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 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.