated at the translational level by symmetrical operator-repressor interactions.

Threonyl‐tRNA synthetase from Escherichia coli represses the translation of its own mRNA by binding to the operator region located upstream from the ribosome binding site. The operator contains two stemloop structures which interact specifically with the homodimeric enzyme. Here, we provide in vitro and in vivo evidence that these two stem‐loop structures are recognized by the enzyme in an analogous way and mimic the anticodon arm of E.coli tRNA(Thr). Determination of the stoichiometry of the different RNA‐threonyl‐tRNA synthetase complexes reveals that two tRNA(Thr) molecules bind to the enzyme whereas only one thrS operator interacts with the homodimeric enzyme. A model is presented in which the two anticodon‐like domains of the operator bind symmetrically to the two tRNA(Thr) anticodon recognition sites (one per subunit) of the dimeric threonyl‐tRNA synthetase. Although symmetrical operator‐repressor interactions in transcriptional control are widespread, this report stresses the importance of such interactions in translational regulation of gene expression.

A novel role for the RNA-binding protein FXR1P in myoblasts cell-cycle progression by modulating p21/Cdkn1a/Cip1/Waf1 mRNA stability.

The Fragile X-Related 1 gene (FXR1) is a paralog of the Fragile X Mental Retardation 1 gene (FMR1), whose absence causes the Fragile X syndrome, the most common form of inherited intellectual disability. FXR1P plays an important role in normal muscle development, and its absence causes muscular abnormalities in mice, frog, and zebrafish. Seven alternatively spliced FXR1 transcripts have been identified and two of them are skeletal muscle-specific. A reduction of these isoforms is found in myoblasts from Facio-Scapulo Humeral Dystrophy (FSHD) patients. FXR1P is an RNA–binding protein involved in translational control; however, so far, no mRNA target of FXR1P has been linked to the drastic muscular phenotypes caused by its absence. In this study, gene expression profiling of C2C12 myoblasts reveals that transcripts involved in cell cycle and muscular development pathways are modulated by Fxr1-depletion. We observed an increase of p21—a regulator of cell-cycle progression—in Fxr1-knocked-down mouse C2C12 and FSHD human myoblasts. Rescue of this molecular phenotype is possible by re-expressing human FXR1P in Fxr1-depleted C2C12 cells. FXR1P muscle-specific isoforms bind p21 mRNA via direct interaction with a conserved G-quadruplex located in its 3′ untranslated region. The FXR1P/G-quadruplex complex reduces the half-life of p21 mRNA. In the absence of FXR1P, the upregulation of p21 mRNA determines the elevated level of its protein product that affects cell-cycle progression inducing a premature cell-cycle exit and generating a pool of cells blocked at G0. Our study describes a novel role of FXR1P that has crucial implications for the understanding of its role during myogenesis and muscle development, since we show here that in its absence a reduced number of myoblasts will be available for muscle formation/regeneration, shedding new light into the pathophysiology of FSHD.