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.