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Accurate splice site selection is critical for fruitful gene expression. Recently, the mammalian EJC was shown to repress competing, cryptic, splice sites (SS). However, the evolutionary generality of this remains unclear. Here, we demonstrate the Drosophila EJC suppresses hundreds of functional cryptic SS, even though most bear weak splicing motifs and are seemingly incompetent. Mechanistically, the EJC directly conceals cryptic splicing elements by virtue of its position-specific recruitment, preventing aberrant SS definition. Unexpectedly, we discover the EJC inhibits scores of regenerated 5’ and 3’ recursive SS on segments that have already undergone splicing, and that loss of EJC regulation triggers faulty resplicing of mRNA. An important corollary is that certain intronless cDNA constructs yield unanticipated, truncated transcripts generated by resplicing. We conclude the EJC has conserved roles to defend transcriptome fidelity by (1) repressing illegitimate splice sites on pre-mRNAs, and (2) preventing inadvertent activation of such sites on spliced segments., Author summary The Exon Junction Complex (EJC) is a conserved multiprotein complex that is deposited ~20–24 nucleotides upstream of exon-exon junctions during mRNA splicing. Although the EJC is well-conserved, many of its overt regulatory requirements differ between species. For example, the mammalian EJC is involved in mRNA surveillance and nonsense mediated decay (NMD), and also suppresses cryptic splicing. On the other hand, the Drosophila EJC does not mediate NMD, and it has multiple roles in promoting splicing of long introns and suboptimal splicing substrates. Here, we unify this by showing that the Drosophila EJC suppresses splicing at hundreds of illegitimate cryptic splice sites, which are presently unannotated in the well-studied Drosophila genome. As in mammals, this role takes advantage of the sequence-independent deposition of the EJC upstream of splice sites, and appears to represent an ancestral function. We expand this concept by showing the necessity of the EJC to prevent resplicing in exonic remnants that inherently regenerate splice sites following canonical splicing. Importantly, cDNA expression constructs evade EJC regulation, and we show that utilization of cDNAs can unintentionally trigger re-splicing into unanticipated products with internal deletions.

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 our recent findings that neural-specific lengthened 3’ UTR isoforms are co-determined by ELAV/Hu factors, we address 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. We corroborate the direct action of Elav in diverse modes of mRNA processing using RRM-dependent Elav-CLIP data from S2 cells. Finally, we provide evidence for conservation in mammalian neurons, which undergo broad programs of distal ALE and APA lengthening, linked to ELAV/Hu motifs downstream of regulated polyadenylation sites. Overall, ELAV/Hu RBPs orchestrate multiple broad programs of neuronal mRNA processing and isoform diversification in Drosophila and mammalian neurons., Author summary ELAV/Hu factors comprise a conserved family of RNA binding proteins (RBPs), many of which are enriched or restricted to neurons. This study shows that overlapping activities of Drosophila ELAV/Hu RBPs determine global patterns of neural alternative splicing, including of cassette exons and alternative last exon (ALE) isoforms. This is supported by both genetic necessity in double mutant CNS, as well as their sufficiency to drive these mRNA processing changes in a non-neuronal setting. The ability of ELAV/Hu RBPs to induce the usage of distal ALE isoforms connects to their recently described functions in global extension of 3’UTRs in neural isoforms. Evidence is provided that switching to distal alternative last exons and to terminal 3’ UTR extensions are also coordinated in mammalian neurons, and show signatures of direct regulation by ELAV/Hu RBPs.

While most miRNA knockouts exhibit only subtle defects, a handful of miRNAs are profoundly required for development or physiology. A particularly compelling locus is Drosophila mir-279, which was reported as essential to restrict the emergence of CO2-sensing neurons, to maintain circadian rhythm, and to regulate ovarian border cells. The mir-996 locus is located near mir-279 and bears a similar seed, but they otherwise have distinct, conserved, non-seed sequences, suggesting their evolutionary maintenance for separate functions. We generated single and double deletion mutants of the mir-279 and mir-996 hairpins, and cursory analysis suggested that miR-996 was dispensable. However, discrepancies in the strength of individual mir-279 deletion alleles led us to uncover that all extant mir-279 mutants are deficient for mature miR-996, even though they retain its genomic locus. We therefore engineered a panel of genomic rescue transgenes into the double deletion background, allowing a pure assessment of miR-279 and miR-996 requirements. Surprisingly, detailed analyses of viability, olfactory neuron specification, and circadian rhythm indicate that miR-279 is completely dispensable. Instead, an endogenous supply of either mir-279 or mir-996 suffices for normal development and behavior. Sensor tests of nine key miR-279/996 targets showed their similar regulatory capacities, although transgenic gain-of-function experiments indicate partially distinct activities of these miRNAs that may underlie that co-maintenance in genomes. Altogether, we elucidate the unexpected genetics of this critical miRNA operon, and provide a foundation for their further study. More importantly, these studies demonstrate that multiple, vital, loss-of-function phenotypes can be rescued by endogenous expression of divergent seed family members, highlighting the importance of this miRNA region for in vivo function., Author Summary Amongst the small number of miRNA knockouts that exhibit substantially overt phenotypes, mutants of Drosophila mir-279 are notable. Previous studies have uncovered its essential requirements in a range of developmental and behavioral assays. Surprisingly, we find that the phenotypes attributed to mir-279 deletions depend on the unanticipated loss of expression of the downstream locus mir-996, whose genomic locus is retained in extant mir-279 mutants. These miRNAs share their seed regions but are divergent elsewhere in the mature sequences. We use precise genetic engineering to show that a single endogenous copy of either mir-279 or mir-996 can fully rescue viability, olfactory neuron, and circadian rhythm defects of double deletion animals. These data and genetic reagents set a new foundation for developmental and behavioral studies of this critical miRNA locus. More generally, these data demonstrate that multiple loss-of-function phenotypes can be rescued by endogenous expression of divergent seed family members, highlighting the importance and potentially sufficiency of this region for in vivo function.

miR-124 is conserved in sequence and neuronal expression across the animal kingdom and is predicted to have hundreds of mRNA targets. Diverse defects in neural development and function were reported from miR-124 antisense studies in vertebrates, but a nematode knockout of mir-124 surprisingly lacked detectable phenotypes. To provide genetic insight from Drosophila, we deleted its single mir-124 locus and found that it is dispensable for gross aspects of neural specification and differentiation. On the other hand, we detected a variety of mutant phenotypes that were rescuable by a mir-124 genomic transgene, including short lifespan, increased dendrite variation, impaired larval locomotion, and aberrant synaptic release at the NMJ. These phenotypes reflect extensive requirements of miR-124 even under optimal culture conditions. Comparison of the transcriptomes of cells from wild-type and mir-124 mutant animals, purified on the basis of mir-124 promoter activity, revealed broad upregulation of direct miR-124 targets. However, in contrast to the proposed mutual exclusion model for miR-124 function, its functional targets were relatively highly expressed in miR-124–expressing cells and were not enriched in genes annotated with epidermal expression. A notable aspect of the direct miR-124 network was coordinate targeting of five positive components in the retrograde BMP signaling pathway, whose activation in neurons increases synaptic release at the NMJ, similar to mir-124 mutants. Derepression of the direct miR-124 target network also had many secondary effects, including over-activity of other post-transcriptional repressors and a net incomplete transition from a neuroblast to a neuronal gene expression signature. Altogether, these studies demonstrate complex consequences of miR-124 loss on neural gene expression and neurophysiology., Author Summary microRNAs are abundant ∼22 nucleotide RNAs inferred to mediate pervasive post-transcriptional control of most genes. Still, relatively little is understood about their endogenous requirements and impact, especially in animal systems. We analyzed a knockout of Drosophila mir-124, which is conserved in sequence and neuronal expression across the animal kingdom, and predicted to have hundreds of mRNA targets. While dispensable for gross neural specification and differentiation, deletion of mir-124 caused short lifespan, increased variation in dendrite numbers, impaired larval locomotion, and aberrant synaptic release at the NMJ. These phenotypes reflect extensive requirements of miR-124 even under optimal culture conditions. Loss of miR-124 broadly upregulated its direct targets but did not support the proposed mutual exclusion model, as its functional target genes were relatively highly expressed in neurons. One notable aspect of the direct miR-124 network was coordinate targeting of five positive components in the retrograde BMP signaling pathway, whose activation in neurons phenocopies loss of miR-124. Derepression of the direct miR-124 target network had many secondary effects, including over-activity of other post-transcriptional repressors and impaired transition from neuroblast to neuronal transcriptome signatures. Altogether, we demonstrate complex requirements for this conserved miRNA on gene expression and neurophysiology.