Phylogenomic instructed target analysis reveals ELAV complex binding to multiple optimally spaced U-rich motifs

ELAV/Hu RNA-binding proteins are gene-specific regulators of alternative pre-mRNA processing. ELAV/Hu family proteins bind to short AU-rich motifs which are abundant in pre-mRNA, making it unclear how they achieve gene specificity. ELAV/Hu proteins multimerize, but how multimerization contributes to decode degenerate sequence environments remains uncertain. Here, we show that ELAV forms a saturable complex on extended RNA. Through phylogenomic instructed target analysis we identify the core binding motif U₅N₂U₃, which is repeated in an extended binding site. Optimally spaced short U₅N₂U₃ binding motifs are key for high-affinity binding in this minimal binding element. Binding strength correlates with ELAV-regulated alternative poly(A) site choice, which is physiologically relevant through regulation of the major ELAV target ewg in determining synapse numbers. We further identify a stem–loop secondary structure in the ewg binding site unwound upon ELAV binding at three distal U motifs. Base-pairing of U motifs prevents ELAV binding, but N⁶-methyladenosine (m⁶A) has little effect. Further, stem–loops are enriched in ELAV-regulated poly(A) sites. Additionally, ELAV can nucleate preferentially from 3′ to 5′. Hence, we identify a decisive mechanism for ELAV complex formation, addressing a fundamental gap in understanding how ELAV/Hu family proteins decode degenerate sequence spaces for gene-specific mRNA processing.