Mechanism of ALE-APA control by ELAV/Hu RBPs is shared with TUTR-APA targets.

(A-B) Validation of new terminal 3’ UTR-APA (TUTR-APA) targets in the S2-GOF system. The 3’ UTR extensions of tai and ctp can be detected in chromatin-associated fractions (A) and in nascent transcripts isolated using 4sU labeling (B). (C) Distal ALE switching induced by GOF of ELAV/Hu RBPs also occurs in chromatin-associated fractions, but is not detected in RNA binding-defective Elav or with the homolog RBP Sxl. (D) Distal ALE switching induced by GOF of ELAV/Hu RBPs is detected in newly-synthesized transcripts. (E-I) Nucleotide and motif analysis of regions surrounding proximal ALE polyadenylation (pA) sites in S2 cells. (E-F) Nucleotide frequency of ALE cleavage sites that are bypassed by ELAV/Hu RBP-GOF (E) or that are unchanged (F) shows enrichment of downstream uridine amongst regulated loci. (G) Elav binding sites (80% match to position weight matrix, PWM80) are enriched downstream of regulated proximal ALE pA sites. (H) De novo search shows the Elav binding site is the most frequent and most significant motif downstream of regulated ALE pA sites. (I) Elav binding sites are correlated with the frequency of bypass as measured by distal isoform induction in the presence of ectopic Elav in S2 cells. (J-N) Nucleotide and motif analysis of regions surrounding proximal ALE pA sites in L1-CNS. (J-K) Nucleotide frequency of ALE cleavage sites that are switched from distal to proximal usage by elav/fne-LOF (J) or that are unchanged (K) shows enrichment of downstream uridine amongst regulated loci. (L) Elav binding sites are enriched downstream of regulated proximal ALE pA sites. (M) De novo search shows the Elav binding site is the most frequent and most significant motif downstream of endogenously regulated ALE pA sites in L1-CNS. (N) Elav binding sites correlate with distal-to-proximal ALE isoform switching in elav/fne mutant L1-CNS.