RNA-binding Protein ELAV Research Articles

Alterations of RNA-binding protein found in neurons in Drosophila neurons and glia influence synaptic transmission and lifespan.

The found in neurons (fne), a paralog of the RNA-binding protein ELAV gene family in Drosophila, is required for post-transcriptional regulation of neuronal development and differentiation. Previous explorations into the functions of the FNE protein have been limited to neurons. The function of fne in Drosophila glia remains unclear. We induced the knockdown or overexpression of fne in Drosophila neurons and glia to determine how fne affects different types of behaviors, neuronal transmission and the lifespan. Our data indicate that changes in fne expression impair associative learning, thermal nociception, and phototransduction. Examination of synaptic transmission at presynaptic and postsynaptic terminals of the larval neuromuscular junction (NMJ) revealed that loss of fne in motor neurons and glia significantly decreased excitatory junction currents (EJCs) and quantal content, while flies with glial fne knockdown facilitated short-term synaptic plasticity. In muscle cells, overexpression of fne reduced both EJC and quantal content and increased short-term synaptic facilitation. In both genders, the lifespan could be extended by the knockdown of fne in neurons and glia; the overexpression of fne shortened the lifespan. Our results demonstrate that disturbances of fne in neurons and glia influence the function of the Drosophila nervous system. Further explorations into the physiological and molecular mechanisms underlying neuronal and glial fne and elucidation of how fne affects neuronal activity may clarify certain brain functions.

A circular RNA derived from FAT atypical cadherin 3 promotes lung cancer progression via forming a regulatory loop with oncogenic ELAV like RNA binding protein 1

Circular RNA (circRNA) is a covalently closed endogenous RNA that participates in disease progression. However, its role in lung cancer is largely undetermined. In the present study, we found an onctogenic circRNA in lung cancer, FAT atypical cadherin 3 (FAT3) circRNA (circ-FAT3) was remarkably upregulated in lung cancer in comparison to paired normal tissues. High circ-FAT3 was closely linked to larger tumour size, lymph node metastasis, later clinical stage, as well as dismal outcome. Stable knockdown of circ-FAT3 inhibited cell proliferation and metastasis both in vitro and in vivo. RNA binding protein ELAV like RNA binding protein 1 (HuR) was found to bind to introns flanking circ-FAT3, promoting the cyclization and generation of circ-FAT3. Further, circ-FAT3 was able to sponge miR-136-5p by acting as a competing endogenous RNA (ceRNA), alleviating the repressive effect of miR-136-5p on HuR mRNA at the transcriptional and post-transcriptional levels. Moreover, circ-FAT3 expression in lung cancer tissues was strongly positively and negatively correlated with HuR and miR-136-5p expression, respectively. Overall, our data reveal the previously uncharacterized regulatory loop of circ-FAT3/miR-136-5p/HuR in lung cancer and provide novel evidence for the importance of circRNA as a ceRNA in tumorigenesis.

Correction: Neural specificity of the RNA-binding protein Elav is achieved by post-transcriptional repression in non-neural tissues.

There were errors published in Development 143, 4474-4485.In Figs 1A-D and 2A,B,H,I,J, the +/+ labels should have been +/–.Owing to production errors, the wrong images were included as Figs 2B′, 3B,B′ and 5C′.The online version of the article has been corrected accordingly. These changes have no impact on the results or the conclusions of the study.We apologise to the authors and readers for this mistake.

Neural specificity of the RNA-binding protein Elav is achieved by post-transcriptional repression in non-neural tissues.

Drosophila Elav is the founding member of the conserved family of Hu RNA-binding proteins (RBPs), which play crucial and diverse roles in post-transcriptional regulation. Elav has long served as the canonical neuronal marker. Surprisingly, although Elav has a well-characterized neural cis-regulatory module, we find endogenous Elav is also ubiquitously transcribed and post-transcriptionally repressed in non-neural settings. Mutant clones of multiple miRNA pathway components derepress ubiquitous Elav protein. Our re-annotation of the elav transcription unit shows not only that it generates extended 3' UTR isoforms, but also that its universal 3' UTR isoform is much longer than previously believed. This longer common 3' UTR includes multiple conserved, high-affinity sites for the miR-279/996 family. Of several miRNA mutants tested, endogenous Elav and a transgenic elav 3' UTR sensor are derepressed in mutant clones of mir-279/996 We also observe cross-repression of Elav by Mei-P26, another RBP derepressed in non-neural miRNA pathway clones. Ubiquitous Elav has regulatory capacity, since derepressed Elav can stabilize an Elav-responsive sensor. Repression of Elav in non-neural territories is crucial as misexpression here has profoundly adverse consequences. Altogether, we define unexpected post-transcriptional mechanisms that direct appropriate cell type-specific expression of a conserved neural RBP.

Alternative polyadenylation coupled to transcription initiation: Insights from ELAV-mediated 3′ UTR extension

Transcription initiation and mRNA maturation were long considered co-occurring but separately regulated events of gene control. In the past decade, gene promoters, the platforms of transcription initiation, have been assigned additional functions such as the regulation of splicing and 3′ end processing. In a recent study, Oktaba and Zhang and al. reveal that neural 3′ UTR extension is dependent on promoter sequences. In Drosophila neurons, promoter regions of a subset of genes recruit the RNA-binding protein ELAV, which is required for subsequent ELAV-mediated alternative polyadenylation. Intriguingly, RNA Polymerase II pausing at promoters seems to facilitate ELAV recruitment. How transcription initiation and alternative polyadenylation, processes separated by an entire gene length, are functionally linked, remains unsolved. In this article, I summarize recent findings and discuss possible mechanisms.

A novel SDS-stable dimer of a heterogeneous nuclear ribonucleoprotein at presynaptic terminals of squid neurons

The presence of mRNAs in synaptic terminals and their regulated translation are important factors in neuronal communication and plasticity. Heterogeneous nuclear ribonucleoprotein (hnRNP) complexes are involved in the translocation, stability, and subcellular localization of mRNA and the regulation of its translation. Defects in these processes and mutations in components of the hnRNP complexes have been related to the formation of cytoplasmic inclusion bodies and neurodegenerative diseases. Despite much data on mRNA localization and evidence for protein synthesis, as well as the presence of translation machinery, in axons and presynaptic terminals, the identity of RNA-binding proteins involved in RNA transport and function in presynaptic regions is lacking. We previously characterized a strongly basic RNA-binding protein (p65), member of the hnRNPA/B subfamily, in squid presynaptic terminals. Intriguingly, in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE), p65 migrated as a 65-kDa protein, whereas members of the hnRNPA/B family typically have molecular masses ranging from 35 to 42kDa. In this report we present further biochemical and molecular characterization that shows endogenous p65 to be an SDS-stable dimer composed of ∼37-kDa hnRNPA/B-like subunits. We cloned and expressed a recombinant protein corresponding to squid hnRNPA/B-like protein and showed its propensity to aggregate and form SDS-stable dimers in vitro. Our data suggest that this unique hnRNPA/B-like protein co-localizes with synaptic vesicle protein 2 and RNA-binding protein ELAV and thus may serve as a link between local mRNA processing and presynaptic function and regulation.

Convergent evolution of neural systems in ctenophores.

Neurons are defined as polarized secretory cells specializing in directional propagation of electrical signals leading to the release of extracellular messengers - features that enable them to transmit information, primarily chemical in nature, beyond their immediate neighbors without affecting all intervening cells en route. Multiple origins of neurons and synapses from different classes of ancestral secretory cells might have occurred more than once during ~600 million years of animal evolution with independent events of nervous system centralization from a common bilaterian/cnidarian ancestor without the bona fide central nervous system. Ctenophores, or comb jellies, represent an example of extensive parallel evolution in neural systems. First, recent genome analyses place ctenophores as a sister group to other animals. Second, ctenophores have a smaller complement of pan-animal genes controlling canonical neurogenic, synaptic, muscle and immune systems, and developmental pathways than most other metazoans. However, comb jellies are carnivorous marine animals with a complex neuromuscular organization and sophisticated patterns of behavior. To sustain these functions, they have evolved a number of unique molecular innovations supporting the hypothesis of massive homoplasies in the organization of integrative and locomotory systems. Third, many bilaterian/cnidarian neuron-specific genes and 'classical' neurotransmitter pathways are either absent or, if present, not expressed in ctenophore neurons (e.g. the bilaterian/cnidarian neurotransmitter, γ-amino butyric acid or GABA, is localized in muscles and presumed bilaterian neuron-specific RNA-binding protein Elav is found in non-neuronal cells). Finally, metabolomic and pharmacological data failed to detect either the presence or any physiological action of serotonin, dopamine, noradrenaline, adrenaline, octopamine, acetylcholine or histamine - consistent with the hypothesis that ctenophore neural systems evolved independently from those in other animals. Glutamate and a diverse range of secretory peptides are first candidates for ctenophore neurotransmitters. Nevertheless, it is expected that other classes of signal and neurogenic molecules would be discovered in ctenophores as the next step to decipher one of the most distinct types of neural organization in the animal kingdom.

RNA metabolism. Promoters drive alternative polyadenylation.

The RNA-binding protein Elav is recruited in the fruit fly to promoter regions, possibly by paused RNA polymerase II, to promote alternative polyadenylation.

ELAV Links Paused Pol II to Alternative Polyadenylation in the Drosophila Nervous System

Alternative polyadenylation (APA) has been implicated in a variety of developmental and disease processes. A particularly dramatic form of APA occurs inthe developing nervous system of flies and mammals, whereby various developmental genes undergo coordinate 3' UTR extension. In Drosophila, the RNA-binding protein ELAV inhibits RNA processing at proximal polyadenylation sites, thereby fostering the formation of exceptionally long 3' UTRs. Here, we present evidence that paused Pol II promotes recruitment of ELAV to extended genes. Replacing promoters of extended genes with heterologous promoters blocks normal 3' extension in the nervous system, while extension-associated promoters can induce 3' extension in ectopic tissues expressing ELAV. Computational analyses suggest that promoter regions of extended genes tend to contain paused Pol II and associated cis-regulatory elements such as GAGA. ChIP-seq assays identify ELAV in the promoter regions of extended genes. Our study provides evidence for a regulatory link between promoter-proximal pausing and APA.

The RNA-binding protein ELAV regulates Hox RNA processing, expression and function within the Drosophila nervous system.

The regulated head-to-tail expression of Hox genes provides a coordinate system for the activation of specific programmes of cell differentiation according to axial level. Recent work indicates that Hox expression can be regulated via RNA processing but the underlying mechanisms and biological significance of this form of regulation remain poorly understood. Here we explore these issues within the developing Drosophila central nervous system (CNS). We show that the pan-neural RNA-binding protein (RBP) ELAV (Hu antigen) regulates the RNA processing patterns of the Hox gene Ultrabithorax (Ubx) within the embryonic CNS. Using a combination of biochemical, genetic and imaging approaches we demonstrate that ELAV binds to discrete elements within Ubx RNAs and that its genetic removal reduces Ubx protein expression in the CNS leading to the respecification of cellular subroutines under Ubx control, thus defining for the first time a specific cellular role of ELAV within the developing CNS. Artificial provision of ELAV in glial cells (a cell type that lacks ELAV) promotes Ubx expression, suggesting that ELAV-dependent regulation might contribute to cell type-specific Hox expression patterns within the CNS. Finally, we note that expression of abdominal A and Abdominal B is reduced in elav mutant embryos, whereas other Hox genes (Antennapedia) are not affected. Based on these results and the evolutionary conservation of ELAV and Hox genes we propose that the modulation of Hox RNA processing by ELAV serves to adapt the morphogenesis of the CNS to axial level by regulating Hox expression and consequently activating local programmes of neural differentiation.

Erect wing regulates synaptic growth in Drosophila by integration of multiple signaling pathways.

BackgroundFormation of synaptic connections is a dynamic and highly regulated process. Little is known about the gene networks that regulate synaptic growth and how they balance stimulatory and restrictive signals.ResultsHere we show that the neuronally expressed transcription factor gene erect wing (ewg) is a major target of the RNA binding protein ELAV and that EWG restricts synaptic growth at neuromuscular junctions. Using a functional genomics approach we demonstrate that EWG acts primarily through increasing mRNA levels of genes involved in transcriptional and post-transcriptional regulation of gene expression, while genes at the end of the regulatory expression hierarchy (effector genes) represent only a minor portion, indicating an extensive regulatory network. Among EWG-regulated genes are components of Wingless and Notch signaling pathways. In a clonal analysis we demonstrate that EWG genetically interacts with Wingless and Notch, and also with TGF-β and AP-1 pathways in the regulation of synaptic growth.ConclusionOur results show that EWG restricts synaptic growth by integrating multiple cellular signaling pathways into an extensive regulatory gene expression network.

The Drosophila RNA-binding protein ELAV is required for commissural axon midline crossing via control of commissureless mRNA expression in neurons

Drosophila ELAV is the founding member of an evolutionarily conserved family of RNA-binding proteins considered as key inducers of neuronal differentiation. Although several ELAV-specific targets have been identified, little is known about the role of elav during neural development. Here, we report a detailed characterization of the elav mutant commissural phenotype. The reduced number of commissures in elav mutant embryos is not due to loss or misspecification of neural cells but results from defects in commissural axon projections across the midline. We establish a causal relationship between the elav mutant commissural phenotype and a reduction in the expression of commissureless, a key component of the Robo/Slit growth cone repulsive signalling pathway. In the nerve cord of elav mutant embryos, comm mRNA expression is strongly reduced in neurons, but not in midline glial cells. Furthermore, specific expression of an elav transgene in posterior neurons of each segment of an elav mutant nerve cord restores comm mRNA expression in these cells, as well as the formation of posterior commissures. Finally, forced expression of comm in specific commissural neuron subsets rescues the midline crossing defects of these neurons in elav mutant embryos, further indicating that elav acts cell autonomously on comm expression.

The Drosophila Fragile X Mental Retardation Protein Controls Actin Dynamics by Directly Regulating Profilin in the Brain

Loss of Fragile X mental retardation protein (FMRP) function causes the highly prevalent Fragile X syndrome [1 and 2]. Identifying targets for the RNA binding FMRP is a major challenge and an important goal of research into the pathology of the disease. Perturbations in neuronal development and circadian behavior are seen in Drosophila dfmr1 mutants. Here we show that regulation of the actin cytoskeleton is under dFMRP control. dFMRP binds the mRNA of the Drosophila profilin homolog and negatively regulates Profilin protein expression. An increase in Profilin mimics the phenotype of dfmr1 mutants. Conversely, decreasing Profilin levels suppresses dfmr1 phenotypes. These data place a new emphasis on actin misregulation as a major problem in fmr1 mutant neurons.

The role of the ELAV homologue EXC-7 in the developmentof the Caenorhabditis elegans excretory canals

The exc mutations of Caenorhabditis elegans alter the position and shape of the apical cytoskeleton in polarized epithelial cells. Mutants in exc-7 form small cysts throughout the tubular excretory canals that regulate organismal osmolarity. We have cloned the exc-7 gene, the closest nematode homologue to the neural RNA-binding protein ELAV. EXC-7 is expressed in the canal for a short time midway through embryogenesis. Cysts in exc-7 mutants do not develop until several hours later, beginning at the time of hatching. We find that the first larval period is when the canal completes the majority of its outgrowth, and adds new apical cytoskeleton at a rapid rate. Ultrastructural studies show that exc-7 mutant defects resemble loss of β H-spectrin (encoded by sma-1) at the distal ends of the excretory canals. In addition, exc-7 mutants exhibit synergistic excretory canal defects with mutations in sma-1, and EXC-7 binds sma-1 mRNA. These data imply that EXC-7 protein may affect expression of sma-1 and other genes to effect proper development of the excretory canals.

The neuron-specific RNA-binding protein ELAV regulates neuroglian alternative splicing in neurons and binds directly to its pre-mRNA.

Drosophila melanogaster neural-specific protein, ELAV, has been shown to regulate the neural-specific splicing of three genes: neuroglian (nrg), erect wing, and armadillo. Alternative splicing of the nrg transcript involves alternative inclusion of a 3'-terminal exon. Here, using a minigene reporter, we show that the nrg alternatively spliced intron (nASI) has all the determinants required to recreate proper neural-specific RNA processing seen with the endogenous nrg transcript, including regulation by ELAV. An in vitro UV cross-linking assay revealed that ELAV from nuclear extracts cross-links to four distinct sites along the 3200 nucleotide long nASI; one EXS is positioned at the polypyrimidine tract of the default 3' splice site. ELAV cross-linking sites (EXSs) have in common long tracts of (U)-rich sequence rather than a precise consensus; moreover, each tract has at least two 8/10U elements; their importance is validated by mutant transgene reporter analysis. Further, we propose criteria for ELAV target sequence recognition based on the four EXSs, sites within the nASI that are (U) rich but do not cross-link with ELAV, and predicted EXSs from a phylogenetic comparison with Drosophila virilis nASI. These results suggest that ELAV regulates nrg alternative splicing by direct interaction with the nASI.

Expression of Neural RNA-Binding Proteins in the Postnatal CNS: Implications of Their Roles in Neuronal and Glial Cell Development

There is an increasing interest in the role of RNA-binding proteins during neural development. Mouse-Musashi-1 (m-Msi-1) is a mouse neural RNA-binding protein with sequence similarity to Drosophila musashi (d-msi), which is essential for neural development. m-Msi-1 is highly enriched in neural precursor cells that are capable of generating both neurons and glia during embryonic CNS development. The present study characterized m-Msi-1-expressing cells in the postnatal and adult CNS. Postnatally, m-Msi-1 was expressed in proliferative neuronal precursors in the external granule cell layer of the cerebellum and in the anterior corner of the subventricular zone of the lateral ventricles. In gliogenesis, the persistent expression of m-Msi-1 was observed in cells of the astrocyte lineage ranging from proliferative glial precursors in the subventricular zone (SVZ) to differentiated astrocytes in the parenchyma. In addition, we showed that m-Msi-1 was still expressed in proliferating cells in the adult SVZ, which may contain neural precursor or stem cells. Another neural RNA-binding protein Hu (the mammalian homolog of a Drosophila neuronal RNA-binding protein Elav) was present in postmitotic neurons throughout the development of the CNS, and its pattern of expression was compared with that of m-Msi-1. These observations imply that these two RNA-binding proteins may be involved in the development of neurons and glia by regulating gene expression at the post-transcriptional level.

Mouse-Musashi-1, a Neural RNA-Binding Protein Highly Enriched in the Mammalian CNS Stem Cell

There is increasing interest in the role of RNA-binding proteins during neural development.DrosophilaMusashi is one of the neural RNA-binding proteins essential for neural development and required for asymmetric cell divisions in theDrosophilaadult sensory organ development. Here, a novel mammalian neural RNA-binding protein, mouse-Musashi-1, was identified based on the homology toDrosophilaMusashi andXenopusNRP-1. In the developing CNS, mouse-Musashi-1 protein was highly enriched in the CNS stem cell. Single-cell culture experiments indicated that mouse-Musashi-1 expression is associated with neural precursor cells that are capable of generating neurons and glia. In contrast, in fully differentiated neuronal and glial cells mouse-Musashi-1 expression is lost. This expression pattern of mouse-Musashi-1 is complementary to that of another mammalian neural RNA-binding protein, Hu (a mammalian homologue of aDrosophilaneuronal RNA-binding protein Elav), that is expressed in postmitotic neurons within the CNS.In vitrostudies indicated that mouse-Musashi-1 possesses binding preferences on poly(G) RNA homopolymer, whereas Hu is known to preferentially bind to short A/U-rich regions in RNA. Based on their differential expression patterns and distinct preferential target RNA sequences, we believe that the mouse-Musashi-1 and Hu proteins may play distinct roles in neurogenesis, either through sequential regulatory mechanisms or differential sorting of mRNA populations during asymmetric division of neural precursor cells.