Multifactorial interplay controls the splicing profile of Alu-derived exons.

Exonization of Alu elements creates primate-specific genomic diversity. Here we combine bioinformatic and experimental methodologies to reconstruct the molecular changes leading to exon selection. Our analyses revealed an intricate network involved in Alu exonization. A typical Alu element contains multiple sites with the potential to serve as 5' splice sites (5'ss). First, we demonstrated the role of 5'ss strength in controlling exonization events. Second, we found that a cryptic 5'ss enhances the selection of a more upstream site and demonstrate that this is mediated by binding of U1 snRNA to the cryptic splice site, challenging the traditional role attributed to U1 snRNA of binding the 5'ss only. Third, we used a simple algorithm to identify specific sequences that determine splice site selection within specific Alu exons. Finally, by inserting identical exons within different sequences, we demonstrated the importance of flanking genomic sequences in determining whether an Alu exon will undergo exonization. Overall, our results demonstrate the complex interplay between at least four interacting layers that affect Alu exonization. These results shed light on the mechanism through which Alu elements enrich the primate transcriptome and allow a better understanding of the exonization process in general.