BIOLOGICAL OVERVIEW

The quantity of a gene product may be controlled in a number of ways: regulation of the gene's rate of transcription initiation, the rate of protein synthesis and degradation, and the stability of the RNA are all factors that determine the level of any given protein in a cell. For years these ideas have reverberated like mantras in molecular biology circles, but evidence for the mechanisms of posttranscriptional regulation were sorely lacking. Recently it has been found that whole classes of cytoplasmic mRNAs can be made inactive, that proteins affect mRNA stability and proteins regulate the structure of mRNA. ELAV is one of a class of proteins that binds to mRNA and presumably has a regulatory function.

ELAV is required for correct differentiation and maintenance of the nervous system. The gene encodes an RNA binding protein expressed in all neurons after birth, qualifying elav as a pan-neural gene. The subcellular distribution of ELAV was investigated using ELAV-specific antibodies and scanning confocal laser microscopy. ELAV is predominantly localized within the nucleus where it concentrates within discrete domains described as dots and webs. To characterize these discrete domains an analysis of Drosophila coiled bodies was initiated. Coiled bodies are non-capsular nuclear bodies that appear to be composed of coiled fibrils. Most coiled bodies disassemble prior to or during mitosis. After cell division, the reassembly of coiled bodies occurs during G1 phase and is preceded by the reformation of nucleoli. Coilin is a 80-kD nuclear protein, identified with autoimmune serum, that is found as an integral component of coiled bodies. The polyclonal antibody R288 raised against human coilin was used to identify coiled bodies in cells of the Drosophila larval central nervous system. Double-labeling immunohistochemistry shows that, similar to vertebrate and plant systems, small nuclear ribonucleoproteins are enriched within these structures. The nuclear distribution of ELAV is reminiscent of the distribution of a number of splicing factors, including snRNPs, snRNAs, U1 70K and U2AF, as well as the snRNA-specific 2,2,7 trimethyl guanosine cap within mammalian nuclei (see Sans fille for more information on Drosophila splicing factors). Further analysis of ELAV reveals that subnuclear domains enriched with this molecule localize within and close to coiled bodies and close to subnuclear domains enriched with splicing factors. Deletion of the ELAV alanine/glutamine-rich amino-terminal auxiliary domain has no discernible effect on localization; proteins produced from elav lethal alleles distribute normally. This morphological study provides the first hint of a role for ELAV in the generation of alternatively spliced neural-specific mRNAs (Yannoni, 1997).

Although the Drosophila erect wing (ewg) gene is broadly transcribed in adults, an unusual posttranscriptional regulation involving alternative and inefficient splicing generates a 116-kDa Ewg protein in neurons, while protein expression elsewhere (or of other isoforms) is below detection at this stage. This posttranscriptional control is important, since broad expression of Ewg can be lethal. Elav is necessary to regulate Ewg protein expression in Elav-null eye imaginal disc clones and Elav is sufficient for Ewg expression in wing disc imaginal tissue after ectopic expression. Analysis of Ewg expression elicited from intron-containing genomic transgenes and cDNA minitransgenes in Elav-deficient eye discs shows that this regulation is dependent on the presence of ewg introns. Analyses of the ewg splicing patterns in wild-type and Elav-deficient eye imaginal discs and in wild-type and ectopic Elav-expressing wing imaginal discs, show that certain neuronal splice isoforms correspond to Elav levels. The data presented in this paper are consistent with a mechanism by which Elav increases the splicing efficiency of ewg transcripts in alternatively spliced regions rather than with a mechanism by which stability of specific splice forms is enhanced by Elav (Koushika, 2000).

The primary transcript of ewg, which has 10 exons, A to J, is alternatively spliced in two regions. Neuron-enriched heads and neuron-poor bodies have different EWG RNA splicing profiles. Heads show enrichment for a transcript encoding a 116-kDa protein, whereas bodies have lower amounts of the transcript that encodes the 116-kDa protein and greater amounts of unprocessed RNA. The head-enriched transcript encoding the 116-kDa protein results from inclusion of exon D and exclusion of exons E and I. Additionally, splicing of introns 3a, 3c, and 6 is inefficient, since these introns are retained in polyadenylated EWG RNA (Koushika, 2000).

Additionally, Elav promotes a neuron-enriched splice isoform of Drosophila armadillo transcript. The neuron-specific arm transcript, n-arm, is generated by an alternative splice event that results from the exclusion of exon 6 of ubiquitous-arm (u-arm). The primer pair used amplifies both u-arm and n-arm transcripts; the 147-bp smaller band corresponds to n-arm, while the 244-bp band corresponds to u-arm. To test if Elav has a role in the formation of n-arm transcripts, RNA from wild-type and elav null allele (edr) eye discs, as well as from wild-type eye discs and wing discs ectopically expressing Elav were analyzed by RT-PCR. The amount of n-arm is reduced in Elav-deficient eye discs, and in the ectopically expressing wing discs expression of n-arm is clearly induced. No change was observed in the band representing u-arm splicing. In summary, the presence of n-arm is correlated with the presence of Elav in both neural and nonneural tissues, implying that arm transcripts are regulated by Elav. Similar data were obtained for splicing of exons VIIa and VIIb of Neuroglian transcripts (Koushika, 2000).

Elav ensures that the correct alternatively spliced protein isoforms of certain genes are generated in neurons. Currently three target genes, ewg, Nrg, and arm have been identified. Both Nrg and arm are vital genes and are broadly transcribed and ubiquitous protein isoforms are broadly expressed, but an additional isoform, encoded by an alternatively spliced transcript, is pan-neurally expressed. The significance of the neural Nrg (n-Nrg) isoform is not known, but the distinct cytoplasmic domain could be important in signaling. The n-Arm isoform differs from the ubiquitous Arm (u-Arm) isoform because it lacks the Wingless interacting domain; moreover, it preferentially interacts with DE-cadherins. Even with these differences in properties, the current evidence suggest that the u-Arm is sufficient to provide the n-Arm function. Perhaps a more detailed phenotypic analysis may reveal a specific role for n-Arm (Koushika, 2000).

ewg, also a vital gene, is broadly transcribed, but the protein product, a likely transcriptional regulator, is almost exclusively neural. In the case of ewg, it is clear that the expression of the 116-kDa protein isoform is essential for viability in the nervous system and that, when expressed in nonneural tissues, it can be lethal. These Elav-regulated genes provide insight into the regulatory role of Elav in neurons. Experiments reported here demonstrate for the first time that the prevalence of neuron-specific ewg, nrg, and arm transcripts positively correlates with Elav levels, and these results are achieved through the increased use of specific splice sites (Koushika, 2000).

ELAV links paused Pol II to alternative polyadenylation in the Drosophila nervous system

Alternative polyadenylation (APA) has been implicated in a variety of developmental and disease processes. A particularly dramatic form of APA occurs in the developing nervous system of flies and mammals, whereby various developmental genes undergo coordinate 3' UTR extension. In Drosophila, the RNA-binding protein ELAV inhibits RNA processing at proximal polyadenylation sites, thereby fostering the formation of exceptionally long 3' UTRs. This study presents evidence that paused Pol II promotes recruitment of ELAV to extended genes. Replacing promoters of extended genes with heterologous promoters blocks normal 3' extension in the nervous system, while extension-associated promoters can induce 3' extension in ectopic tissues expressing ELAV. Computational analyses suggest that promoter regions of extended genes tend to contain paused Pol II and associated cis-regulatory elements such as GAGA. ChIP-seq assays identify ELAV in the promoter regions of extended genes. This study provides evidence for a regulatory link between promoter-proximal pausing and APA (Oktaba, 2014).

ELAV is an RNA-binding protein that has been shown to bind to U-rich regions in target mRNAs, including neuroglian and erect wings. Recently, the Hox gene Ultrabithorax (Ubx) was shown to be bound by ELAV through similar elements to regulate alternative splicing, but ELAV was not found to bind to predicted binding sites in the Ubx 3' UTR. Similarly, this study also failed to identify specific ELAV recognition sequences within extended 3' UTRs. The present study investigated how ELAV is selectively recruited to appropriate targets during neurogenesis (Oktaba, 2014).

The activities of synthetic reporter genes were exanubed in transgenic embryos to determine whether extended 3' UTRs are sufficient for the selective recruitment of ELAV in vivo. Transgenes contain the Drosophila synthetic core promoter (DSCP) attached to a GFP coding sequence followed by the entire extended 3' UTR of elav, one of the targets of ELAV. If elav 3' UTR sequences are sufficient to recruit ELAV, then this transgene should produce mRNAs containing extended 3' UTRs (Oktaba, 2014).

Expression of 3' UTR sequences was monitored via double labeling assays with GFP coding sequences to distinguish transgene mRNAs from endogenous elav transcripts. Expression of the transgene was confirmed by colocalization of GFP with a probe directed against the short 3' UTR. However, colocalization of GFP with extended sequences was not observed, indicating that mRNAs produced from the transgene lack 3' extensions. The only signals containing 3' extensions corresponded to endogenous elav mRNAs (Oktaba, 2014).

Additional experiments were done to determine why the transgene fails to produce extended transcripts. The possibility that the GFP coding sequence somehow inhibits expression of extended sequences by creating GFP transgenes lacking proximal poly(A) signals was excluded. Such constructs no longer depended on ELAV for 3' extension and were found to produce mRNAs containing extended 3' UTR sequences when expressed in ectopic tissues lacking ELAV (Oktaba, 2014).

To test whether promoter sequences play a role in ELAV recruitment, the DSCP sequence was swapped with a 333 base pairs (bp) genomic DNA fragment encompassing the native elav promoter region, consisting of 92 bp upstream and 241 bp downstream of the (TSS). Strikingly, colocalization of GFP and extension sequences was observed indicating expression of the elav 3' UTR extension, as seen for the endogenous locus (Oktaba, 2014).

To confirm that 3' extension depends on native promoter regions of extended genes, a construct bearing the fully extended brat 3' UTR downstream of GFP was also tested, using three different promoters: the DSCP, the native promoter producing the short form of brat, and the native promoter producing the extended form of brat. Only the brat promoter associated with endogenous extension mediated expression of transgenic transcripts containing 3' UTR extensions. These observations suggest that the promoter regions of extended genes are essential for the ELAV-mediated expression of 3' UTR extensions (Oktaba, 2014).

The preceding results suggest that promoter sequences are important for the synthesis of 3' extensions in the developing nervous system. Their importance was determined by examining nonneural tissues. Ectopic ELAV can drive 3' UTR extension in ectopic tissues from endogenous loci. Attempts were made to determine whether ectopic ELAV could also induce ectopic 3' extensions from transgenic DNAs (Oktaba, 2014).

Both the GFP-elav transgene and ELAV protein were expressed in muscle cells using a Mef2-Gal4 driver. In this context, mRNA expression from the reporter is easily distinguished from endogenous elav expression, which occurs only in the nervous system. The DSCP fails to generate 3' UTR extensions, and only endogenous elav transcripts in the CNS were detected. In contrast, the GFP-elav transgene containing the native elav promoter produced transcripts with extended 3' UTRs in muscle tissue. Quantification of transgene expression in dissected muscle tissue using quantitative PCR (qPCR) shows that both promoters drive robust transgene expression (GFP signal), but only the native promoter drives expression of extension sequences. Similarly, the second brat promoter, but not the DSCP, was also able to drive expression of an extended brat 3' UTR in muscle cells (Oktaba, 2014).

Whether the promoter sequence from one extended gene could promote extension of the 3' UTR of another such gene was also tested. Indeed, a GFP transgene containing the elav promoter and brat extended 3' UTR exhibited ELAV-mediated APA. These observations suggest a link between transcription initiation and ELAV-mediated APA (Oktaba, 2014).

To determine whether the promoter regions of extended genes share common sequence motifs, 252 neural-specific transcripts produced by 219 different genes exhibiting 3' UTR extensions were examined. The most significantly enriched motif is the GAGA element, which occurs in nearly half of all extended genes. To investigate the functional significance of the GAGA element in promoters of extended genes, whether 3' UTR extension is diminished in animals lacking the GAGA-binding protein, Trithorax-like (Trl) was tested. For all six genes examined, the ratio between extension sequences and coding sequences was reduced between 15% and 75% in Trl mutant flies. These observations suggest that the GAGA motifs in the promoters of extended genes are important for proper 3' UTR extension (Oktaba, 2014).

The GAGA element is a motif commonly found in the promoter regions of genes containing paused Pol II. Paused Pol II is a pervasive feature of gene regulation in metazoan development, and at least 10%-30% of all genes in Drosophila contain paused Pol II. It is thought that paused promoters are poised for rapid activation and thereby exhibit synchronous induction in the different cells of a tissue. Another function of promoter pausing might be to ensure proper recruitment of essential factors for RNA elongation and processing (Oktaba, 2014).

Most extended genes contain paused Pol II, based on whole genome Pol II ChIP-seq assays. Some extended genes express both short and long isoforms from the same promoter (for example elav), while others (e.g., brat) employ different promoters for the different isoforms. In the latter case, only the promoter driving the extended isoform contains paused Pol II (Oktaba, 2014).

To determine whether paused Pol II might be associated with the formation of 3' UTR extensions, the overall Pol II pausing index (PI) of extended genes and various control genes was examined. Extended transcripts were found to be derived from significantly more paused promoters than any of the control groups, including neural-specific (but nonextended) genes. Thus, there is a clear association between Pol II pausing and 3' UTR extension, which transcends the general pausing seen for neural-specific gene expression. Extended transcripts are also strongly paused in muscle cells, where they are not actively transcribed and where ELAV is not expressed. Thus, Pol II pausing at extended genes occurs independently of ELAV (Oktaba, 2014).

The preceding analyses raise the possibility that ELAV is selectively recruited to the promoter regions of extended genes. To test this hypothesis, ChIP-seq assays were performed using anti-ELAV antibodies. ELAV is an RNA-binding protein that directly binds and inhibits proximal poly(A) elements of target transcripts. It was therefore reasoned that it should be possible to identify the genome-wide distribution of ELAV by crosslinking ELAV/RNA complexes to associated DNA templates. ELAV ChIP-seq assays were conducted with nuclei obtained from 6-8 hr and 10-12 hr embryos. These stages were selected based on previous observations regarding the timing of 3' extensions in the nervous system (Oktaba, 2014).

6,879 genomic regions bound by ELAV were identified in 6-8 hr embryos and 8,076 regions in 10-12 hr embryos. There is a striking enrichment of ELAV in the promoter regions of extended genes. For example, argonaute1 (ago1) produces multiple APA isoforms driven from three different promoters. The two promoters that produce extended transcripts display ELAV peaks, whereas the promoter that expresses the short (ubiquitous) isoform does not. High levels of ELAV are also found at 3' poly(A) sites, consistent with previous RNA immunoprecipitation assays (Oktaba, 2014).

The ChIP-seq data were combined into a 'meta-gene' plot that provides simple visualization of key sites of ELAV binding. There is a significant enrichment of ELAV at the promoter regions of extended genes as compared with neural-specific nonextended genes. A distinct ELAV peak is seen near the TSS, although ELAV binding continually increases across the 5' UTR and peaks at ~300 bp downstream of the start site (Oktaba, 2014).

ELAV not only binds to promoter regions, but also to 3' UTRs and introns of extended genes. ELAV is strikingly depleted from coding sequences. As expected, binding markedly increases in the vicinity of proximal poly(A) sites and remains high across extended regions where there are additional poly(A) elements (Oktaba, 2014).

A meta-gene analysis was also performed of previously published Pol II ChIP-seq data. Pol II binding is highly enriched in the promoter regions of extended genes, which is consistent with earlier evidence that such genes tend to contain paused Pol II. The Pol II binding profile did not otherwise differ from nonextended neural-specific genes. It is possible that ELAV binds to both nascent transcripts and associated DNA templates, since ELAV is usually detected at distal poly(A) sites of extended genes prior to full transcriptional extension (Oktaba, 2014).

This study has presented evidence that paused Pol II fosters selective recruitment of ELAV and coordinates expression of extended 3' UTR sequences during neurogenesis. The basis for selective recruitment of ELAV is a bit of a mystery since it has been shown to interact with broadly distributed low-complexity RNA sequences (e.g., U-rich). Increased interaction between paused promoters and termination regions might help promote 3' extension, for example, by bringing ELAV to the promoter via gene looping. The observed association of ELAV with the paused promoter regions of extended genes provides a foundation for selectivity and also strengthens the link between transcription initiation and 3' cleavage. It is improbable that paused Pol II is sufficient for recruitment of ELAV, since not all paused genes exhibit APA. It is therefore likely that additional sequence elements, for example, in extended 3' UTRs, are essential for recruitment. ELAV proteins are highly conserved, and it is easy to imagine that the regulation of 3' extension in the vertebrate CNS depends on selective promoter recruitment as seen in Drosophila (Oktaba, 2014).

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).

ELAV/Hu RNA binding proteins determine multiple programs of neural alternative splicing

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 (Elav, 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 alternative last exon (ALE) and alternative polyadenylation (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).

The vast majority of genes in higher eukaryotes are subject to a variety of alternative processing mechanisms that diversify the functional outputs of the transcriptome. The usage of alternative promoters, engagement of distinct internal and/or last exons by alternative splicing, and the utilization of alternative polyadenylation signals (PAS), can collectively generate transcript isoforms that differ in 5' UTRs, coding exons, and/or 3' UTRs. These regulatory regimes have broad consequences for differential regulation of isoforms as well as to broaden the protein outputs of an individual locus, and are aberrant in disease and cancer (Lee, 2021).

While all of these regulatory concepts are applicable to all tissues and cells, the nervous system is well-known for exuberant deployment of alternative isoforms. For example, vertebrate brains encode the most diverse and conserved sets of alternative splicing events. An extreme example is alternative splicing of the Drosophila immunoglobulin superfamily gene Dscam, which can generate ~38,000 distinct proteins. Indeed, experimental manipulations demonstrate that a high diversity of isoforms is functionally required for neuronal wiring. The nervous system also exhibits broad usage of other unusual splicing programs. For example, mammalian brains preferentially express a suite of short "microexons" (encoding <10aa), which appear to be dysregulated in neurological disease. Back-splicing that generates circular RNAs can be considered as another type of alternative splicing, and this class of species is most abundant and diverse in the nervous system. Finally, Drosophila and vertebrate CNS exhibit the longest 3' UTRs of any tissue (Lee, 2021).

Splicing and polyadenylation are catalyzed by large multisubunit RNA processing complexes, the former by the spliceosome and the latter by the cleavage and polyadenylation (CPA) machinery. As may be expected, direct experimental manipulations of core spliceosome or polyadenylation factors have broad impacts to deregulate isoform choice. The levels of many core splicing and polyadenylation factors differ across development and/or tissue or cell type, suggesting that their endogenous modulation may in part be linked to alternative mRNA processing. Reciprocally, trans-acting factors can impinge on the activities of splicing or polyadenylation machineries, either positively or negatively, to confer alternative processing (Lee, 2021).

The diversity of molecular processing mechanisms is best understood for alternative splicing. Most of these strategies influence the definition of exons or introns, resulting in differential inclusion or exclusion of sequences in mature mRNAs. The SR family of RNA binding proteins (RBPs) provide a paradigm for proteins that bind exonic sequences to promote their inclusion, but certain SR factors bind intronic sequences to promote exon exclusion. Many other non-SR proteins can regulate splicing (e.g., Nova, ELAV/Hu factors, PTBP proteins, MBNL proteins, etc.), and they generally mediate their effects by binding in the vicinity of splice sites. Notably, several of these have position-specific effects. For example, RBP binding overlapping a splice site can block exon inclusion, but intronic binding of the same factor can also promote exon inclusion (Lee, 2021).

Regarding APA, there are numerous ways that core CPA and trans-acting factors that influence sites of 3' cleavage, and these can have distinct impacts depending on location within the gene. When multiple pA sites are differentially utilized in the same terminal 3' UTR exon ("TUTR-APA", also referred to as tandem APA), this results in inclusion or exclusion of cis-regulatory sites within nested 3' UTRs. The length of alternative 3' UTRs can be quite substantial (>10 kb), and there may be sequence-independent effects of 3' UTR length variants. Another general class of APA events occurs within regions that are internal to the last coding exon of the longest gene model. These might occur within internal exons or introns, and have variously been termed upstream region-APA, intronic APA, coding sequence-APA (CDS-APA) or alternative last exon (ALE)-APA; the latter designation will be used in this study (Lee, 2021).

If internal polyadenylation impairs the stability of the alternative transcript, this can result in loss-of-function of that isoform. However, ALE-APA can also produce alternative stable transcripts, which encode distinct protein isoforms. In some cases, internally polyadenylated isoforms yield proteins that lack C-terminal domains of "full-length" counterparts, which might inactivate them and/or encode dominant negative or neomorphic activities. On the other hand, there are many examples of loci with distinct ALE isoforms that harbor different activities, analogous to distinct internal splice isoforms. Indeed, early characterizations of alternative polyadenylation involved ALE isoforms that switch the localization of IgM heavy chain products from secreted to membrane-bound, or generate distinct protein isoforms of calcitonin/CGRP. Beyond distinct coding potential, different ALE isoforms can also be subject to regulated subcellular localization, particularly within neurons. Although less is understood about trans-acting factors that influence APA, several RBPs (e.g., PABPN1, CELF1, etc.) operate in a conceptually similar manner to splicing regulators. Namely, several RBPs modulate 3'-end selection by binding in the vicinity of cleavage sites to oppose the action of the CPA machinery (Lee, 2021).

The ELAV/Hu family of RBPs is conserved across metazoans and play broad roles in RNA biogenesis, including alternative splicing, APA, target stability, translation, and localization. In Drosophila, Elav was proposed as a direct regulator of both splicing and APA. Notably, there is a conceptual mechanistic link between these regulatory processes. The first direct target of Elav characterized was erect wing (ewg), and it controls splicing of its alternative last exons. Elav also regulates the splicing of certain internal exons, but unlike other strategies for splicing control mentioned above, Elav controls neural-specific ewg by modulating cleavage and polyadenylation. In particular, Elav suppresses cleavage at the 3' end of an internal ewg terminal exon isoform, thereby permitting transcription to extend to the distal terminal exon. This mechanism also applies to neural-specific of neuroglian (nrg). An analogous function for Elav was reported to promote neural-specific 3' UTR lengthening at select genes bearing tandem polyA signals within the same 3' UTR. In this setting, it was proposed that association of Elav with a proximal polyA signal inhibits processing by the CPA machinery, thereby permitting transcription into distal 3' UTR segments (Lee, 2021).

These studies focused on regulation of a few specific Drosophila genes, but broader impacts of Elav and/or its paralogs on the mRNA processing were not yet addressed. In work published during review of this manuscript, the following observations were made. First, although Elav has long been considered to be embryonic lethal, this study found that elav deletion mutants are nominally viable as 1st instar larvae. With access to elav null larval CNS it was found that its complete loss was compatible with expression of many neural APA 3' UTR extensions. Second, it was found using gain-of-function strategies, that all three Drosophila ELAV/Hu members (Elav, Fne, Rbp9) have similar capacities to induce a neural 3' UTR extension landscape in an ectopic setting (S2 cells). Third, it was found that functional redundancy is endogenously relevant, because elav/fne double mutant larval CNS exhibit a severe loss of neural 3' UTR extension landscape. Fourth, the functional overlap of Elav and Fne involves a regulatory interplay, because Elav represses fne alternative splicing that switches it from a cytoplasmic to a nuclear isoform. The hierarchical role of Elav and Fne in neural mRNA processing was also reported in a contemporary study by Carrasco, 2020 (Lee, 2021 and references therein).

This study exploited gain-of-function and loss-of-function genomic datasets to study the impact of Drosophila ELAV/Hu factors on alternative splicing, including both internal exons as well as terminal exons. The set of cassette exons and alternative 5' or 3' splice sites that are regulated by Elav and Fne was broadened from just a few to many hundreds. Moreover, it was shown that overlapping activities of ELAV/Hu factors are necessary and sufficient to define a broad program of neural ALE splicing. Genomic analyses reveal mechanistic parallels between neural ALE splicing and neural 3' UTR lengthening, demonstrating that these are analogous processes that operate in a directional manner on transcripts to promote the inclusion of distal exonic sequences in neurons. Finally, these findings were extended to mammals and provide evidence for coincident shifts towards usage of distal ALE isoforms and extended tandem 3' UTRs during directed neuronal differentiation, coupled to enrichment of ELAV/Hu motifs at bypassed pA sites. This indicates conservation and coordination of these two RNA processing pathways across metazoan neurons (Lee, 2021).

Mammalian ELAV/Hu RBPs have been extensively connected to alternative splicing of cassette exons, but only to selected 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. Endogenous relevance was shown by demonstrating that dual deletion of elav and fne causes reciprocal changes to splice isoform accumulation. Notably, the endogenous breadth of splicing control by ELAV/Hu RBPs was revealed 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, the 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 it was not possible to obtain triple mutant larvae of Drosophila ELAV/Hu members, 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 were observed upstream of excluded exons. However, enrichment of ELAV/Hu RBP motifs were also observed 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 is noted 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 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 PAS 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).

In concurrent work, this study 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 was extended to reveal broad catalogs of directional ALE isoform switches by ELAV/Hu factors. Using mechanistic tests and genomic analyses of de novo motif and RRM-dependent Elav CLIP maps it is not possible 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 for ELAV/Hu RBPs to promote isoform diversity by suppression of processing sites used outside of the nervous system are suggested (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, evidence is provided 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).

Repression of the Hox gene abd-A by ELAV-mediated Transcriptional Interference

Intergenic transcription is a common feature of eukaryotic genomes and performs important and diverse cellular functions. This study investigates the iab-8 ncRNA from the Drosophila Bithorax Complex and shows that this RNA is able to repress the transcription of genes located at its 3' end by a sequence-independent, transcriptional interference mechanism. Although this RNA is expressed in the early epidermis and CNS, this study found that its repressive activity is limited to the CNS, where, in wild-type embryos, it acts on the Hox gene, abd-A, located immediately downstream of it. The CNS specificity is achieved through a 3' extension of the transcript, mediated by the neuronal-specific, RNA-binding protein, ELAV. Loss of ELAV activity eliminates the 3' extension and results in the ectopic activation of abd-A. Thus, a tissue-specific change in the length of a ncRNA is used to generate a precise pattern of gene expression in a higher eukaryote (Castro Alvarez, 2021).

Several noncoding RNAs (ncRNAs) have been identified from the Hox clusters of different species; a few of these have been shown to play key roles in gene regulation. One of these ncRNAs is the 92 Kb, spliced and polyadenylated transcript called the iab-8 ncRNA. Located within the Drosophila Bithorax Complex (BX-C), the iab-8 ncRNA originates from a promoter located about 4.5Kb downstream of the Abd-B transcription unit and continues until within about 1 Kb of the abd-A promoter. In situ hybridization experiments show that it is transcribed specifically in the very posterior epidermis of the embryo from the cellular blastoderm stage. From later embryonic stages, its expression becomes limited to parasegments (PS) 13 and 14 of the CNS. Loss of the iab-8 ncRNA has been shown to result in both male and female sterility, likely due to problems in the innervation of muscles important for reproduction. Much of its function has been attributed to a microRNA located between its sixth and seventh exons, called miR-iab-8 (miRNA). miR-iab-8 targets multiple transcripts including the Ubx and abd-A homeotic genes and their cofactors hth and exd. Indeed female sterility has been directly linked to ectopic hth, Ubx and abd-A in the CNS (Castro Alvarez, 2021).

In the embryonic CNS, abd-A expression is normally limited to PS7-12. Studies have shown that the restriction of abd-A expression from PS13 in the CNS is dependent upon expression of the iab-8 ncRNA. Although, the miR-iab-8 miRNA plays a part in the repression of abd-A in PS13, a deletion of the miRNA template sequence only results in a mild derepression of abd-A in PS13. On the contrary, mutations preventing the production of the iab-8 ncRNA cause a complete de-repression of abd-A, such that the abd-A expression pattern in PS13 mimics that of PS12), suggesting the existence of a second repression mechanism. This study explored the mechanism by which the iab-8 ncRNA represses abd-A. Using deletions spanning different regions of the iab-8 transcript, this study failed to identify specific parts of the transcript that can account for the additional repression of abd-A by the iab-8 ncRNA. Furthermore, it was found that the iab-8 transcript can repress an exogenous reporter gene placed downstream of its sequence. Based on these findings, it is concluded that it is the act of transcription that is necessary for repression, rather than the sequence transcribed (a phenomenon called transcriptional interference). Examination of the iab-8 transcript in the CNS, shows that there is a 3' extension made specifically within the CNS. This elongated transcript seems to be essential for abd-A down-regulation and requires the neuronal-specific, RNA-binding protein ELAV (or its paralogue FNE) for its creation. Overall, this work suggests that ELAV mediates a 3' extension of the iab-8 ncRNA that, in turn, allows it to specifically repress abd-A expression in the posterior CNS through transcriptional interference (Castro Alvarez, 2021).

Previous papers reported that the main transcript of the iab-8 ncRNA terminates ~1 kb upstream of the abd-A transcription unit. This finding was based on 3'RACE experiments performed on RNA isolated from relatively early embryos (6-12 hours). This study shows that, in later embryos, where the iab-8 ncRNA is restricted to the CNS, the iab-8 transcript extends well into the abd-A transcription unit. In every condition tested, it was seen that abd-A is repressed when there is an extended transcript. However, outside of the sequences required for normal transcription and RNA processing, the iab-8 transcript, itself, does not seem to require any specific sequences to mediate this repression. Based on these findings, and the cis nature of this repression, it is concluded that the act of transcribing the extended iab-8 ncRNA is what represses abd-A expression in PS13 of the CNS. This type of inhibition is called transcriptional interference (Castro Alvarez, 2021).

In transcriptional interference the transcription of one gene spreading over the coding or regulatory sequences of another gene is able to downregulate the target gene's expression. The mechanisms mediating transcriptional interference seem to depend on the relative position of both promoters. In the case of the iab-8 ncRNA and the repression of abd-A, there exist tandem promoters, where the genes are transcribed in the same direction and the upstream transcript transcribes over portions of the downstream gene (promoter, enhancers, transcription unit). Studies performed in single cell organisms (yeast and bacteria) suggest that there are two main mechanisms mediating transcriptional interference of tandem promoters. The first is called the 'sitting duck' mechanism, where an initiating RNA polymerase or activating transcription factors are knocked off of the target gene by the passing polymerase. The potential second mechanism to mediate transcriptional interference is called the 'occlusion' mechanism, where activating transcription factors (or RNA polymerase itself) for the downstream gene are prevented from binding to their binding sites by the passing RNA polymerase or by the modified chromatin structure following the passage of an elongating polymerase. Thus far, it is not possible to distinguish between these two mechanisms in this system. However, both mechanisms have been shown to be dependent upon the strength of the silencing transcript's promoter relative to the target transcript promoter. The stronger the promoter activity from the upstream gene, the stronger the repression of the downstream gene. In the case of transcriptional interference by the iab-8 ncRNA, it is believed that its level of transcription is approximately equivalent to that of abd-A. Indeed, using an abd-A intronic probe to compare levels (a probe not subject to possible stabilization of the exonic probes of the abd-A coding mRNA), a similar level is seen of transcription from both the iab-8 (PS13 and 14) and abd-A promoters (PS7 through PS12). Given the slower nature of transcription initiation vs transcriptional elongation, this high level of transcription might favor downstream gene repression (Castro Alvarez, 2021).

From work on mammalian cells, it has long been known that the final exon of coding genes often promotes termination by the recruitment of the termination machinery to the poly(A) site [26]. Although in recent years, ELAV has been studied as a protein whose function lies in extending the 3'UTRs of neuron-specific genes by altering the selection of poly-A signals, RT-PCR results suggest that, here, ELAV may play a role in the alternative splicing of the final exons of the iab-8 transcript. This function in alternative splicing is consistent with the role described for ELAV as an RNA binding protein involved in the alternative splicing of the neuronal isoforms of the Nrg and fne gene products. In fact, ELAV family members have been shown to be particularly important for splicing into a terminal exon. Thus, ELAV might extend the iab-8 ncRNA by suppressing the ability of the iab-8 transcript to splice into its normal terminal exon. This would then prevent the transcribing RNA polymerase from terminating, causing it to continue transcribing until it finds a new terminal exon. Published ChIP-seq experiments (where nascent transcripts were cross-linked to the genomic DNA along with proteins) on ELAV from early and later embryos support this interpretation. According to these results, there is additional ELAV binding at the junction between intron 7 and exon 8 of the iab-8 transcript in later embryos when iab-8 is expressed only in the CNS (Castro Alvarez, 2021).

Interestingly, among the spliced fusions between iab-8 and abd-A, this study found one isoform that contains the abd-A ATG sequence. This would seem counter-productive, if the function of transcriptional interference is to prevent abd-A expression. Although it is not possible to judge the amount of this specific transcript based on the current experiments, previous results have suggested that exons one and two of the iab-8 ncRNA act as translational repressors. Indeed, the male specific abdominal (MSA) RNA, which is identical to the iab-8 ncRNA except that the first two exons of iab-8 are replaced by an alternative first exon, actually codes for a peptide whose coding sequence lies in the shared last exon. A GFP fusion to this peptide and other reporters placed in the iab-8 sequence have shown that these proteins are never expressed in the CNS, but can be expressed in the male accessory gland, where MSA is expressed. Thus, even if this form is produced in a significant quantity, it seems that the embryos have further buffered themselves against ectopic abd-A, by repressing its translation (Castro Alvarez, 2021).

Lastly, it is of note that even in the elav, fne, miR-iab-8 triple mutant, the derepression of abd-A, while strong, may not be complete. There are a few cells, that still seem to repress abd-A in the posterior CNS. At the moment, this result cannot be explained. It is believed that some of this change may be due to fact that elav, fne, miR-iab-8 mutant nerve cords are very much abnormal and may have certain cellular defects. It was noticed, for example that these nerve cords were more difficult to dissect as they were to the extremely fragile relative to wild type. However, it is also possible that there are additional factors that allow transcriptional readthrough in these embryos or perform a repressive function on abd-A by another mechanism. Interestingly, RT-qPCR results still seem to detect a low level of transcriptional readthrough even in elav, fne double mutants, hinting that some transcriptional interference might occur even in the absence of these factors. One possible candidate to mediate this transcriptional readthrough is the rbp9 gene, the third elav homologue in Drosophila. Like elav and fne, rbp9 that is expressed in neurons and has been shown to be capable of promoting 3'RNA extensions when ectopically expressed in cultured cells (Castro Alvarez, 2021).

As a mechanism of transcriptional repression, transcriptional interference has mostly been found in organisms with compact genomes like yeast and bacteria. Because most of the multicellular eukaryotes studied in the lab have much larger genomes, containing a large proportion of 'non-essential' DNA, transcriptional interference has often been disregarded as a common mechanism for gene repression. However, due to co-regulation and/or gene duplication events, eukaryotic genes may be more compact at certain locations than generally assumed. This is very evident in the HOX gene clusters where there are numerous examples of tightly packed or overlapping transcription units. With all of these examples of overlapping transcription units and possible transcriptional interference, it is interesting to ask if this association could relate to an ancient gene regulatory mechanism. Within the Hox genes there is a known phenomenon called posterior dominance. According to the principle of posterior dominance, the more posterior Hox gene expressed in a segment generally plays the dominant role in patterning the segment. In Drosophila, this is often seen by down-regulation of the more-anterior gene. It is interesting to note that in the most studied Hox clusters, the Hox genes are organized on the chromosome in a way in which each Hox gene is located directly 3' to the next more-posterior segment specifying Hox gene. If it is considered that the Hox clusters are thought to have arisen from successive gene duplication events and after such duplication events, the two genes should have equal regulatory potential, then how could the upstream gene consistently take on a more dominant role? Transcriptional interference provides a possible explanation for this. According to this model, the upstream gene might have a slight advantage over the downstream gene due to transcriptional interference. This advantage, although potentially weak in many cases, could then be intensified and fixed through evolving cross-regulatory interactions. In the case being studied, the finding that ectopic abd-A in the posterior CNS leads to female sterility would help to drive such interactions (Castro Alvarez, 2021).

Although this phenomenon has been studied in a HOX cluster, other situations might exist where genes are located in similar tight configurations that induce transcriptional interference. An interesting bioinformatic analysis of nested genes in Drosophila suggests that transcriptional interference might be a natural consequence of tight, tandem gene arrangement. There is a significantly lower number of nested genes transcribed from the same strand in the Drosophila genome. Furthermore, nested genes in the same orientation contained fewer or no introns. Examining the expression data of the tandem, nested genes showed that these genes were often downregulated in tissues where the upstream gene was expressed, leading to s suggestion that the genetic arrangement of the genes might lead to transcriptional interference through mechanisms like unnatural splicing. This is very similar situation to what was found in the Hox complex and may hint that transcriptional interference exists at other loci displaying a similar arrangement of genes. Examining the mechanism that mediates transcriptional interference at model loci like iab-8 may help to define the conditions necessary for transcriptional interference to occur and potential lead to the identification other loci regulated in similar fashion (Castro Alvarez, 2021).

GENE STRUCTURE

Bases in 5' UTR - 491

Exons - three

PROTEIN STRUCTURE

Amino Acids - 519

Structural Domains

The protein contains three repeats of a pair of sequences defined as RNA-binding consensus sequences, an octopeptide (RNP1), and an appropriately spaced hexapeptide, RNP2 (Yao, 1991).

The neuron specific Drosophila Elav protein belongs to the Elav family of RNA binding proteins, characterized by three highly conserved RNA recognition motifs; an N-terminal domain, and a hinge region between the second and third RNA recognition motifs. The role of the Elav hinge in localization and function has been examined in vivo using transgenes encoding Elav hinge deletions. Subcellular localization of the hinge mutant proteins reveals that residues between amino acids 333-374 are necessary for nuclear localization. This delineated sequence has no significant homology to classical nuclear localization sequences, but it is similar to the recently characterized nucleocytoplasmic shuttling sequence, the HNS, from a human Elav family member, HuR. However, this defined sequence is insufficient for nuclear localization as tested using hinge-GFP fusion proteins. Functional assays have revealed that mutant proteins that fail to localize to the nucleus are unable to provide Elav vital function, but their function is significantly restored when translocated into the nucleus by a heterologous nuclear localization sequence tag (Yannoni, 1999).

date revised: 1 March 2024

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