Drosophila has multiple ELAV family members

The sequences of the two Drosophila proteins, ELAV and RBP9, are similarly related to each of the four classes of human ELAV sequences (52% - 54% amino acid identity in the case of ELAV and 56% - 61% amino acid identity for RBP9). On the basis of their strict neuronal nuclear localization, it can be inferred that they are more related to the ELAV-C or -D types, although ELAV is singled out by its systematic and exclusive neuronal expression. Together, the different subcellular localizations and the diversity of in vitro binding specificities underline differences in the function of ELAV-A, -B, -C, and -D proteins, but the strong structural conservation indicates the possibility that all mediate the same general function in RNA metabolism, possibly triggering differentiation of specific cell types, both for the exclusively neuronal and the ubiquitous members of the family (Samson, 1998 and references).

ELAV homologs in yeast

The stability of several oncogene, cytokine, and growth factor transcripts is tightly regulated by signaling pathways through an ARE (AU-rich element) present in their 3'-UTRs. A yeast transcript, TIF51A, has been identified whose stability is regulated through its AU-rich 3'-UTR. The mammalian TNFalpha and c-fos AREs regulate turnover of a reporter yeast transcript in a similar manner. AREs stabilize the transcript in glucose media and function as destabilizing elements in media lacking glucose or when the Hog1p/p38 MAP kinase pathway is inhibited. Significantly, both yeast and mammalian AREs promote deadenylation-dependent decapping in the yeast system. Furthermore, the yeast ELAV homolog, Pub1p, regulates the stability mediated by the TNFalpha ARE. These results demonstrate that yeast possess a regulatable mechanism for ARE-mediated decay and suggest conservation of this turnover process from yeast to humans (Vasudevan, 2001).

C. elegans ELAV family proteins

The exc mutations of C. 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 organizmal osmolarity. The exc-7 gene, the closest nematode homolog to the neural RNA-binding protein ELAV, has been cloned. 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. 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 small ßH-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 (Fujita, 2003).

The increased defects in canal structure seen when exc-7 is mutated in a sma-1 null background indicate that the loss of EXC-7 function must affect other genes besides sma-1. Since exc-7 mutations also show similar synergistic effects with exc-3 mutations, EXC-7 may also affect expression of this (as yet uncloned) gene. Since the observed defects of exc-7 mutation are relatively mild, and the gene is expressed in many cells besides the excretory cell, EXC-7 likely affects expression of additional genes, as do the vertebrate ELAVs. Further studies on other genes causing synergistic effects with ELAV homolog mutations may identify other genes needed in large amounts during specific stages of C. elegans development that are regulated by EXC-7 or other of the nine nematode ELAV homologs (Fujita, 2003).

While there is evidence that distinct protein isoforms resulting from alternative pre-mRNA splicing play critical roles in neuronal development and function, little is known about molecules regulating alternative splicing in the nervous system. Using C. elegans as a model for studying neuron/target communication, this study reports that unc-75 mutant animals display neuroanatomical and behavioral defects indicative of a role in modulating GABAergic and cholinergic neurotransmission but not neuronal development. unc-75 encodes an RRM domain-containing RNA binding protein that is exclusively expressed in the nervous system and neurosecretory gland cells. UNC-75 protein, as well as a subset of related C. elegans RRM proteins, localizes to dynamic nuclear speckles; this localization pattern supports a role for the protein in pre-mRNA splicing. Human orthologs of UNC-75, whose splicing activity has recently been documented in vitro, are expressed nearly exclusively in brain and when expressed in C. elegans, rescue unc-75 mutant phenotypes and localize to subnuclear puncta. Furthermore, the subnuclear-localized EXC-7 protein, the C. elegans ortholog of the neuron-restricted Drosophila ELAV splicing factor, acts in parallel to UNC-75 to also affect cholinergic synaptic transmission. In conclusion, a new neuronal, putative pre-mRNA splicing factor, UNC-75, has been identified and it has been shown that UNC-75, as well as the C. elegans homolog of ELAV, are each required for the fine tuning of synaptic transmission. These findings thus provide a novel molecular link between pre-mRNA splicing and presynaptic function (Loria, 2003).

The vertebrate orthologs of UNC-75, CELF3/BrunoL1, CELF4/BrunoL4, and CELF5/BrunoL5, have been shown to be involved in splicing in an in vitro assay. To investigate whether the function of these proteins is conserved (a notion that was expected from the level of primary sequence similarity), UNC-75 and its human orthologs were compared in more detail. mRNA samples derived from a variety of different human tissues were hybridized with probes specific to three of the four human orthologs. CELF3/BrunoL1, CELF4/BrunoL4, and CELF5/BrunoL5 each show highly similar expression patterns that are largely restricted to the nervous system. Within the nervous system, every region tested shows expression of CELF3/BrunoL1, CELF4/BrunoL4, and CELF5/BrunoL5. Thus, the pan-neuronal expression of the human orthologs of UNC-75 mirrors the pan-neuronal expression of C. elegans UNC-75 (Loria, 2003).

Functional comparison of UNC-75 and EXC-7 proteins was extended by analyzing exc-7 null mutant animals. In contrast to fly Elav, which severely affects neuronal development and viability, exc-7 mutants are viable and show no locomotory or defecation defects. Also in contrast to fly Elav, exc-7 is only expressed in a subset of neurons in the nervous system, several of which are cholinergic neurons. The development and morphology of several cholinergic neuron classes were assessed by using cell-specific gfp markers; no obvious defects were found. Moreover, synaptic vesicles in the cholinergic SAB neurons cluster normally in exc-7 null mutants, leading to the conclusion that EXC-7 has no significant impact on neuronal development. However, when cholinergic motorneuron function was tested in more detail, it was found that exc-7(rh252) animals show a synaptic transmission defect similar to unc-75 (Loria, 2003).

The relation of the ric phenotype of unc-75 and exc-7 was assessed. If these two genes act in a similar process, their null phenotypes should not enhance one another. It was found, however, that the synaptic transmission defect of the double mutant is significantly enhanced compared to the single mutants. Moreover, although exc-7 mutant animals show no locomotory defects on their own, unc-75; exc-7 double mutant animals are smaller and appear significantly more uncoordinated than unc-75 single mutants. Lastly, it was found that the ric phenotype of exc-7 null mutants is not rescued by an elevation in ambient temperature. This lack of temperature sensitivity is similar to that of unc-17 mutants, which are affected in synaptic vesicle loading. It is concluded that unc-75 and exc-7 have nonredundant and distinct roles in cholinergic synaptic transmission and likely regulate the pre-mRNA splicing of a distinct set of target genes (Loria, 2003).

Identification and characterization of ELAV family members

Hel-N1, a human neural RNA-binding protein, shares significant homology with Drosophila ELAV. Hel-N1 binds in vitro to 3' untranslated regions of mRNAs, encoding c-myc, c-fos, g/mcsf, and the transcriptional repressor, Id (Gao, 1994). Other elav homologs have been discovered in fish, frogs and mammals. Some are neural specific, while others are found ubiquitously (Good, 1995).

The Hu antigens are composed of a family of neuronal-specific, RNA-binding proteins encoded by at least three distinct genes. All three gene products, HuD, HuC/ple21, and Hel-N1, are human homologs of Elav. Although the three proteins are very similar in structure, they are differentiated by alternative splicing of their mRNAs. The Hu antigens bind avidly to the AU-rich element resident in many mRNAs that regulate cell proliferation. This interaction suggests that the Hu antigens promote neuronal differentiation by suppressing the neuroblast cell cycle. Such a mechanism provides a plausible model for the role of the Hu antigens in tumorigenesis, neuronal differentiation, and paraneoplastic neurologic disorders (Liu, 1995).

A Xenopus laevis elav homolog, Xel-1, like the other elav-related genes, encodes a putative RNA-binding protein that contains three RNA Recognition Motifs and is solely expressed in the nervous system. Xel-1 is most likely the Xenopus homologue of Hel-N1, one of the three known human genes related to elav. Xel-1 is not expressed in early neural precursors but rather in differentiating neurons of the central nervous system, as well as in the cranial and the spinal ganglion cells. Xel-1 thus appears to be an early differentiation marker for both the central and the peripheral nervous system of Xenopus (Perron, 1995).

The translational activation of several maternal mRNAs in Xenopus laevis is dependent on cytoplasmic poly(A) elongation. Messages harboring the UUUUUAU-type cytoplasmic polyadenylation element (CPE) in their 3' untranslated regions (UTRs) undergo polyadenylation and translation during oocyte maturation. This CPE is bound by the protein CPEB, which is essential for polyadenylation. mRNAs that have the poly(U)12-27 embryonic-type CPE (eCPE) in their 3' UTRs undergo polyadenylation and translation during the early cleavage and blastula stages. A 36-kDa eCPE-binding protein in oocytes and embryos has been identified by UV cross-linking. This 36-kDa protein is ElrA, a member of the ELAV family of RNA-binding proteins. The proteins are identical in size; antibody directed against ElrA immunoprecipitates the 36-kDa protein, and the two proteins have the same RNA binding specificity in vitro. C12 and activin receptor mRNAs, both of which contain eCPEs, are detected in immunoprecipitated ElrA-mRNP complexes from eggs and embryos. In addition, this in vivo interaction requires the eCPE. Although a number of experiments have failed to define a role for ElrA in cytoplasmic polyadenylation, the expression of a dominant negative ElrA protein in embryos results in an exogastrulation phenotype (Wu, 1997).

Drosophila and vertebrate elav/Hu genes are involved in the development and the maintenance of the nervous system. They all encode proteins that contain three RNA recognition motifs (RRM) and are thus expected to play a role in RNA metabolism. Drosophila ELAV and RBP9 proteins have been reported to be exclusively distributed in the nuclei of neurons, whereas known human Hu proteins display a bipartite nuclear and cytoplasmic distribution. A member of this family has been isolated from Xenopus. Xel-1 is exclusively expressed in neural tissues from the early tailbud stage onward. The subcellular distribution of exogenous XEL-1 protein in neural tissues differs with developmental stage. In the neural tube at the neurula stage, where endogenous Xel-1 is not expressed, exogenous tagged XEL-1 protein is localized in both the nucleus and the cytoplasm. At the tailbud stage, where endogenous Xel-1 is expressed, exogenous tagged XEL-1 protein is localized essentially in the cytoplasm of neural tube cells. In contrast, exogenous Drosophila ELAV protein localizes to the nucleus at all stages in Xenopus embryos. The variability in the subcellular localization of ELAV/Hu proteins in different species may have functional implications (Perron, 1997).

Three chicken Hu/elav family RNA-binding protein genes have been identified. cHuD and cHuC are expressed specifically in neurons of both the central and peripheral nervous systems. Although cHuA is expressed in a wide variety of tissues, including neurogenic precursor cells, it is transiently down-regulated, and is then re-expressed in maturing neurons. Misexpression of cHuD in cultured neural crest cells results in a dramatic increase in the proportion of cells exhibiting neuronal morphology, molecular markers for neurons, and neurotrophin dependence. All Hu proteins can bind the AU-rich element of 3'UTR of transcription factors and cytokines, such as c-fos, c-myc, Id, GM-CSF, and others. Hu proteins may bind to mRNA products and post-transcriptionally modulate the expression of genes that regulate proliferation, differentiation and survival. In the present studies, no significant effect was observed on cell proliferation by misexpression of Hu proteins, at least not those in cultured neural crest cells. However, ectopic expression of cHuD in such cells results in elevated apoptosis. These data confirm that cHuD protein is involved in regulating neuronal differentiation (Wakamatsu, 1997).

Three new zebrafish Elav/Hu homologs (HuA, HuD and HuG) have been isolated and characterized. While HuA and HuG showed weak and ubiquitous expressions, HuD, as well as HuC, are specifically expressed in the neuronal cells. The first expression of HuD is detectable at the 10-somite stage, that is, several hours later than HuC. After 24 h of embryonic development, although HuD and HuC expressions overlap overall, the cells expressing HuD are restricted to subsets of the HuC-positive neuronal cells in the brain and spinal cord. These differentially regulated spatial and temporal expression patterns imply distinct roles for HuC and HuD in neuronal determination and neuronal differentiation, respectively (H. C. Park, 2000).

mHuA (Elavl1) belongs to a highly conserved family of genes encoding RNA-binding proteins and has been linked to cell growth and proliferation through its regulation of mRNA stability. An RNase protection assay has been used to demonstrate that the mHuA transcript is relatively abundant in a range of mouse tissues, with the highest levels being found in lung and embryonic stem cells. An 18 kb DNA fragment has been cloned and mapped that encompasses the 5' end of the mHuA gene. The genomic organization in this region is similar to the neural-restricted family members, Hel-N1 (ElavL2) and mHuD (Elavl4). The first exon is lengthy and untranslated, and the second exon, which includes the methionine start site, ends between the ribonucleoprotein motifs of the first RNA binding domain. Mapping of the mHuA transcript by primer extension demonstrates three potential transcription-initiation sites that are detected consistently among different tissues and cell lines. Analysis of the sequence flanking these sites reveals the presence of transcriptional elements including TATA, CREB, c-ets, and AP1 sites. Transfection analysis of this promoter region using a luciferase-reporter-gene assay indicates strong transcriptional activity both in HeLa and in mouse macrophage (RAW) cells; this is consistent with the ubiquitous expression pattern of mHuA. Thus, while the genomic organization of mHuA is similar to the neural-restricted members of the Elav family, the promoter element differs substantially both by sequence analysis and transcriptional activity in non-neural cell types (King, 2000b).

The chordate central nervous system has been hypothesized to originate from either a dorsal centralized, or a ventral centralized, or a noncentralized nervous system of a deuterostome ancestor. In an effort to resolve these issues, the hemichordate Saccoglossus kowalevskii was examined and the expression of orthologs of genes that are involved in patterning the chordate central nervous system was examined. All 22 orthologs studied are expressed in the ectoderm in an anteroposterior arrangement nearly identical to that found in chordates. Domain topography is conserved between hemichordates and chordates despite the fact that hemichordates have a diffuse nerve net, whereas chordates have a centralized system. It is proposed that the deuterostome ancestor may have had a diffuse nervous system, which was later centralized during the evolution of the chordate lineage (Lowe, 2003).

The adult S. kowalevskii has tripartite, tricoelomic organization. At the anterior is the muscular proboscis or prosome, used for burrowing and collecting food particles. It contains the heart, kidney, a section of the dorsal nerve cord, and the protocoel. The middle region, which is the collar or mesosome, contains the mouth, a section of dorsal nerve cord formed by neurulation, the paired mesocoels, and the base of the stomochord, which projects forward into the prosome. The posterior region or metasome contains the gill slits, the remainder of the dorsal nerve cord, the entire ventral nerve cord, paired metacoels, gonads, a long through-gut, and terminal anus. At juvenile stages, a ventral post-anal extension (called a tail or sucker) is present (Lowe, 2003).

Gastrulation entails uniform and simultaneous inpocketing of the vegetal half of the hollow blastula. As the blastopore closes, a gumdrop-shaped gastrula is formed. As the embryo lengthens, two circumferential grooves indent and divide the length into prosome, mesosome, and metasome regions. Mesodermal coeloms outpouch from the gut anteriorly and laterally. The first gill slit pair appears externally by day 5, and the animal bends from the dorsal side. The hatched juvenile elongates and adds further pairs of gill slits successively. The animal is nearly bilaterally symmetric, except that the prosome excretory pore (the proboscis pore) from the kidney is reliably on the left, defining a left-right asymmetry (Lowe, 2003).

The hemichordate adult nervous system is not centralized but is a diffuse intraepidermal, basiepithelial nerve net. Nerve cells are interspersed with epidermal cells and account for 50% or more of the cells in the proboscis and collar ectoderm and a lower percentage in the metasome. Axons form a meshwork at the basal side of the epidermis. The two nerve cords are through-conduction tracts of bundled axons and are not enriched for neurogenesis. This general organizational feature of the nervous system has been largely underemphasized in recent literature that focuses on possible homologies between chordate and hemichordate nerve cords (Lowe, 2003).

Although neurons are dispersed throughout the epidermis in the adult, it has not been demonstrated that neurogenesis in the embryo is uniform. To determine the site of neurogenesis, the domains of expression were localized for three orthologs of pan-neural genes of chordates and Drosophila -- nrp/ musashi, sox1/2/3/ soxneuro, and hu/elav. The first two are markers of proliferating neuron precursors, whereas the third is a marker of differentiating neurons. All are expressed in the neural plate of various chordates, but not in the epidermis. nrp/musashi and sox1/2/3 /soxneuro are expressed in the entire ectoderm of the early S. kowalevskii embryo (except for the ciliated band, which all probes except emx fail to stain). In later stages, the expression remains strong in the prosome and declines in the metasome, correlating with Bullock's observation of decreasing neuron density posteriorly. In sections, weak expression of nrp/musashi can be detected in the posterior endoderm, possibly correlated with a sparse endodermal nerve net. Hu/elav exhibits similar diffuse staining throughout the ectoderm in early stages. Additionally, Hu/elav staining remains strong along the posterior dorsal midline at later stages, in a punctate pattern perhaps reflecting a concentration of early-differentiating nerves at this site. In sagittal sections of embryos, hu/elav expression appears localized toward the basal side of the ectoderm (basiepithelial); it is absent from the mesoderm. Thus, S. kowalevskii shows pervasive neurogenesis with no large, contiguous nonneurogenic subregion, as occurs in chordates (Lowe, 2003).

HuD expression and localization

Mammalian ELAV proteins bind to polyadenylated messenger RNAs and have specificity for AU-rich sequences. Preferred binding sites in vitro include the AUUUA pentamer and related sequences present in the 3' untranslated regions of many growth regulatory mRNAs. Human ELAV (hELAV) proteins have been implicated in post-transcriptional regulation of gene expression by their effects on the stability and translatability of growth regulatory mRNAs. The intracellular localization of ELAV proteins has been examined in neurons and in tumor cells of neuronal origin using indirect immunofluorescence, confocal microscopy and biochemical separation. Mammalian neuronal ELAV proteins are found predominantly in the cytoplasm of cells in mRNP complexes termed alpha complexes which, when associated with polysomes, form large and high density beta complexes. Puromycin, cytochalasin or EDTA treatments disrupt beta complexes causing the release of alpha complexes, which then appear, by confocal microscopy, as large hELAV mRNP granules associated with microtubules. Association of partially purified hELAV mRNP alpha complexes with microtubules was confirmed by in vitro reconstitution assays. Colchicine treatment of cells suggests that association of hELAV mRNP alpha complexes with microtubules is also necessary for the formation of beta complexes. These data suggest a model in which a subset of mRNAs is associated with microtubules as ELAV mRNP particles (alpha complexes) that, in turn, associate with polysomes to form a translational apparatus (beta complex) through polysomes, associated with the microfilament cytoskeletal network. hELAV proteins in these mRNP granules may affect post-transcriptional regulation of gene expression via the intracellular transport, localization and/or translation of growth regulatory mRNAs (Antic, 1998).

The expression of mRNA for the neuronal antigen HuD (Elavl4) associated with paraneoplastic encephalomyelitis and sensory neuronopathy was evaluated in the developing and adult rat nervous system. Using RNase protection assay and non-radioactive in situ hybridization histochemistry, HuD is found to be expressed at high levels at the earliest time point observed (E15), but declines significantly during the first postnatal week to levels that are maintained into adulthood. In the adult, HuD expression becames restricted primarily to large pyramidal-like neurons. Exceptions of note are many smaller neurons within a variety of thalamic nuclei. Expression of HuD coincides with terminal differentiation of all neuronal structures evaluated, regardless of the timing of their development, providing correlative evidence for a role in neuronal differentiation or the maintenance of neuronal phenotype. The marked restriction of HuD mRNA expression with maturity suggests that its functional role in adult neurons varies significantly throughout the CNS (Clayton, 1998).

ELAV proteins are implicated in regulating the stability and translation of cytokine and growth regulatory mRNAs such as GM-CSF, IL-2, c-myc, c-fos and GLUT1 by binding to their AU-rich 3'UTRs. The tissue-specific ELAV protein HuB (aka. Hel-N1) is predominantly cytoplasmic and has been shown to stabilize GLUT1 and c-myc mRNAs and to increase their translation following ectopic expression in 3T3-L1 cells. The most widely expressed mouse ELAV protein, mHuA, is predominately nuclear in cultured NIH-3T3 cells, but is localized in the cytoplasm during early G1 of the cell cycle. Therefore, much like the primarily cytoplasmic HuB, HuA becomes temporally localized in the cytoplasm where it can potentially regulate the stability or translation of bound mRNAs. Stimulation of mouse spleen cells using either mitogenic or sub-mitogenic levels of anti-CD3/CD28 results in a dramatic increase in the level of HuA. Upregulation of HuA corresponds to previously documented increases in cytokine expression, which are due to increased mRNA stability following T cell activation. Consistent with these findings, HuA is down regulated in quiescent cells and upregulated in 3T3 cells following serum stimulation. The increase of murine HuA during the cell cycle closely resembles that of cyclin B1 which peaks in G2/M. These data indicate that mammalian ELAV proteins function during cell growth and differentiation due in part to their effects on posttranscriptional stability and translation of multiple growth regulatory mRNAs. This supports the hypothesis that ELAV proteins can function as transacting factors that affect a default pathway of mRNA degradation involved in the expression of growth regulatory proteins (Atasoy, 1998).

HuD and neurogenesis

Hu genes encode a large number of alternatively spliced transcripts to produce a series of related neuron-specific RNA binding proteins. Despite this complexity, several ordered features of Hu expression have been discerned. In the embryo, specific Hu genes are expressed in a hierarchy during early neurogenesis. In the E16 developing cortex, mHuB is induced in very early postmitotic neurons exiting the ventricular zone; mHuD is expressed in migrating neurons of the intermediate zone, and mHuC is expressed in mature cortical plate neurons. Such a hierarchy suggests distinct functional roles for each gene in developing neurons. In the adult, all neurons express some set of Hu mRNA and protein. However, specific patterns are evident such that individual neuronal types in the hippocampus, cerebellum, olfactory cortex, neocortex, and elsewhere express from one to several Hu genes. The complexity of potential protein variants within a gene family and of different Hu family members within a neuron suggests a diverse array of function. Given the strong homologies among the Hu proteins, the Drosophila neurogenic gene elav, and the Drosophila splicing factor Sxl, it is predicted that different combinations of Hu proteins determine different neuron-specific aspects of post-transcriptional RNA regulation. The findings of specific developmental patterns of expression and the correlation between immune targeting of the Hu proteins and adult neurodegenerative disease suggest that the Hu proteins are critical in both the proper development and function of mature neurons (Okano. 1997)

In the postembryonic zebrafish forebrain, subpial locations of neurogenesis do exist in the early cerebellar external granular layer, and -- unusually among vertebrates -- in the primordial pretectal (M1) and preglomerular (M2) anlagen as shown with BrdU/Hu-immunocytochemistry and in situ hybridization of neuroD. Hu is a neuronal protein expressed in proliferating neurogenic cells. An intermediate BrdU incubation time of 12-16 h reveals, in addition to proliferative ventricularly located cells, those in M1 and M2. This BrdU saturation-labeling shows, in conjunction with a Hu-assay demonstrating earliest neuronal differentiation, that proliferating cells in M1 and M2 represent neuronal progenitors. This is demonstrated by single BrdU-labeled and double BrdU-/Hu-labeled cells in these aggregates. Further, expression of NeuroD, a marker for freshly determined neuronal cells, confirms this unusual subpial postembryonic forebrain neurogenesis (Mueller, 2002).

HuD mutants

Hu proteins are mammalian embryonic lethal abnormal visual system (ELAV)-like neuronal RNA-binding proteins that contain three RNA recognition motifs. Although Drosophila Elav is required for the correct differentiation and survival of neurons, the roles played by the Hu genes in the mammalian nervous system remain largely unknown. To explore the in vivo functions of mouse Hu proteins, they were overexpressed in rat pheochromocytoma PC12 cells, where they induce a neuronal phenotype in the absence of nerve growth factor. The functions of various forms of mHuB and mHuC bearing point mutations or deletions have been characterized. Mutants of mHuC lose biologic activity as well as RNA-binding activity if amino acid exchanges take place in the RNP1 domain of the first or second RNA recognition motifs (RRMs). In addition, the mutants containing only the third RRM fail to induce the neuronal phenotype in PC12 cells and inhibit the biologic activity of cotransfected wild-type mHuB and mHuC, thus acting as a dominant-negative form. However, these mutants can not suppress the nerve growth factor-induced differentiation of PC12 cells. Wild-type and dominant-negative Hu were misexpressed in E9.5 mouse embryos, by using electroporation into the neural tube at the level of the rhombencephalon. mHuB and mHuC induce the ectopic expression of neuronal markers, whereas the dominant-negative forms of mHuB and mHuC suppress the differentiation of central nervous system motor neurons. It has been suggested that Hu proteins are required for neuronal differentiation in the mammalian nervous system (Akamatsu, 1999).

HuD forms a multimeric complex

Hu proteins are RNA-binding proteins that are the vertebrate homologs of Drosophila ELAV, and are implicated in stabilization or enhanced translation of specific mRNAs with AU-rich elements (AREs) in the 3'-untranslated region. Using the yeast two-hybrid system, it has been shown that neuron-specific Hu proteins can interact with themselves. Immuno precipitation assays demonstrate that the interaction between Hu proteins occurs in mammalian cells and is strongly enhanced in the presence of cellular RNA. Furthermore, using in situ chemical crosslinking assays, it was found that HuD, one of the neuron-specific Hu proteins, multimerizes in cells. The crosslinked HuD multimers retain specific RNA-binding ability and can interact with additional Hu proteins. Consistent with this biochemical property, HuD showed granular distribution in two neurogenic cell lines. These results suggest that the RNA-bound form of HuD multimerizes cooperatively to form a specific granular structure that may serve as a site of post-transcriptional regulation of ARE-containing mRNAs (Kasashima, 2003).

Characterization of the interaction between HuD and AU-rich RNA

Human neuron-specific RNA-binding protein HuD belongs to the family of Hu proteins and consists of two N-terminal RNA recognition motifs (RRM1 and -2), a hinge region, and a C-terminal RRM (RRM3). Hu proteins can bind to AU-rich elements in the 3' untranslated regions of unstable mRNAs, causing the stabilization of certain transcripts. The interaction between HuD and prototype mRNA instability elements of the sequence UU(AUUU)(n)AUU were studied using equilibrium methods and real-time kinetics. A single molecule of HuD requires at least three AUUU repeats to bind tightly to the RNA. Deletion of RRM1 reduced the K(d) by 2 orders of magnitude and caused a decrease in the association rate and a strong increase in the dissociation rate of the RNA-protein complex, as expected when a critical RNA-binding domain is removed. In contrast, deletion of either RRM2 or -3, which only moderately reduces the affinity, causes marked increases in the association and dissociation rates. The slower binding and stabilization of the complex observed in the presence of all three RRMs suggest that a change in the tertiary structure occurs during binding. The individual RRMs bind poorly to the RNA (RRM1 binds with micromolar affinity, while the affinities of RRM2 and -3 are in the millimolar range). However, the combination of RRM1 and either RRM2 or RRM3 in the context of the protein allows binding with a nanomolar affinity. Thus, the three RRMs appear to cooperate not only to increase the affinity of the interaction but also to stabilize the formed complex. Kinetic effects, similar to those described in this study, could play a role in RNA binding by many multi-RRM proteins and may influence the competition between proteins for RNA-binding sites and the ability of RNA-bound proteins to be transported intracellularly (S. Park, 2000).

Hu proteins have been shown to bind to AU-rich elements (AREs) in the 3'-untranslated region of unstable mRNAs. They can thereby inhibit the decay of labile transcripts by antagonizing destabilizing proteins that target these AU-rich sequences. The sequence preferences of HuD were examined to elucidate the possible role of HuD in counteracting mRNA decay. Using repeats of the prototype destabilizing sequence UU(AUUU)nAUU, it has been shown that all three HuD RNA-binding domains participate in binding to AU-tracts that can be as short as 13 residues, depending on the position of the remaining As. Removal of the A residues, resulting in a poly(U)-tract, increase the affinity of HuD for RNA, suggesting that the presence of As in destabilizing elements might favor the recruitment of other proteins and/or prevent HuD from binding too tightly to AREs. In vitro selection experiments with randomized RNAs confirm the preference of HuD for poly(U). RNA binding analysis of the related protein HuB shows a similar preference for poly(U). In contrast, tristetraprolin, an mRNA destabilizing protein, strongly prefers AU-rich RNA. Many labile mRNAs contain U-tracts in or near their AREs. Individual AREs may thus differentially affect mRNA half-life by recruiting a unique complement of stabilizing and destabilizing factors (Park-Lee, 2003).

ELAV homologs target 3' UTRs of mRNAs

Continued: ELAV Evolutionary homologs part 2/2

elav: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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