Memory consolidation in honey bees is enhanced by down-regulation of Down syndrome cell adhesion molecule and changes its alternative splicing.

Down syndrome cell adhesion molecule (Dscam) gene encodes a cell adhesion molecule required for neuronal wiring. A remarkable feature of arthropod Dscam is massive alternative splicing generating thousands of different isoforms from three variable clusters of alternative exons. Dscam expression and diversity arising from alternative splicing have been studied during development, but whether they exert functions in adult brains has not been determined. Here, using honey bees, we find that Dscam expression is critically linked to memory retention as reducing expression by RNAi enhances memory after reward learning in adult worker honey bees. Moreover, alternative splicing of Dscam is altered in all three variable clusters after learning. Since identical Dscam isoforms engage in homophilic interactions, these results suggest a mechanism to alter inclusion of variable exons during memory consolidation to modify neuronal connections for memory retention.

CMTr mediated 2'-O-ribose methylation status of cap-adjacent nucleotides across animals.

Cap methyltransferases (CMTrs) O methylate the 2' position of the ribose (cOMe) of cap-adjacent nucleotides of animal, protist, and viral mRNAs. Animals generally have two CMTrs, whereas trypanosomes have three, and many viruses encode one in their genome. In the splice leader of mRNAs in trypanosomes, the first four nucleotides contain cOMe, but little is known about the status of cOMe in animals. Here, we show that cOMe is prominently present on the first two cap-adjacent nucleotides with species- and tissue-specific variations in Caenorhabditis elegans, honeybees, zebrafish, mouse, and human cell lines. In contrast, Drosophila contains cOMe primarily on the first cap-adjacent nucleotide. De novo RoseTTA modeling of CMTrs reveals close similarities of the overall structure and near identity for the catalytic tetrad, and for cap and cofactor binding for human, Drosophila and C. elegans CMTrs. Although viral CMTrs maintain the overall structure and catalytic tetrad, they have diverged in cap and cofactor binding. Consistent with the structural similarity, both CMTrs from Drosophila and humans methylate the first cap-adjacent nucleotide of an AGU consensus start. Because the second nucleotide is also methylated upon heat stress in Drosophila, these findings argue for regulated cOMe important for gene expression regulation.

CMTr cap-adjacent 2'-O-ribose mRNA methyltransferases are required for reward learning and mRNA localization to synapses.

Cap-adjacent nucleotides of animal, protist and viral mRNAs can be O-methylated at the 2' position of the ribose (cOMe). The functions of cOMe in animals, however, remain largely unknown. Here we show that the two cap methyltransferases (CMTr1 and CMTr2) of Drosophila can methylate the ribose of the first nucleotide in mRNA. Double-mutant flies lack cOMe but are viable. Consistent with prominent neuronal expression, they have a reward learning defect that can be rescued by conditional expression in mushroom body neurons before training. Among CMTr targets are cell adhesion and signaling molecules. Many are relevant for learning, and are also targets of Fragile X Mental Retardation Protein (FMRP). Like FMRP, cOMe is required for localization of untranslated mRNAs to synapses and enhances binding of the cap binding complex in the nucleus. Hence, our study reveals a mechanism to co-transcriptionally prime mRNAs by cOMe for localized protein synthesis at synapses.

Parallel evolution of a splicing program controlling neuronal excitability in flies and mammals.

Alternative splicing increases neuronal transcriptomic complexity throughout animal phylogeny. To delve into the mechanisms controlling the assembly and evolution of this regulatory layer, we characterized the neuronal microexon program in Drosophila and compared it with that of mammals. In nonvertebrate bilaterians, this splicing program is restricted to neurons by the posttranscriptional processing of the enhancer of microexons (eMIC) domain in Srrm234. In Drosophila, this processing is dependent on regulation by Elav/Fne. eMIC deficiency or misexpression leads to widespread neurological alterations largely emerging from impaired neuronal activity, as revealed by a combination of neuronal imaging experiments and cell type-specific rescues. These defects are associated with the genome-wide skipping of short neural exons, which are strongly enriched in ion channels. We found no overlap of eMIC-regulated exons between flies and mice, illustrating how ancient posttranscriptional programs can evolve independently in different phyla to affect distinct cellular modules while maintaining cell-type specificity.

Channel nuclear pore protein 54 directs sexual differentiation and neuronal wiring of female reproductive behaviors in Drosophila.

BACKGROUND: Female reproductive behaviors and physiology change profoundly after mating. The control of pregnancy-associated changes in physiology and behaviors are largely hard-wired into the brain to guarantee reproductive success, yet the gene expression programs that direct neuronal differentiation and circuit wiring at the end of the sex determination pathway in response to mating are largely unknown. In Drosophila, the post-mating response induced by male-derived sex-peptide in females is a well-established model to elucidate how complex innate behaviors are hard-wired into the brain. Here, we use a genetic approach to further characterize the molecular and cellular architecture of the sex-peptide response in Drosophila females. RESULTS: Screening for mutations that affect the sensitivity to sex-peptide, we identified the channel nuclear pore protein Nup54 gene as an essential component for mediating the sex-peptide response, with viable mutant alleles leading to the inability of laying eggs and reducing receptivity upon sex-peptide exposure. Nup54 directs correct wiring of eight adult brain neurons that express pickpocket and are required for egg-laying, while additional channel Nups also mediate sexual differentiation. Consistent with links of Nups to speciation, the Nup54 promoter is a hot spot for rapid evolution and promoter variants alter nucleo-cytoplasmic shuttling. CONCLUSIONS: These results implicate nuclear pore functionality to neuronal wiring underlying the sex-peptide response and sexual differentiation as a response to sexual conflict arising from male-derived sex-peptide to direct the female post-mating response.

Dynamically expressed single ELAV/Hu orthologue elavl2 of bees is required for learning and memory.

Changes in gene expression are a hallmark of learning and memory consolidation. Little is known about how alternative mRNA processing, particularly abundant in neuron-specific genes, contributes to these processes. Prototype RNA binding proteins of the neuronally expressed ELAV/Hu family are candidates for roles in learning and memory, but their capacity to cross-regulate and take over each other's functions complicate substantiation of such links. Honey bees Apis mellifera have only one elav/Hu family gene elavl2, that has functionally diversified by increasing alternative splicing including an evolutionary conserved microexon. RNAi knockdown demonstrates that ELAVL2 is required for learning and memory in bees. ELAVL2 is dynamically expressed with altered alternative splicing and subcellular localization in mushroom bodies, but not in other brain regions. Expression and alternative splicing of elavl2 change during memory consolidation illustrating an alternative mRNA processing program as part of a local gene expression response underlying memory consolidation.

Hakai is required for stabilization of core components of the m6A mRNA methylation machinery.

N6-methyladenosine (m6A) is the most abundant internal modification on mRNA which influences most steps of mRNA metabolism and is involved in several biological functions. The E3 ubiquitin ligase Hakai was previously found in complex with components of the m6A methylation machinery in plants and mammalian cells but its precise function remained to be investigated. Here we show that Hakai is a conserved component of the methyltransferase complex in Drosophila and human cells. In Drosophila, its depletion results in reduced m6A levels and altered m6A-dependent functions including sex determination. We show that its ubiquitination domain is required for dimerization and interaction with other members of the m6A machinery, while its catalytic activity is dispensable. Finally, we demonstrate that the loss of Hakai destabilizes several subunits of the methyltransferase complex, resulting in impaired m6A deposition. Our work adds functional and molecular insights into the mechanism of the m6A mRNA writer complex.

Channel nuclear pore complex subunits are required for transposon silencing in Drosophila.

The nuclear pore complex (NPC) is the principal gateway between nucleus and cytoplasm that enables exchange of macromolecular cargo. Composed of multiple copies of ~30 different nucleoporins (Nups), the NPC acts as a selective portal, interacting with factors which individually license passage of specific cargo classes. Here we show that two Nups of the inner channel, Nup54 and Nup58, are essential for transposon silencing via the PIWI-interacting RNA (piRNA) pathway in the Drosophila ovary. In ovarian follicle cells, loss of Nup54 and Nup58 results in compromised piRNA biogenesis exclusively from the flamenco locus, whereas knockdowns of other NPC subunits have widespread consequences. This provides evidence that some Nups can acquire specialised roles in tissue-specific contexts. Our findings consolidate the idea that the NPC has functions beyond simply constituting a barrier to nuclear/cytoplasmic exchange as genomic loci subjected to strong selective pressure can exploit NPC subunits to facilitate their expression.

Transposons are genetic sequences, which, when active, can move around the genome and insert themselves into new locations. This can potentially disrupt the information required for cells to work properly: in reproductive organs, for example, transposon activity can lead to infertility. Many organisms therefore have cellular systems that keep transposons in check. Animal cells comprise two main compartments: the nucleus, which contains the genetic information, and the cytosol, where most chemical reactions necessary for life take place. Molecules continually move between nucleus and cytosol, much as people go in and out of a busy train station. The connecting 'doors' between the two compartments are called Nuclear Pore Complexes (NPCs), and their job is to ensure that each molecule passing through reaches its correct destination. Recent research shows that the individual proteins making up NPCs (called nucleoporins) may play other roles within the cell. In particular, genetic studies in fruit flies suggested that some nucleoporins help to control transposon activity within the ovary ­ but how they did this was still unclear. Munafò et al. therefore set out to determine if the nucleoporins were indeed actively silencing the transposons, or if this was just a side effect of altered nuclear-cytosolic transport. Experiments using cells grown from fruit fly ovaries revealed that depleting two specific nucleoporins, Nup54 and Nup58, re-activated transposons with minimal effects on most genes or the overall health of the cells. This suggests that Nup54 and Nup58 play a direct role in transposon silencing. Further, detailed analysis of gene expression in Nup54- and Nup58-lacking cells revealed that the product of one gene, flamenco, was indeed affected. Normally, flamenco acts as a 'master switch' to turn off transposons. Without Nup54 and Nup58, the molecule encoded by flamenco could not reach its dedicated location in the cytosol, and thus could not carry out its task. These results show that, far from being mere 'doorkeepers' for the nucleus, nucleoporins play important roles adapted to individual tissues in the body. Further research will help determine if the same is true for other organisms, and if these mechanisms can help understand human diseases.

Srrm234, but not canonical SR and hnRNP proteins, drive inclusion of Dscam exon 9 variable exons.

Alternative splicing of pre-mRNA is a major mechanism to diversify protein functionality in metazoans from a limited number of genes. The Drosophila melanogaster Down syndrome cell adhesion molecule (Dscam) gene, which is important for neuronal wiring and phagocytosis of bacteria, can generate up to 38,016 isoforms by mutually exclusive alternative splicing in four clusters of variable exons. However, it is not understood how a specific exon is chosen from the many variables and how variable exons are prevented from being spliced together. A main role in the regulation of Dscam alternative splicing has been attributed to RNA binding proteins (RBPs), but how they impact on exon selection is not well understood. Serine-arginine rich (SR) proteins and hnRNP proteins are the two main types of RBPs with major roles in exon definition and splice site selection. Here, we analyzed the role of SR and hnRNP proteins in Dscam exon 9 alternative splicing in mutant Drosophila melanogaster embryos because of their essential function for development. Strikingly, loss or overexpression of canonical SR and hnRNP proteins even when multiple proteins are depleted together, does not affect Dscam alternative exon selection very dramatically. Conversely, noncanonical SR protein Serine-arginine repetitive matrix 2/3/4 (Srrm234) is a main determinant of exon inclusion in the Dscam exon 9 cluster. Since long-range base-pairings are absent in the exon 9 cluster, our data argue for a small complement of regulatory factors as main determinants of exon inclusion in the Dscam exon 9 cluster.

Plasmid-based gap-repair recombineered transgenes reveal a central role for introns in mutually exclusive alternative splicing in Down Syndrome Cell Adhesion Molecule exon 4.

Alternative splicing is a key feature of human genes, yet studying its regulation is often complicated by large introns. The Down Syndrome Cell Adhesion Molecule (Dscam) gene from Drosophila is one of the most complex genes generating vast molecular diversity by mutually exclusive alternative splicing. To resolve how alternative splicing in Dscam is regulated, we first developed plasmid-based UAS reporter genes for the Dscam variable exon 4 cluster and show that its alternative splicing is recapitulated by GAL4-mediated expression in neurons. We then developed gap-repair recombineering to very efficiently manipulate these large reporter plasmids in Escherichia coli using restriction enzymes or sgRNA/Cas9 DNA scission to capitalize on the many benefits of plasmids in phiC31 integrase-mediated transgenesis. Using these novel tools, we show that inclusion of Dscam exon 4 variables differs little in development and individual flies, and is robustly determined by sequences harbored in variable exons. We further show that introns drive selection of both proximal and distal variable exons. Since exon 4 cluster introns lack conserved sequences that could mediate robust long-range base-pairing to bring exons into proximity for splicing, our data argue for a central role of introns in mutually exclusive alternative splicing of Dscam exon 4 cluster.

Zc3h13/Flacc is required for adenosine methylation by bridging the mRNA-binding factor Rbm15/Spenito to the m6A machinery component Wtap/Fl(2)d.

N6-methyladenosine (m6A) is the most abundant mRNA modification in eukaryotes, playing crucial roles in multiple biological processes. m6A is catalyzed by the activity of methyltransferase-like 3 (Mettl3), which depends on additional proteins whose precise functions remain poorly understood. Here we identified Zc3h13 (zinc finger CCCH domain-containing protein 13)/Flacc [Fl(2)d-associated complex component] as a novel interactor of m6A methyltransferase complex components in Drosophila and mice. Like other components of this complex, Flacc controls m6A levels and is involved in sex determination in Drosophila We demonstrate that Flacc promotes m6A deposition by bridging Fl(2)d to the mRNA-binding factor Nito. Altogether, our work advances the molecular understanding of conservation and regulation of the m6A machinery.

Nucleotide modifications in messenger RNA and their role in development and disease.

Modified nucleotides in messenger RNA (mRNA) have been discovered over 40 years ago, but until recently little was known about which transcripts contain them and what their function is. High-throughput sequencing approaches revealed a dynamic landscape of the 'Epitranscriptome' for many mRNA modifications in various organisms from yeast to humans. Meanwhile, also many genes encoding mRNA-modifying enzymes and auxiliary proteins have been identified yielding functional insights by reverse genetics into their role in development and disease.

m6A potentiates Sxl alternative pre-mRNA splicing for robust Drosophila sex determination.

N6-methyladenosine (m6A) is the most common internal modification of eukaryotic messenger RNA (mRNA) and is decoded by YTH domain proteins. The mammalian mRNA m6A methylosome is a complex of nuclear proteins that includes METTL3 (methyltransferase-like 3), METTL14, WTAP (Wilms tumour 1-associated protein) and KIAA1429. Drosophila has corresponding homologues named Ime4 and KAR4 (Inducer of meiosis 4 and Karyogamy protein 4), and Female-lethal (2)d (Fl(2)d) and Virilizer (Vir). In Drosophila, fl(2)d and vir are required for sex-dependent regulation of alternative splicing of the sex determination factor Sex lethal (Sxl). However, the functions of m6A in introns in the regulation of alternative splicing remain uncertain. Here we show that m6A is absent in the mRNA of Drosophila lacking Ime4. In contrast to mouse and plant knockout models, Drosophila Ime4-null mutants remain viable, though flightless, and show a sex bias towards maleness. This is because m6A is required for female-specific alternative splicing of Sxl, which determines female physiognomy, but also translationally represses male-specific lethal 2 (msl-2) to prevent dosage compensation in females. We further show that the m6A reader protein YT521-B decodes m6A in the sex-specifically spliced intron of Sxl, as its absence phenocopies Ime4 mutants. Loss of m6A also affects alternative splicing of additional genes, predominantly in the 5' untranslated region, and has global effects on the expression of metabolic genes. The requirement of m6A and its reader YT521-B for female-specific Sxl alternative splicing reveals that this hitherto enigmatic mRNA modification constitutes an ancient and specific mechanism to adjust levels of gene expression.

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. Our 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.

Multiple pathways mediate the sex-peptide-regulated switch in female Drosophila reproductive behaviours.

Male-derived sex-peptide (SP) induces profound changes in the behaviour of Drosophila females, resulting in decreased receptivity to further mating and increased egg laying. SP can mediate the switch in female reproductive behaviours via a G protein-coupled receptor, SPR, in neurons expressing fruitless, doublesex and pickpocket. Whether SPR is the sole receptor and whether SP induces the postmating switch in a single pathway has not, to our knowledge been tested. Here we report that the SP response can be induced in the absence of SPR when SP is ectopically expressed in neurons or when SP, transferred by mating, can access neurons through a leaky blood brain barrier. Membrane-tethered SP can induce oviposition via doublesex, but not fruitless and pickpocket neurons in SPR mutant females. Although pickpocket and doublesex neurons rely on G(o) signalling to reduce receptivity and induce oviposition, G(o) signalling in fruitless neurons is required only to induce oviposition, but not to reduce receptivity. Our results show that SP's action in reducing receptivity and inducing oviposition can be separated in fruitless and doublesex neurons. Hence, the SP-induced postmating switch incorporates shared, but also distinct circuitry of fruitless, doublesex and pickpocket neurons and additional receptors.

ELAV-mediated 3'-end processing of ewg transcripts is evolutionarily conserved despite sequence degeneration of the ELAV-binding site.

Regulation of alternative mRNA processing by ELAV (embryonic lethal abnormal visual system)/Hu proteins is mediated by binding to AU-rich elements of low complexity. Since such sequences diverge very rapidly during evolution, it has not been clear if ELAV regulation is maintained over extended phylogenetic distances. The transcription factor Erect wing (Ewg) is a major target of ELAV in Drosophila melanogaster and coordinates metabolic gene expression with regulation of synaptic plasticity. Here, we demonstrate evolutionary conservation of ELAV regulation of ewg despite massive degeneration of its binding site and of associated elements in the regulated intronic 3'-end processing site in distantly related Drosophila virilis. In this species, the RNA-binding part of ELAV protein is identical to D. melanogaster. ELAV expression as well as expression and regulation of ewg are also conserved. Using in vitro binding assays and in vivo transgene analysis, we demonstrate, however, that the ELAV-binding site of D. virilis is fully functional in regulating alternative splicing of ewg intron 6 in D. melanogaster. Known features of the ELAV-binding site, such as the requirement of multiple poly(U) motifs spread over an extended binding site of ∼150 nt and a higher affinity to the 3' part of the binding site, are conserved. We further show that the 135-bp ELAV-binding site from D. melanogaster is sufficient for ELAV recruitment in vivo. Hence, our data suggest that ELAV/Hu protein-regulated alternative RNA processing is more conserved than anticipated from the alignment of degenerate low-complexity sequences.

Differential activity of EWG transcription factor isoforms identifies a subset of differentially regulated genes important for synaptic growth regulation.

The vast majority of genes in the human genome is alternatively spliced. The functional consequences of this type of post-transcriptional gene regulation that is particularly prominent in the brain, however, remains largely elusive. Here we analyzed the role of alternative splicing in the transcription factor erect wing (ewg) in Drosophila and dissect its function through differential rescue with transgenes encoding different isoforms. Transgenes expressing the SC3 ORF isoform fully rescue viability and synaptic growth defects. In contrast, transgenes expressing the ∆DJ isoform, that lack exons D and J, have a lower activity as inferred from their expression levels and exert reduced rescue of viability and synaptic growth defects. By comparison of the gene expression profile of ewg(l1) mutants rescued either by the SC3 ORF or the ∆DJ transgene, we identified a set of genes whose expression is exclusively restored by the SC3 isoform. These genes are mostly involved in regulating gene expression while a core function of EWG is indicated by the regulation of metabolic genes by both isoforms. In conclusion, we demonstrated that differential rescue with different isoform encoding transgenes of the transcription factor EWG identifies a unique set of genes associated with synaptic growth regulation.

Determinants of ELAV gene-specific regulation.

How RNA-binding proteins recognize their complement of targets in a complex cellular environment remains poorly understood. Sequence degeneracy and redundancy of short motifs at genomic scales have mostly eluded predictions of specific target genes for gene-specific ELAV (embryonic lethal abnormal visual system)/Hu proteins that bind ubiquitous AU-rich motifs. Using the genetic tools of Drosophila, we have analysed binding properties of ELAV in vitro and ELAV-dependent regulation of its major target ewg (erect wing) in neurons. These studies reveal that an integral part of ELAV gene-specific regulation involves combinatorial binding to variably spaced short U-rich motifs on an extensive binding site.

Regulation of the ELAV target ewg: insights from an evolutionary perspective.

ELAV (embryonic lethal abnormal visual system)/Hu family proteins are prototype RNA-binding proteins with binding preferences for AU-rich regions. Due to frequent occurrence of AU-rich motifs in introns and untranslated regions, it is poorly understood how gene-specific RNA-binding proteins, such as ELAV/Hu family members, recognize their complement of target RNAs in a complex cellular environment. The powerful genetic tools of Drosophila make the fruitfly an excellent model to study alternative mRNA processing in vivo in a developing organism. Recent sequencing of 12 Drosophila genomes will provide a novel resource to enhance our understanding of how gene-specific regulation of mRNA processing is achieved by ELAV/Hu family proteins.