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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.

ELAV-Mediated 3′-End Processing of ewg Transcripts Is Evolutionarily Conserved Despite Sequence Degeneration of the ELAV-Binding Site