InteractiveFly: GeneBrief

found in neurons : Biological Overview | References

BIOLOGICAL OVERVIEW

ELAV/Hu factors are conserved RNA binding proteins (RBPs) that play diverse roles in mRNA processing and regulation. The founding member, Drosophila Elav, was recognized as a vital neural factor 35 years ago. Nevertheless, little was known about its impacts on the transcriptome, and potential functional overlap with its paralogs. Building on recent findings that neural-specific lengthened 3' UTR isoforms are co-determined by ELAV/Hu factors, this study addressed their impacts on splicing. While only a few splicing targets of Drosophila are known, ectopic expression of each of the three family members (Fne and Rbp9) alters hundreds of cassette exon and alternative last exon (ALE) splicing choices. Reciprocally, double mutants of elav/fne, but not elav alone, exhibit opposite effects on both classes of regulated mRNA processing events in larval CNS. While manipulation of Drosophila ELAV/Hu RBPs induces both exon skipping and inclusion, characteristic ELAV/Hu motifs are enriched only within introns flanking exons that are suppressed by ELAV/Hu factors. Moreover, the roles of ELAV/Hu factors in global promotion of distal ALE splicing are mechanistically linked to terminal 3' UTR extensions in neurons, since both processes involve bypass of proximal polyadenylation signals linked to ELAV/Hu motifs downstream of cleavage sites. This study corroborates the direct action of Elav in diverse modes of mRNA processing using RRM-dependent Elav-CLIP data from S2 cells. Finally, evidence is provided for conservation in mammalian neurons, which undergo broad programs of distal ALE and APA lengthening, linked to ELAV/Hu motifs downstream of regulated polyadenylation sites. Overall, ELAV/Hu RBPs orchestrate multiple broad programs of neuronal mRNA processing and isoform diversification in Drosophila and mammalian neurons (Lee, 2021).

Mammalian ELAV/Hu RBPs have been extensively connected to alternative splicing of cassette exons, but only to selected alternative polyadenylation (APA) events. In contrast, only a handful of Drosophila genes were known to be alternative splicing targets of Elav, of which only two loci (Dscam1 and arm) harbor regulated cassette exons. Thus, it was unclear to what extent there are conserved utilities of this RBP family in mRNA processing (Lee, 2021).

This study shows that all three ELAV/Hu members specifying hundreds of alternative splicing events. We show endogenous relevance, by demonstrating that dual deletion of elav and fne causes reciprocal changes to splice isoform accumulation. Notably, this study revealed the endogenous breadth of splicing control by ELAV/Hu RBPs by analyzing dissected larval CNS, which contains more mature neurons than embryos and also removes the expression of non-neuronal isoforms outside of the nervous system from consideration. In particular, elav null L1-CNS has only mild effects on alternative splicing, despite its lethality, and analysis of fne nulls showed no effects on specific targets. Thus, the combined activity of ELAV/Hu RBPs, likely involving a hierarchial suppression of Fne nuclear localization via exon-exclusion of fne splicing by Elav, is critical to broadly determine neuronal mRNA isoforms (Lee, 2021).

Until now, evidence for roles of Rbp9 in mRNA processing is based on ectopic expression. Even though Drosophila ELAV/Hu RBPs exhibit distinct subcellular preferences, all of them exhibit similar binding capacities in vitro, and have overlapping regulatory capacities in ectopic assays. Since triple mutant larvae of Drosophila ELAV/Hu members could not be attained, it was not possible to assay nervous system devoid of this RBP family. This may require creative conditional genetics to achieve the requisite conditions, especially in pupal and/or adult stages, when Rbp9 is expressed at much higher levels in the nervous system (Lee, 2021).

Substantial differences were observed in the flanking intronic content of exon classes that are regulated ELAV/Hu RBPs. Their exclusion targets are substantially enriched for characteristic U-rich ELAV/Hu binding motifs, and have elevated Elav-CLIP signal, but such features were not observed with their inclusion targets. In general, little is known of the mechanism of splicing control by ELAV/Hu RBPs. In mammals, exclusion of a Fas cassette exon by HuR was reported to involve competition with U2AF65 at the upstream 3' splice site. A competition model is potentially consistent with the fly data, since substantially higher density of ELAV/Hu RBP motifs was observed upstream of excluded exons. However, this study also observe enrichment of ELAV/Hu RBP motifs downstream, although to a lesser extent. For exons that are preferentially included in the presence of ELAV/Hu members, they might still depend on binding that is below the sensitivity of these analyses. Another possibility is that these exons might involve additional regulatory factors, which is hinted at by enrichment for A-rich motifs located downstream of regulated exons. It was notde that PABP, PABP2 (PABPN1), ZC3H14/dNab2, and hnRNP-Q (Syncrip) proteins associate with qualitatively similar A-rich motifs, and include known neuronal splicing regulators. The discovery of extensive ELAV/Hu-mediated cassette exon targets, including the finding that individual ELAV/Hu proteins can robustly induce exon exclusion and inclusion in an ectopic context, provides a framework for future mechanistic dissection (Lee, 2021).

Many studies in the literature have treated ALEs and tandem UTRs separately, since ALEs may be regulated by splicing while tandem UTRs are only regulated by alternative polyadenylation. Nevertheless, distal ALE and downstream tandem APA usage are correlated in mammals, with directionality toward more distal/longer isoforms in neurons. The underlying mechanisms have not been specifically defined. It is known that telescripting, suppression of premature cleavage and polyadenylation, via U1 snRNP suppresses premature 3'-end cleavage and polyadenylation. While this can occur in intronic regions and terminal 3' UTRs, the dominant usage of this mechanism seems to be for U1 to inhibit the usage of cryptic polyadenylation signals that are especially abundant within long introns, and U1/telescripting has not yet been shown to have a broad impact on endogenous tissue-specific implementation of 3' isoforms (Lee, 2021).

Drosophila Elav was linked to both isoform regulatory programs, since it was originally shown to promote distal ALE switching by suppressing 3' end usage of proximal internal last exons at ewg and nrg and later shown to mediate neuronal 3' extension of select loci. Likewise, regulation of APA was shown for all four Hu proteins in suppressing an intronic polyA site in the calcitonin/CGRP gene and HuR autoregulates by APA. In addition, HuR regulates 3'-end processing of several membrane proteins. This individual cases set the possibility that ELAV/Hu RBPs may coregulate these programs (Lee, 2021).

This work work has established that the three Drosophila ELAV/Hu members (Elav/Fne/Rbp9) are individually sufficient to induce the neural extended 3' UTR landscape, and that endogenous overlapping activities of Drosophila Elav and its paralog Fne are critical to determine the extended 3' UTR landscape of the larval CNS, as also shown in the embryo. This study extends this to reveal broad catalogs of directional alternative last exon (ALE) isoform switches by ELAV/Hu factors. Using mechanistic tests and genomic analyses of de novo motif and RRM-dependent Elav CLIP maps this study was now able to unify the rationale for distinct neuronal mRNA processing programs. In particular, Drosophila ELAV/Hu RBPs are necessary and sufficient to specify broad switching to distal alternative last exons, analogous to broad lengthening of terminal 3' UTRs via usage of distal pA sites. In both settings, ELAV/Hu RBPs suppress proximal pA sites via downstream U-rich sequences/ELAV motifs downstream of cleavage sites, and promote distal isoform usage by acting within newly-synthesized, chromatin-associated transcripts. Since this study also found that ELAV/Hu proteins are broadly involved in exon exclusion, via overt enrichment of their sites near regulated exons, broad analogies are suggested for ELAV/Hu RBPs to promote isoform diversity by suppression of processing sites used outside of the nervous system (Lee, 2021).

Importantly, it is suggested that similar regulatory rationale applies to the implementation of both neuronal ALE and APA in mammalian neurons. In particular, this study provides evidence that ELAV/Hu RBPs are poised to regulate both classes of 3' ends using similar mechanisms (i.e. polyA bypass mediated through U-rich sequences). Mammalian ELAV/Hu factors are well-known to mediate diverse regulatory outputs, ranging from mRNA stability and translation, to splicing and terminal APA regulation of selected loci. However, they are not yet documented to have broad roles in directional selection of alternative last exons or pA sites within terminal 3' UTRs. This genomic analyses now lends strong support to this notion (Lee, 2021).

Given that Elav paralogs have strongly compensatory activity that masks the effects of single elav mutants, and only double mutants of mammalian neural Elav factors have been examined to date, it is suggested that other multiple-knockout conditions may reveal greater collective impacts of ELAV/Hu factors on the neural transcriptome. More generally, the data argue that these classes of 3' ends can be broadly coregulated and that they may be just two versions of the same process (with splicing playing a comparative minor role in ALE regulation compared to polyadenylation). This may underlie the observation that global ALE-APA and TUTR-APA utilization are broadly correlated in mammals, and may be coregulated by other RBPs (Lee, 2021).

ELAV and FNE determine neuronal transcript signatures through exon-activated rescue

The production of alternative RNA variants contributes to the tissue-specific regulation of gene expression. In the animal nervous system, a systematic shift toward distal sites of transcription termination produces transcript signatures that are crucial for neuron development and function. This study reports that, in Drosophila, the highly conserved protein ELAV globally regulates all sites of neuronal 3' end processing and directly binds to proximal polyadenylation sites of target mRNAs in vivo. An endogenous strategy of functional gene rescue was uncovered that safeguards neuronal RNA signatures in an ELAV loss-of-function context. When not directly repressed by ELAV, the transcript encoding the ELAV paralog FNE acquires a mini-exon, generating a new protein able to translocate to the nucleus and rescue ELAV-mediated alternative polyadenylation and alternative splicing. It is proposed that exon-activated functional rescue is a more widespread mechanism that ensures robustness of processes regulated by a hierarchy, rather than redundancy, of effectors (Carrasco, 2020).

Most metazoan genes express multiple transcript isoforms through the use of alternative polyadenylation (poly(A)) sites that signal transcription termination. Alternative cleavage and polyadenylation (APA) generates mRNA isoforms that differ in their coding sequence (CDS-APA) or, more commonly, their 3' untranslated region (3' UTR-APA). Because 3' UTRs control mRNA fate through regulation of translation, degradation, and subcellular localization, APA profoundly impacts gene expression and the resulting cell behavior. Disrupted patterns of polyadenylation as well as specific APA events have been associated with human diseases, including cancer, autoimmune disorders, and neuropathological diseases (Carrasco, 2020).

Widespread changes in 3' end isoform usage also occur in a tissue-specific manner. In animals from flies to humans, hundreds of genes undergo a shift toward the distal poly(A) site exclusively in neurons, giving rise to sometimes extremely long 3' UTRs. Systematic changes in poly(A) site usage are understood to be caused by alterations in the expression of core 3' end processing factors. However, neuronal 3' UTR extension occurs in an exquisitely synchronous, specific, and robust manner, indicating that other, neuron-specific regulators are involved (Carrasco, 2020).

Neuronal ELAV-like proteins are highly conserved RNA-binding proteins (RBPs) that serve as gold-standard markers for neuronal commitment across model organisms. In flies and mammals, neuronal ELAV/ Hu proteins have been shown to regulate transcript stability, alternative splicing, CDS-APA , and, more recently, UTR-APA of individual genes. While ELAV/Hu proteins are prominent for their role in numerous neurological diseases and are required for neuronal differentiation, their molecular function is not well understood. This study postulates that ELAV represents the central effector of neuron-specific transcriptome signatures in vivo (Carrasco, 2020).

This study demonstrates that two neuronal proteins, ELAV and FNE, globally mediate neuron-specific alternative 3' end processing, thereby shaping the distinct identity of the complex neuronal transcriptome. The drastic physiological consequences of aberrant neuronal APA are immediately evident in cases in which protein-coding sequences are affected, effectively causing the loss of essential neuron-specific proteins such as EWG and giant Ankyrin. The effects of aberrant 3' UTR extension, which constitutes the majority of ELAV/nFNE-mediated APA events, are less well understood. Accumulating evidence indicates that long, neuron-specific 3' UTR isoforms perform specific and important functions in neurogenesis, both globally and individually. The finding that ELAV/nFNE mediate neuronal APA and/or alternative splicing (AS) in hundreds of genes showcases the impact of ELAV-family proteins in neurogenesis and neuronal function. In mammals, ELAV/Hu proteins, though best known for their role in mRNA stabilization in the cytoplasm, also act in AS and APA; it will be interesting to study a global loss of neuronal APA in the mammalian brain. (Carrasco, 2020).

The ELAV/nFNE genetic interaction described in this study is the first documented example of exon-activated rescue. It is proposed that this mode of context-specific protein activation ensures robustness of other biological processes that depend on one central regulator. Such regulators must hold the potential to alter the coding isoform of a secondary effector; candidates include splicing and APA factors, but can be expanded to transcription factors, chromatin regulators, and RNA editing and modification enzymes (Carrasco, 2020).

Interestingly, the n-fne mini-exon is conserved. In other insects, including some distantly related Drosophila species, nFNE homologs are naturally expressed and coexist with FNE and ELAV. In mammals, neuronal ELAV proteins are both nuclear and cytoplasmic, and hinge region exons regulate protein localization. In those species, nFNE and ELAV homologs coexist in wild-type conditions, and exon-activated functional rescue may occur under normal circumstances, arguing that redundancy, rather than functional rescue, is at play. In D. melanogaster, functional redundancy between ELAV proteins seems to have been evolutionarily suppressed in favor of hierarchization. Spatial compartmentalization, and more generally, specialization of a protein into a main effector may increase specificity and synchrony of systematic processes like neuronal APA. In such a hierarchy, the activation of a substitute effector represents a safeguarding mechanism to ensure function (Carrasco, 2020).

Overlapping activities of ELAV/Hu family RNA binding proteins specify the extended neuronal 3' UTR landscape in Drosophila

The tissue-specific deployment of highly extended neural 3' UTR isoforms, generated by alternative polyadenylation (APA), is a broad and conserved feature of metazoan genomes. However, the factors and mechanisms that control neural APA isoforms are not well understood. This study shows that three ELAV/Hu RNA binding proteins (Elav, Rbp9, and Fne) have similar capacities to induce a lengthened 3' UTR landscape in an ectopic setting. These factors promote accumulation of chromatin-associated, 3' UTR-extended, nascent transcripts, through inhibition of proximal polyadenylation site (PAS) usage. Notably, Elav represses an unannotated splice isoform of fne, switching the normally cytoplasmic Fne toward the nucleus in elav mutants. This study used genomic profiling to reveal strong and broad loss of neural APA in elav/fne double mutant CNS, the first genetic background to largely abrogate this distinct APA signature. Overall, this study demonstrates how regulatory interplay and functionally overlapping activities of neural ELAV/Hu RBPs drives the neural APA landscape (Wei, 2020).

The 3' untranslated region (UTR) is the major hub for post-transcriptional control and harbors elements that direct regulation by RNA binding proteins (RBPs), miRNAs, and RNA modifications. Such regulatory elements can be rendered conditional by alternative polyadenylation (APA), which yields 3' UTR diversity from an individual locus. Most eukaryotic genes accumulate distinct 3' UTR isoforms, and this can be influenced by differentiation status, tissue identity, and environmental and metabolic conditions. Moreover, APA is broadly disregulated in disease and cancer and may help to drive aberrant gene expression states (Wei, 2020).

Many tissues generate characteristic APA landscapes, implying that developmental factors regulate 3' UTR programs. A striking example involves the nervous system, where many hundreds of genes express substantially longer 3' UTRs compared to other tissues. Many of these neural 3' UTR extensions are extremely lengthy, and stable isoforms bearing 20 kb 3' UTRs have been documented in flies and mice by Northern blot. Despite the breadth and conservation of this phenomenon and functional studies that link neural-specific 3' UTRs to splicing choice, transcript localization, local translation, and miRNA regulation, relatively little is known of mechanisms that determine neural-extended 3' UTR isoforms (Wei, 2020).

Several identified APA mechanisms modulate the levels or activities of cleavage and polyadenylation factors. For example, interaction of U1 snRNP with poly(A) factors plays a major role in inhibiting premature 3' end processing. Other mechanisms that impact poly(A) site choice include recruitment of poly(A) factors at promoters and RNA Pol II speed. However, there is growing appreciation that local recruitment of RBPs can affect poly(A) site recognition or regulate later steps to inhibit cleavage and polyadenylation (Wei, 2020).

Among RBPs with roles in APA are certain members of the ELAV/Hu family, of which there are four in human (HuR and HuB-D) and three in Drosophila (Elav, Fne, and Rbp9). All are expressed in neurons, but HuB and RBP9 are also expressed in gonads and HuR is ubiquitous. Drosophila Elav was shown to regulate APA at erect wing (ewg), where it binds U-rich motifs distal of the cleavage site and inhibits 3' end processing. Likewise, all four mammalian Hu proteins suppress an intronic poly(A) site in calcitonin/CGRP, and HuR autoregulates by APA. In addition, HuR regulates 3' end processing of several membrane proteins. Given the predominant neuronal expression of many ELAV/Hu members, these proteins are candidate regulators of CNS-specific 3' UTR extensions. Elav mediates neural 3' UTR extensions of certain genes, but the breadth of Elav involvement in the neuronal APA landscape has not been investigated (Wei, 2020).

To gain a comprehensive understanding of ELAV/Hu RBPs in 3' UTR isoform regulation, genomic approaches were applied, using gain and loss-of-function genetics. Surprisingly, it was found that elav knockouts are not strictly embryonic lethal, as long believed, nor is Elav essential for most neural 3' UTR extensions to accumulate. Using a heterologous system this study found all three Drosophila ELAV/Hu RBPs (Elav, Fne, and Rbp9) have similar capacities to broadly induce a neural 3' UTR extension landscape. They do so by promoting bypass of proximal polyadenylation signals (PAS) in nascent transcripts. Although Elav is normally the predominant nuclear Hu factor in Drosophila, this study found that in elav-null CNS, the normally cytoplasmic Fne protein becomes substantially nuclear, owing to induction of a previously unrecognized splice isoform. Accordingly, genomic analyses of elav/fne double mutant CNS reveal strong loss of neural 3' UTR extensions. Overall, this study demonstrates critical overlapping roles for ELAV/Hu RBPs to generate the neural-extended 3' UTR landscape (Wei, 2020).

The accumulation of substantially extended 3' UTR isoforms in the nervous system represents a broad and conserved phenomenon. This phenomenon was associated with activity of Elav, a neuronally enriched RBP that has been shown to block proximal PAS usage by binding to U-rich sequences. However, the evidence was limited to a handful of loci. Therefore, the endogenous contribution of Hu RBPs to the general neural 3' UTR extended landscape, and the mechanism of their regulatory impacts, were largely unknown. Indeed, initial studies challenged the notion that Elav alone is critical for this process, since analysis of full knockout elav larval CNS showed they still broadly express neural 3' UTR extensions (Wei, 2020).

This study resolved this conundrum with two main lines of evidence. First, it was shown that a family of neural Hu family RBPs in Drosophila all have capacity to broadly induce neural 3' UTR extensions, largely by promoting the bypass of proximal PAS to permit continued transcription of extension regions. Second, it was revealed that there is substantial endogenous functional overlap of the Hu RBPs Elav and Fne in broadly driving endogenous neural 3' UTR lengthening. Since Fne proteins accumulate modestly in embryos, later time points were essential to better reveal their genetic interactions. Although many cells and tissues exhibit characteristic 3' UTR profiles, the mechanisms are little known. This work reveals the first demonstration of wholesale loss of a tissue-specific APA landscape, revealed upon co-deletion of elav and fne (Wei, 2020).

Many hundreds of genes acquire distinct presumably regulatory capacity as a result of neural APA, which can add miRNA and RBP sites and change overall 3' UTR structures. However, until experimental interventions are performed, it is difficult to say how important these extensions are for normal gene regulation, cell behavior, or organismal phenotype. Recently, CRISPR engineering was used to show that neural 3' UTR extension of homothorax contains an array of binding sites for miR-iab-4/8 that control its protein output and are critical for normal adult behavior (Garaulet, 2020). In particular, deletion of the mir-iab-4/8 locus, surgical mutation of their binding sites in the homothorax 3' UTR, and specific deletion of the homothorax neural 3' UTR extension all derepress Homothorax in a specific region of the abdominal ventral nerve cord and induce defective virgin female behavior (Garaulet, 2020). Notably, the current data show that the homothorax 3' UTR extension is largely maintained in elav mutant CNS but is completely lost in elav/fne double mutant CNS. Thus, ELAV/Hu-RBPs are upstream regulators to this newly recognized behavioral switch, and their combinatorial activities are presumably relevant to other neural-specific 3' UTR biology, since they maintain hundreds of neural 3' UTR extensions (Wei, 2020).

ELAV family proteins have been assigned gene-specific roles in regulating RNA processing at all levels, including alternative splicing, APA, target stability, translation, and subcellular mRNA localization. It was initially thought that individual ELAV/Hu family members would adopt distinct RNA processing functions based on cellular localization. Despite a preferred cellular localization, however, they shuttle between the nucleus and the cytoplasm, and localization also depends on cell type. Accordingly, Drosophila Fne and Rbp9 can regulate the Elav targets ewg, nrg, and arm (Zaharieva, 2015). Such functional overlap was not anticipated as Fne and Rbp9 are normally cytoplasmic (Zaharieva, 2015). The current data suggest that modest levels of nuclear ELAV/Hu proteins can promote genomically widespread neural 3' UTR extensions, since Fne comprises a small fraction of total ELAV/Hu proteins in larval CNS. Conversely, while Elav is largely utilized as a nuclear marker, this study documented it also has ubiquitous cytoplasmic accumulation, so it may conceivably overlap with cytoplasmic Fne/Rbp9 activities (Wei, 2020).

Complex regulatory interactions among the Drosophila Hu factors have been documented, since misexpression of Fne results in downregulation of endogenous Elav and Fne (Samson, 2003), and misexpression of a NLS-tagged nuclear variant of Rbp9 results in relocalization of endogenous Elav into the cytoplasm (Zaharieva, 2015). This study now documents multiple additional cross-regulatory mechanisms that control total nuclear levels of ELAV/Hu proteins in Drosophila. First, Elav represses fne transcript levels, which may be associated with the strong control of fne neural 3' UTR extension by Elav. Second Fne represses an alternative splice isoform of Fne that is preferentially localized to the nucleus. This Fne microexon, while not previously annotated, is deeply conserved in insects and may reflect the sole ELAV/Hu protein in other arthropods that is likely to carry out both nuclear and cytoplasmic activities (Samson, 2008). By contrast, even though Drosophila elav is the only lethal member of the family, it is intronless and is presumably a derived retrogene copy that originated in the Drosophilid ancestor. The Fne microexon inserts sequence adjacent to the octapeptide in the hinge region, which is known to be involved in nuclear localization. As the hinge region is not sufficient for nuclear localization, other parts of the ELAV/Hu protein may also contribute to its subcellular control (Wei, 2020).

Cross-over in their regulatory functions is facilitated by the highly overlapping in vitro target specificities of ELAV/Hu factors, including Elav/Fne/Rbp9 (Ray, 2013). Consistent with this, Elav/Fne/Rbp9-repressed cleavage sites were found to be enriched for similar U-rich motifs. Interestingly, the same motif was identified as a high-affinity initiator for forming a larger and saturable megadalton Elav complex (Soller, 2005). In addition, the same motif is the main conserved element in Drosophila virilis about 100 bp distal of the regulated poly(A) site in an otherwise very distinct extended binding sequence in ewg (Wei, 2020).

These data suggest that Rbp9 may also play a role in neural APA, since it has very similar gain-of-function activities as Elav and Fne. However, its impact may be masked by the earlier accumulation of Elav and Fne proteins in neurons. Because of apparent embryonic lethality of available elav/fne/rbp9 triple mutant genotypes, iy was not possible to analyze this genotype at a developmentally relevant post-embryonic time point (i.e., in 2nd instar larval CNS when Rbp9 protein is more detectably accumulated). As it is suspected that simple RNAi approaches will be insufficient to eliminate the relevant activities, FLP-out systems or somatic CRISPR might be investigated to bypass early lethality of elav mutants (Wei, 2020).

The ELAV/Hu protein Found in neurons regulates cytoskeletal and ECM adhesion inputs for space-filling dendrite growth

Dendritic arbor morphology influences how neurons receive and integrate extracellular signals. This study shows that the ELAV/Hu family RNA-binding protein Found in neurons (Fne) is required for space-filling dendrite growth to generate highly branched arbors of Drosophila larval class IV dendritic arborization neurons. Dendrites of fne mutant neurons are shorter and more dynamic than in wild-type, leading to decreased arbor coverage. These defects result from both a decrease in stable microtubules and loss of dendrite-substrate interactions within the arbor. Identification of transcripts encoding cytoskeletal regulators and cell-cell and cell-ECM interacting proteins as Fne targets using TRIBE further supports these results. Analysis of one target, encoding the cell adhesion protein Basigin, indicates that the cytoskeletal defects contributing to branch instability in fne mutant neurons are due in part to decreased Basigin expression. The ability of Fne to coordinately regulate the cytoskeleton and dendrite-substrate interactions in neurons may shed light on the behavior of cancer cells ectopically expressing ELAV/Hu proteins (Alizzi, 2020).

Dendrite branching is a dynamic process that depends heavily on the regulation of cytoskeletal organization and dendrite-substrate interactions. Class IV da neurons depend on Fne throughout development as the rapidly growing epidermis demands both the extension of existing branches and elaboration of new branches in order to maintain coverage. The results of this study indicate that Fne regulates targets involved in cytoskeletal organization and dendrite-ECM interactions, leading to stable branch growth and arbor elaboration. Whereas terminal branches in wild-type neurons are largely stable by the end of larval development, these branches remain highly dynamic in fne^- neurons likely due to weakened interactions with the ECM, although additional effects on actin dynamics cannot be ruled out. These weakened dendrite-ECM interactions as well as the decrease in stable microtubules in fne^- neurons prevent long-term branch stabilization and, consequently, field coverage is not maintained as the larva grows (Alizzi, 2020).

The ability of both loss and overexpression of fne to cause space-filling morphology defects despite their opposing effects on microtubule content is consistent with previous work showing that increased and decreased microtubule stability in class IV da neurons can cause similar effects on branching. Loss or overexpression of fne also has consequences during pupariation, when class IV da neuron dendrites are pruned back to the soma. As microtubule breakdown is an important first step in pruning and is linked to dendrite thinning and destabilization of the dendritic membrane, the pruning defects in fne mutant and overexpressing neurons may arise from the same effects of Fne on microtubule composition observed in larval neurons. Destabilization of microtubules in fne- neurons could lead to a premature initiation of the pruning process. Conversely, increased microtubule stability in fneOE neurons could lead to a delay in this process (Alizzi, 2020).

The identification of RNAs encoding cytoskeletal regulators and cell adhesion molecules like Bsg as targets of Fne provides insight into to how Fne may coordinate inputs to dendrite patterning. The requirement for Bsg in both the neuron and epidermis for proper class IV da neuron morphogenesis supports a role for Bsg as an effector of Fne in mediating dendrite communication with the overlying epidermal cells. Recent work has implicated epidermally-derived signals in class IV da neuron space-filling morphology. Among these, Syndecan, a heparin sulfate proteoglycan (HSPG) on the surface of epidermal cells, promotes microtubule stabilization in higher order dynamic branches in order to promote the space-filling morphology of class IV da neurons, although how Syndecan communicates with the neuron is currently unknown. In T cells, the mammalian homolog of Bsg, cluster of differentiation 147 (CD147), forms a complex with Syndecan-1 in cis. Similarly, epidermal Bsg could interact with epidermal Syndecan and bind to neuronal Bsg to signal from the epidermis to the neuron to promote branch stabilization. The finding that Bsg is required for space-filling growth but not for regulating terminal branch dynamics fits well with results from previous work on Syndecan showing that the microtubule stabilization promoted by HSPG is required for long-term branch stabilization but not short-term branching dynamics and suggests that the two molecules may interact. Furthermore, the finding that knockdown of bsg reduces stable microtubule content similarly to mutation of fne and can partially ameliorate the increase in stable microtubules caused by fne overexpression supports the idea that Fne activation of Bsg expression during larval growth facilitates dendrite-epidermal communication that in turn impacts the dendritic microtubule cytoskeleton. This role of Fne in epidermal-neuronal control of space-filling dendrite growth was not revealed by the initial phenotypic analysis, but only through TRIBE identification of Fne target RNAs (Alizzi, 2020).

RNA-binding proteins typically have numerous targets and the TRIBE data indicate that this is the case for Fne. Thus, it is not surprising that regulation of bsg accounts for only a subset of defects observed in fne^- neurons. Evidence that Bsg colocalizes with integrins in cultured Drosophila cells and in the Drosophila retina, and coimmunoprecipitates with integrin from mammalian cells [50] initially suggested that Bsg might function in the integrin-dependent adhesion of class IV da neurons to the ECM. The results, however, do not support such a role, as dendrite crossings-which are a consequence of loss of dendrite-ECM adhesion-were not affected by neuronal bsg RNAi. Although the high false-negative rate of TRIBE may have precluded the identification of mys as a target of Fne, the analysis did identify several candidates that could be effectors of dendrite-ECM adhesion. One potential candidate is sema-1b, since another semaphorin, sema-2a, has been shown to promote integrin-mediated dendrite-ECM adhesion in class IV da neurons. Another candidate, 14-3-3ζ, has been shown to directly interact with L1 cell adhesion molecule (L1CAM) to limit neurite outgrowth in mice. In class IV da neurons, the L1CAM homolog Neuroglian is a component of the enclosure complex, suggesting that excess 14-3-3ζ in fne^- neurons might lead to increased enclosure and loss of contact with the ECM. Further analysis of these targets, and their interactions with Fne, could elucidate the ways in which Fne mediates dendrite growth along the ECM (Alizzi, 2020).

Although previous work has uncovered many transcriptional regulators of class IV da neuron development, less is known about the post-transcriptional mechanisms that govern the morphology of these neurons. In contrast to transcriptional regulation, post-transcriptional regulation allows for rapid and localized control. Such features are particularly important in neurons, which must respond rapidly to a variety of extracellular and developmental cues and whose dendrites can extend long distances from the soma. By regulating numerous, functionally-related transcripts, a single RBP can efficiently promote synchronized control over multiple inputs that impact neuronal patterning. In this manner, Fne may ensure that both the cytoskeletal organization and dendrite-substrate interactions required for stable, space-filling dendrite growth are regulated in tandem. How Fne acts on its targets, however, is poorly understood. Although Elav/Hu proteins have been shown to function at almost every stage of RNA metabolism, the somatic, cytoplasmic localization of Fne in class IV da neurons suggests that it functions in regulating transcript stabilization or translation. Furthermore, as Fne contains only RNA recognition motifs and no other known functional domains, it likely acts by recruiting other proteins to its target transcripts. Identification of the protein interacting partners of Fne will shed light on the molecular mechanism(s) by which Fne controls its various targets (Alizzi, 2020).

The space-filling defects observed in fne^- class IV da neurons are similar to the neuronal defects observed in HuD knockout mice. Loss of HuD led to decreased total branch length and reduced arbor complexity in neurons in the lower layer of the neocortex and the CA3 region of the hippocampus, indicating a role for HuD in the expansion of these neurons early in development [31]. These results suggest a common role for the two homologous proteins. The defects observed when fne is overexpressed may shed light on the phenotype of metastatic cancers including small cell lung carcinoma (SCLC) and neuroblastoma that express the neuron-specific HuD protein. SCLC cells take on properties of migrating neuroblasts, extending microtubule-rich axon-like projections that increase their ability to metastasize. Furthermore, integrin-mediated ECM interactions have been previously shown to increase metastasis and migration in SCLC cells and play an important role in neuroblast migration. In class IV da neurons, fne overexpression prevented branching along the main dendrites and forced branching to occur at the periphery of the arbor, likely through increased microtubule stabilization and altered dendrite-ECM contacts. Ectopic expression of fne in epithelial cells led to alterations in integrin expression and distribution. Additionally, expression of fne caused these normally cuboidal epithelial cells to become squamous, suggesting that the changes in cytoskeletal composition and cell-ECM interactions coordinated by Fne promote cell spreading. The ability of Fne to regulate both cytoskeletal organization and integrin-mediated cell-ECM interactions suggests that HuD may drive similar processes that produce the neuron-like morphology and migratory properties of SCLC cells. Thus, whereas the cellular behaviors promoted by Fne support the unique space-filling morphology of class IV da neurons, they could result in untoward effects in epithelial cells when expression of Fne homologs is dysregulated (Alizzi, 2020).

Sxl_-dependent, tra/tra2-independent alternative splicing of the Drosophila melanogaster X-Linked gene found in neurons

Somatic sexual determination and behavior in Drosophila melanogaster are under the control of a genetic cascade initiated by Sex lethal (Sxl). In the female soma, SXL RNA binding-protein regulates the splicing of transformer (tra) transcripts into a female-specific form. The RNA binding protein TRA and its cofactor TRA2 function in concert in females, whereas SXL, TRA and TRA2 are thought not to function in males. To better understand sex-specific regulation of gene expression, this study analyzed male and female head transcriptome datasets for expression levels and splicing, quantifying sex-biased gene expression via RNA-Seq and qPCR. The data uncouples the effects of Sxl and tra/tra2 in females in the sex-biased alternative splicing of head transcripts from the X-linked locus found in neurons (fne), encoding a pan-neuronal RNA-binding protein of the ELAV family. FNE protein levels are down regulated by Sxl in female heads, also independently of tra/tra2. It is argued that this regulation may have important sexually dimorphic consequences for the regulation of nervous system development or function (Sun, 2015).

The Drosophila sex determination hierarchy is the classical model of developmentally regulated alternative splicing. To identify genes expressed differentially in males and females, head samples were chosen, thereby eliminating large numbers of events restricted to gonadal differentiation. Moreover, the neurons, enriched in heads, are the site of extensive regulation at the level of alternative splicing (Sun, 2015).

In addition to dsx and fru, canonical regulators of Drosophila sex determination, we identified and further characterized the expression of fne and tango13 as genes expressed in a sex-biased manner. This study found that tango13 sex-specific expression responds to tra, tra2, and Sxl mutations in females as expected if under the control of the canonical sex determination pathway. An intriguing feature is the absence of reciprocity in the regulation of the mutually exclusive tango13-a and tango13-b splice forms, because tango13-a levels are reduced in tra, tra2, and Sxl mutants, but tango13-b levels are not. This observation suggests that tra and tra2 could possibly have an impact on the levels/stability of the tango13-a transcript rather than on the alternative splicing of tango13 RNA per se. The impact of tra/tra2 alleles on the expression of tango13-a is similar to that on the expression of dsx-F, consistent with regulation downstream of the sex determination pathway (Sun, 2015).

In contrast to fru, dsx, and tango13, the expression of fne is independent of TRA and TRA2. Crucially, fne splicing nevertheless depends on Sxl function in female heads: Sxl^- pseudomales switch to a male mode of fne alternative splicing, consistent with a role for SXL in promoting, directly or indirectly, the formation of the fne-b isoform at the expense of fne-a in normal females. Further, although fne alternative splicing is male-like in XX Sxl pseudomales, FNE protein levels are also upregulated two-fold to three-fold compared to CS males and females. Both male-like splicing and increased FNE protein levels in the pseudomales are reverted by the introduction of a Sxl⁺ minigene, confirming the specificity of Sxl in the control of both the splicing and protein levels. These data thus show that a Sxl-dependent, tra/tra2-independent mechanism regulates fne expression in females (Sun, 2015).

Canton-S (CS) female and male pools of fne RNA yield similar amounts of FNE protein in the two sexes. However, XX SxlM1,fΔ33 / Sxl^f7M1 pseudomales have a male-like pool of fne RNA and two-fold to three-fold increased FNE protein levels compared to CS. Because fne is an X-linked gene, its expression is presumably influenced by the canonical dosage compensation pathway, which could be responsible for the upregulation of FNE levels in XX Sxl_- pseudomales. However, according to the canonical model, higher fne transcript levels would be expected in pseudomales than in males and females, but that is not the case. Additional mechanisms must be at play (Sun, 2015).

First, increased FNE protein levels in XX Sxl_- pseudomales compared to wild-type males do not result from increased transcript levels. Because males and pseudomales share similar spliced pools of fne RNA, their distinct FNE outputs necessarily result from a regulatory mechanism that operates independently of the effects of Sxl on alternative splicing. Formally, this mechanism appears to stimulate the translation of fne transcripts in XX individuals. Second, increased FNE protein levels, concomitant with changes in alternative splicing but not associated with changes in transcript levels, as in XX Sxl_- pseudomales compared to wild-type females, are consistent with the existence of a Sxl_-dependent mechanism that downregulates FNE protein levels in XX females. Only the XX-dependent upregulation would persist in Sxl_- pseudomales, hence their increased FNE level. It is conceivable that fne regulation by Sxl occurs via direct binding of Sxl to fne transcripts. An interesting alternative as a means to regulate its splicing is the possibility that the impact of Sxl on fne expression occurs indirectly (possibly via an hormonal axis), since the extensive impact of the germline on the expression of somatic genes has been documented (Sun, 2015).

fne encodes an RNA-binding protein concentrated in the soma of neurons and present throughout development. It is necessary for the normal development of the mushroom bodies of males and females, and it is involved in the regulation of male courtship. It is intriguing that the expression of pan-neuronal fne is regulated in a sex-biased manner under the control of Sxl (Sun, 2015).

In addition to its role in the development of the germline, Sxl is involved in several regulatory pathways in the soma. It responds to a cell autonomous signal (number of X chromosomes) and is crucial both for the sexual development of somatic cells and for dosage compensation in males. SXL, but not TRA or TRA2, is also required independently of the somatic sex determination pathway for the development of a subset of sexually dimorphic neurons, with consequences on female ovulation. Additional phenotypes independent of the canonical somatic sex determination pathway but dependent on Sxl are the control of the sexually dimorphic body size of flies and the sex-specific bristle number on the A5 sternite. The latter occurs through general downregulation of the Notch pathway by SXL in multiple tissues . Thus, the Sxl_-regulated expression of fne fits within the context of Sxl acting in parallel with the canonical Sxl_-tra/tra2 cascade, constituting an example of its impact on tissues that do not show obvious sexual dimorphism (Sun, 2015).

fne is a member of a fairly new multigene family restricted to dipterans. The birth of this family predates the role of SXL in sex determination, which is restricted to the drosophilids. Based on RNA-Seq data and the Flybase models, sex-specific alternative splicing has not been reported for either of the other two paralogues in this family, elav (embryonic lethal abnormal visual system, X linked) or rbp9 (RNA binding protein 9, second chromosome). elav is the result of a retrotransposition and is likely to have acquired new cis-regulatory elements in the process. It autoregulates via a posttranscriptional mechanism involving its 3' UTR. It is unclear whether the Sxl_-dependent regulation of fne is an ancestral property that has been lost for rbp9 or was recently acquired. Nevertheless, sex-specific alternative splicing provides fne with the ability to be differentially regulated in females, which may have an important impact on sex-specific nervous system function or development, for which there are numerous instances of a role for Sxl. Within the context of the canonical sex determination pathway, Sxl regulates the expression of fru and dsx, two transcription factors crucial for behavior and nervous system function. SXL also controls, via an independent pathway, specific aspects of female behavior (Evans and Cline 2013). Still outside of the context of the canonical sex determination pathway, Sxl regulates the neurogenic locus Notch. Further, in Drosophila virilis, SXL protein accumulates in the male developing nervous system, consistent with a role there . Thus, the control exerted by Sxl on pan-neuronal fne outside of the context of the canonical sex determination pathway may be part of the heritage of an SXL ancestral function more focused on the nervous system than on sexual differentiation (Sun, 2015).

Concentration and localization of coexpressed ELAV/Hu proteins control specificity of mRNA processing

Neuronally coexpressed ELAV/Hu proteins comprise a family of highly related RNA binding proteins which bind to very similar cognate sequences. How this redundancy is linked to in vivo function and how gene-specific regulation is achieved have not been clear. Analysis of mutants in Drosophila ELAV/Hu family proteins ELAV, FNE, and RBP9 and of genetic interactions among them indicates that they have mostly independent roles in neuronal development and function but have converging roles in the regulation of synaptic plasticity. Conversely, ELAV, FNE, RBP9, and human HuR bind ELAV target RNA in vitro with similar affinities. Likewise, all can regulate alternative splicing of ELAV target genes in nonneuronal wing disc cells and substitute for ELAV in eye development upon artificially increased expression; they can also substantially restore ELAV's biological functions when expressed under the control of the elav gene. Furthermore, ELAV-related Sex-lethal can regulate ELAV targets, and ELAV/Hu proteins can interfere with sexual differentiation. An ancient relationship to Sex-lethal is revealed by gonadal expression of RBP9, providing a maternal fail-safe for dosage compensation. These results indicate that highly related ELAV/Hu RNA binding proteins select targets for mRNA processing through alteration of their expression levels and subcellular localization but only minimally by altered RNA binding specificity (Zaharieva, 2015).

Deletion of the Drosophila neuronal gene found in neurons disrupts brain anatomy and male courtship

The fne (found-in-neurons) locus encodes one of the three paralogs of the ELAV gene family of Drosophila melanogaster. Members of this family are found throughout metazoans and encode RNA-binding proteins with primarily neuronal localization, but with remarkably diverse functions given their high level of amino acid sequence conservation. The first identified member of the family, elav of Drosophila is a vital gene. Mutations in the second Drosophila elav paralog, rbp9, are viable but female sterile. No alleles of fne were previously available. FNE protein is normally present in the cytoplasm of all neurons throughout development. This study describes the generation and characterization of fne^null mutations by homologous recombination. In contrast to elav and similar to rbp9, fne^null mutants are viable, but exhibit a specific and fully penetrant fusion of the beta-lobes in their mushroom bodies (MB), a paired neuropil of the central brain involved in a variety of complex behaviors. Mutant males have reduced courtship indices, but normal short- and long-term courtship memory. These data show that fne has specific functions which are non-overlapping with the other two family members, namely in courtship behavior and in the development of the adult MB. The data further show that courtship memory does not require intact beta-lobes in the MB (Zanini, 2012).

found in neurons, a third member of the Drosophila elav gene family, encodes a neuronal protein and interacts with elav

elav, a gene necessary for neuronal differentiation and maintenance in Drosophila, encodes the prototype of a family of conserved proteins involved in post-transcriptional regulation. This study identified found in neurons (fne), a gene encoding a new ELAV paralogue. FNE was shown to bind RNA in vitro. fne transcripts are present throughout development and contain long untranslated regions. Transcripts and proteins are restricted to neurons of the CNS and PNS during embryogenesis. These features are reminiscent of elav. However, fne expression is delayed compared to elav's, and FNE protein appears cytoplasmic, while ELAV is nuclear. GAL4-directed overexpression of fne in neurons leads to a reduction of stable transcripts produced from both the fne and elav endogenous loci, suggesting that fne autoregulates and also regulates elav (Samson, 2003).

fne encodes a predicted protein with three RNA recognition motifs, RRMs. In vitro binding assays demonstrate the RNA binding capability of FNE. Comparison with ELAV in the same assays indicates that the proteins have distinct nucleic acid binding properties, although they both show a high affinity for polyuridilyc acid. Conceptual translation of the Drosophila genomic sequence relying upon the prediction of best splicing sites, generates a shorter protein (Accession AAF48215) missing 18 residues between RNP-2 and RNP-1 of RRM1 of the FNE encoded by cDNA-28h, suggesting that there might be a second form of FNE protein. New fne cDNA/EST sequences will be necessary to verify this possibility. No alternative forms of ELAV have been reported, but do exist for RBP9 and for some of the vertebrate paralogs (Samson, 2003).

Strikingly, similarity is higher among the four vertebrate ELAV proteins than it is among the three fly proteins. In addition, FNE/RBP9 resemble vertebrate ELAV as well as, if not better than, Drosophila ELAV. These observations underline the complex evolutionary relationships among the different ortho-/paralogues. One scenario is that fne and rbp9 derive from a duplication of an ancestral gene that had already diverged from elav, possibly before the separation of vertebrates and invertebrates. The human genes might thus derive from two rounds of duplication of the fne/rbp9 ancestor. The functional relationships among the different members of the family within and between species remain unclear, but the specific differences seen in their subcellular localization and in their nucleic acid binding properties in vitro are suggestive of different or complex functions (Samson, 2003).

Similar to elav, fne transcripts contain long untranslated regions. The identified fne cDNA contains a 1068 nucleotide-long ORF, spanning 2.8 kb of genomic sequence. Transcripts whose sizes range from 4 to at least 8 kb, reveal the presence of unusually long untranslated region(s) in fne RNA. Genome analysis predicts fne untranslated regions of up to 500 nucleotides 5' and up to 6 kb 3' to the gene. The significance of unusually long UTRs in the case of fne is not known. The elav gene contains a 6 kb long 3' UTR whose important role in normal elav function has been demonstrated. In general, 3' UTRs act in cis to regulate mRNA translation, stability, and localization through association with regulatory proteins or with antisense RNA. 3' UTRs also play a role in trans in myoblast growth and differentiation. These possibilities remain to be explored in the case of fne and elav (Samson, 2003).

fne is expressed in most, if not all neurons of the central and peripheral nervous system during embryogenesis. Consistent with the enrichment of fne transcripts in heads and the similarity between elav and fne transcript patterns, it is believed that fne expression remains specific to the nervous system in later stages. FNE protein appears in embryonic neurons shortly after ELAV, and it is also produced in elav^null embryos. As opposed to ELAV, FNE is essentially cytoplasmic. The apparent subcellular localization of proteins only partly reflects their presence and function in cell compartments. For instance, HuR appears nuclear but shuttles between nuclei and the cytoplasm, where it is thought to be involved in mRNA stabilization. Nevertheless, the different subcellular localizations of FNE and ELAV suggest different molecular functions (Samson, 2003).

Overexpression of fne in neurons causes a significant developmental arrest during larval stages. ELAV ectopic expression in the wing discs is known to lead to neo-function. Although the possibility that fne overexpression causes neo-function cannot be excluded, the elav promotor was chosen to restrict overexpression within normal fne cellular and temporal specificities and mimic a hypermorphic mutation. Indeed, the properties of the GAL4 expressing lines predict that the onset of fne overexpression is delayed compared to its normal onset, and that depending upon lines, the specificity of fne overexpression differs. This probably accounts at least in part for the differences in the severity of the phenotypes that are observed in different genetic combinations (Samson, 2003).

Overexpression of fne in neurons causes a decrease in the stable levels of both endogenous elav and fne transcripts in embryos. Although it is not possible to exclude that this reflects the occurrence of embryonic neuronal death, this possibility is not favored because anti-ELAV immunocytochemistry shows apparently normal CNS and PNS in embryos. Rather, this data suggests that fne overexpression decreases the level of expression of the endogenous fne locus by autoregulation, and that of the elav locus by feedback regulation (since fne is expressed later than elav). It is not clear whether alteration of the stable transcript patterns has a significant repercussion on protein expression during embryonic stages, since both ELAV and FNE appear to be stable proteins. It may be that the effect is delayed, consistent with the larval lethality that was observed (Samson, 2003).

It is possible that fne autoregulates and regulates elav via direct binding to the RNAs produced by these genes. Consistent with this possibility, both ELAV and FNE were found to bind in vitro to a sequence present in the elav 3' UTR. A role for RBP9 in transcript downregulation has been previously reported. Additional experiments will be necessary to determine the mechanism by which FNE affects elav expression in neurons (Samson, 2003).

Search PubMed for articles about Drosophila Fne

Alizzi, R. A., Xu, D., Tenenbaum, C. M., Wang, W. and Gavis, E. R. (2020). The ELAV/Hu protein Found in neurons regulates cytoskeletal and ECM adhesion inputs for space-filling dendrite growth. PLoS Genet 16(12): e1009235. PubMed ID: 33370772

Carrasco, J., Rauer, M., Hummel, B., Grzejda, D., Alfonso-Gonzalez, C., Lee, Y., Wang, Q., Puchalska, M., Mittler, G. and Hilgers, V. (2020). ELAV and FNE determine neuronal transcript signatures through exon-activated rescue. Mol Cell 80(1): 156-163. PubMed ID: 33007255

Garaulet, D. L., Zhang, B., Wei, L., Li, E. and Lai, E. C. (2020). miRNAs and neural alternative polyadenylation specify the virgin behavioral state. Dev Cell 54(3): 410-423 e414. PubMed ID: 32579967

Lee, S., Wei, L., Zhang, B., Goering, R., Majumdar, S., Wen, J., Taliaferro, J. M. and Lai, E. C. (2021). ELAV/Hu RNA binding proteins determine multiple programs of neural alternative splicing. PLoS Genet 17(4): e1009439. PubMed ID: 33826609

Samson, M. L. and Chalvet, F. (2003). found in neurons, a third member of the Drosophila elav gene family, encodes a neuronal protein and interacts with elav. Mech Dev 120(3): 373-383. PubMed ID: 12591606

Soller, M. and White, K. (2005). ELAV multimerizes on conserved AU4-6 motifs important for ewg splicing regulation. Mol Cell Biol 25(17): 7580-7591. PubMed ID: 16107705

Sun, X., Yang, H., Sturgill, D., Oliver, B., Rabinow, L. and Samson, M. L. (2015). Sxl^-dependent, tra/tra2-independent alternative splicing of the Drosophila melanogaster X-Linked gene found in neurons. G3 (Bethesda) 5(12):2865-74. PubMed ID: 26511498

Wang, H., Zeng, F., Liu, Q., Liu, H., Liu, Z., Niu, L., Teng, M. and Li, X. (2013). The structure of the ARE-binding domains of Hu antigen R (HuR) undergoes conformational changes during RNA binding. Acta Crystallogr D Biol Crystallogr 69(Pt 3): 373-380. PubMed ID: 23519412

Wei, L., Lee, S., Majumdar, S., Zhang, B., Sanfilippo, P., Joseph, B., Miura, P., Soller, M. and Lai, E. C. (2020). Overlapping activities of ELAV/Hu family RNA binding proteins specify the extended neuronal 3' UTR landscape in Drosophila. Mol Cell 80(1): 140-155. PubMed ID: 33007254

Zaharieva, E., Haussmann, I. U., Brauer, U. and Soller, M. (2015). Concentration and localization of coexpressed ELAV/Hu proteins control specificity of mRNA processing. Mol Cell Biol 35(18): 3104-3115. PubMed ID: 26124284

Zanini, D., Jallon, J. M., Rabinow, L. and Samson, M. L. (2012). Deletion of the Drosophila neuronal gene found in neurons disrupts brain anatomy and male courtship. Genes Brain Behav 11(7): 819-827. PubMed ID: 22741816

date revised: 2 January 2023

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