Edouard W. Khandjian

RNA-binding proteins in human genetic disease

RNA-binding proteins (RBPs) are key components in RNA metabolism, regulating the temporal, spatial and functional dynamics of RNAs. Altering the expression of RBPs has profound implications for cellular physiology, affecting RNA processes from pre-mRNA splicing to protein translation. Recent genetic and proteomic data and evidence from animal models reveal that RBPs are involved in many human diseases ranging from neurologic disorders to cancer. Here we review the emerging evidence showing the involvement of RBPs in many disease networks and conclude that defects in RNA metabolism caused by aberrations in RBPs might underlie a broader spectrum of complex human disorders.

A novel function for fragile X mental retardation protein in translational activation.

Fragile X syndrome, the most frequent form of inherited mental retardation, is due to the absence of Fragile X Mental Retardation Protein (FMRP), an RNA-binding protein involved in several steps of RNA metabolism. To date, two RNA motifs have been found to mediate FMRP/RNA interaction, the G-quartet and the “kissing complex,” which both induce translational repression in the presence of FMRP. We show here a new role for FMRP as a positive modulator of translation. FMRP specifically binds Superoxide Dismutase 1 (Sod1) mRNA with high affinity through a novel RNA motif, SoSLIP (Sod1 mRNA Stem Loops Interacting with FMRP), which is folded as three independent stem-loop structures. FMRP induces a structural modification of the SoSLIP motif upon its interaction with it. SoSLIP also behaves as a translational activator whose action is potentiated by the interaction with FMRP. The absence of FMRP results in decreased expression of Sod1. Because it has been observed that brain metabolism of FMR1 null mice is more sensitive to oxidative stress, we propose that the deregulation of Sod1 expression may be at the basis of several traits of the physiopathology of the Fragile X syndrome, such as anxiety, sleep troubles, and autism.

Dicer-Derived MicroRNAs Are Utilized by the Fragile X Mental Retardation Protein for Assembly on Target RNAs

In mammalian cells, fragile X mental retardation protein (FMRP) has been reported to be part of a microRNA (miRNA)-containing effector ribonucleoprotien (RNP) complex believed to mediate translational control of specific mRNAs. Here, using recombinant proteins, we demonstrate that human FMRP can act as a miRNA acceptor protein for the ribonuclease Dicer and facilitate the assembly of miRNAs on specific target RNA sequences. The miRNA assembler property of FMRP was abrogated upon deletion of its single-stranded (ss) RNA binding K-homology domains. The requirement of FMRP for efficient RNA interference (RNAi) in vivo was unveiled by reporter gene silencing assays using various small RNA inducers, which also supports its involvement in an ss small interfering RNA (siRNA)-containing RNP (siRNP) effector complex in mammalian cells. Our results define a possible role for FMRP in RNA silencing and may provide further insight into the molecular defects in patients with the fragile X syndrome.

The fragile X syndrome: exploring its molecular basis and seeking a treatment

Fragile X syndrome (FXS) - the leading cause of inherited mental retardation - is an X-linked disease caused by loss of expression of the FMR1 (fragile X mental retardation 1) gene. In addition to impairment of higher-cognitive functions, FXS patients show a variety of physical and other mental abnormalities. FMRP, the protein encoded by the FMR1 gene, is thought to play a key role in translation, trafficking and targeting of mRNA in neurons. To better understand FMRPs functions, the protein partners and mRNA targets that interact with FMRP have been sought. These and functional studies have revealed links with processes such as cytoskeleton remodelling via the RhoGTPase pathway and mRNA processing via the RNA interference pathway. In this review, we focus on recent insights into the function of FMRP and speculate on how the absence of FMRP might cause the clinical phenotypes seen in FXS patients. Finally, we explore potential therapies for FXS.

Disordered RNA chaperone proteins: from functions to disease

Abstract.RNA chaperones are ubiquitous proteins that play pivotal roles in cellular RNA metabolism and RNA virus replication. Here we propose that they act by organizing complex and highly dynamic networks of RNA-RNA, RNA-protein and protein-protein interactions. How this is achieved and how their malfunction may lead to disease will be discussed through the examples of human immunodeficiency virus type 1 nucleocapsid protein (NCp7), the fragile X mental retardation protein and the prion protein.

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 FMRPs 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.

A novel function for the survival motoneuron protein as a translational regulator

SMN1, the causative gene for spinal muscular atrophy (SMA), plays a housekeeping role in the biogenesis of small nuclear RNA ribonucleoproteins. SMN is also present in granular foci along axonal projections of motoneurons, which are the predominant cell type affected in the pathology. These so-called RNA granules mediate the transport of specific mRNAs along neurites and regulate mRNA localization, stability, as well as local translation. Recent work has provided evidence suggesting that SMN may participate in the assembly of RNA granules, but beyond that, the precise nature of its role within these structures remains unclear. Here, we demonstrate that SMN associates with polyribosomes and can repress translation in an in vitro translation system. We further identify the arginine methyltransferase CARM1 as an mRNA that is regulated at the translational level by SMN and find that CARM1 is abnormally up-regulated in spinal cord tissue from SMA mice and in severe type I SMA patient cells. We have previously characterized a novel regulatory pathway in motoneurons involving the SMN-interacting RNA-binding protein HuD and CARM1. Thus, our results suggest the existence of a potential negative feedback loop in this pathway. Importantly, an SMA-causing mutation in the Tudor domain of SMN completely abolished translational repression, a strong indication for the functional significance of this novel SMN activity in the pathology.

Fragile X related protein 1 isoforms differentially modulate the affinity of fragile X mental retardation protein for G-quartet RNA structure

Fragile X syndrome, the most frequent form of inherited mental retardation, is due to the absence of expression of the Fragile X Mental Retardation Protein (FMRP), an RNA binding protein with high specificity for G-quartet RNA structure. FMRP is involved in several steps of mRNA metabolism: nucleocytoplasmic trafficking, translational control and transport along dendrites in neurons. Fragile X Related Protein 1 (FXR1P), a homologue and interactor of FMRP, has been postulated to have a function similar to FMRP, leading to the hypothesis that it can compensate for the absence of FMRP in Fragile X patients. Here we analyze the ability of three isoforms of FXR1P, expressed in different tissues, to bind G-quartet RNA structure specifically. Only the longest FXR1P isoform was found to be able to bind specifically the G-quartet RNA, albeit with a lower affinity as compared to FMRP, whereas the other two isoforms negatively regulate the affinity of FMRP for G-quartet RNA. This result is important to decipher the molecular basis of fragile X syndrome, through the understanding of FMRP action in the context of its multimolecular complex in different tissues. In addition, we show that the action of FXR1P is synergistic rather than compensatory for FMRP function.

Expression of dopamine D1-receptor mRNA in the Carotid body of adult rabbits, cats and rats

Dopamine is a major neurotransmitter in the carotid body of several animal species and its functional role at the level of peripheral arterial chemoreflex pathway is attributed to the presence of the dopamine D2-receptors. We present evidence that the dopamine D1-receptor mRNA is also expressed in the carotid body of adult rabbits, cats and rats. A DNA fragment of 611 bp of this receptor was first isolated from rabbit. The nucleic acid sequence of this fragment was found to be 84.5% identical to that of rat. This specific 611 bp fragment was used as a probe to detect, either by Northern analysis or by the reverse transcription-polymerase chain reaction, the dopamine D1-receptor mRNA. The results revealed the presence of dopamine D1-receptor transcript in the carotid body as well as in the petrosal ganglion and the superior cervical ganglion from the three animal models studied here. The physiological significance of dopamine D1-receptor expression in the carotid body is discussed.

Muscle specific fragile X related protein 1 isoforms are sequestered in the nucleus of undifferentiated myoblast.

BackgroundThe family of Fragile X Mental Retardation Proteins is composed of three members: Fragile Mental Retardation 1, Fragile X Related 1 and X Related 2 proteins. These proteins are associated with mRNPs within translating ribosomes and have the capacity to shuttle between the nucleus and the cytoplasm. Great attention has been given to FMRP due to its implication in human hereditary mental retardation while FXR1P and FXR2P have only recently been studied.ResultsUsing antibodies directed against several epitopes of FXR1P, we have detected protein isoforms generated by small peptides pocket inserts. Four isoforms of MW 70, 74, 78, 80 kDa are widely distributed in mouse organs, while in striated muscles these isoforms are replaced by proteins of 82 and 84 kDa containing an extra pocket of 27 aa. Expression of these muscle isoforms is an early event during in vitro differentiation of myoblasts into myotubes and correlates with the activation of muscle-specific genes. However, while FXR1P82,84 are associated with cytoplasmic mRNPs in myotubes, they are sequestered in the nuclei of undifferentiated myoblasts. These observations suggest that, in addition to a cytoplasmic function yet to be defined, FXR1P82,84 may play a nuclear role in pre-mRNA metabolism.ConclusionsThe pattern of subcellular partitioning of FXR1P isoforms during myogenesis is unique among the family of the FXR proteins. The model system described here should be considered as a powerful tool for ongoing attempts to unravel structure-function relationships of the different FMR family members since the potential role(s) of FXR1P as a compensatory factor in Fragile X syndrome is still elusive.