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

Abstract

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 39-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 39 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.

Concentration and Localization of Coexpressed ELAV/Hu Proteins Control Specificity of mRNA Processing

“…An alternative route to bind target genes has been suggested through recruitment at the promoter and deposition by elongating RNA polymerase II (Pol II) (73). Our data from using heterologous promoters for expressing ewg and nrg reporters, however, argue against this possibility for these genes but might affect a minority of large genes (40,48,74).…”

“…Next, we determined the RNA binding specificities of ELAV family members in vitro using the well-characterized ELAV binding sequence in the ewg gene (pA-I), which comprises 135 bp (20,36,40). For these binding experiments, we generated recombinant proteins in Escherichia coli for ELAV, FNE, and RBP9 and also for human HuR because it is functionally closest to ELAV family proteins in Drosophila ( Fig.…”

“…Similar results were obtained for Drosophila ELAV proteins (ELAV, FNE, and RBP9) as well as for human HuR when the extended ELAV binding site in the ewg gene was used. In this ELAV target RNA, a number of short U-rich motifs are interspersed along the 135-nt binding site, but they do not have a fixed position, eluding RNA secondary structure to contribute to target selectivity (20,36,40). Likewise, when artificially expressed in nonneuronal larval wing disc cells or during eye development, ELAV/Hu proteins can regulate neuron-specific splicing of ELAV target genes and substitute for ELAV in eye development.…”

“…Further details about mutant alleles and gene expression patterns can be found in FlyBase (www.flybase.org). Transgenic flies were obtained by C31-mediated transformation as described previously (40) using landing sites at 57F (RBP9 genomic construct, PBac{y ϩ -attP-3B}VK22), 76A (UAS construct, PBac{y ϩ -attP-3B}VK00002), and 55C (genomic rescue construct, P{y[lsqb]ϩt7. 7[rsqb]ϭCaryP}attP1).…”

Acute Thiamethoxam exposure in Apis mellifera : Absence of both stress-induced changes in mRNA splicing and synergistic effects of common fungicide and herbicide

“…ELAV regulates alternative splicing by binding to AU-rich motifs, which are abundant in introns and untranslated regions 51 . The Drosophila genome has three members of the ELAV family of proteins, while the honeybee genome encodes only one ELAV protein 30,52,53 . ELAV proteins have prominent roles in regulating synaptic plasticity [53][54][55][56][57] .…”