Coupling between alternative polyadenylation and alternative splicing is limited to terminal introns

Alternative polyadenylation (APA) has been implicated as an important regulator of gene expression. In some cases APA is known to couple with alternative splicing to influence last intron removal, however it is unknown whether APA events influence alternative splicing decisions at upstream exons. A genome-wide approach was used to examine the correlation between APA and upstream alternative splicing. CstF64 knockdown in HeLa cells coupled with Pas-Seq was used to trigger and identify APA events. APA genes were then evaluated for changes in alternative splicing using RNA-seq analyses of the same knockdown samples. Although a significant number of alternative splicing events were identified, no general correlation between APA and upstream alternative splicing events were observed. These results suggest that the coupling and diversification achieved between APA and alternative splicing in general, is fixed to defining the last exon. iClip-Seq experiments identified CstF64 binding to be predominantly in intronic regions. Interestingly, the genome-wide binding analysis also showed that CstF64 density is elevated upstream of skipped exons indicating a potential role for CstF64 in alternative splicing. We conclude that while the influence of APA on alternative splicing is generally limited to terminal introns, CstF64 binding to nascent pre-mRNA may contribute to modulate splicing patterns.