Conserved intronic secondary structures with concealed branch sites regulate alternative splicing of poison exons

Alternative splicing (AS) generates large amounts of RNA isoforms and dramatically increases the complexities of transcriptomes and proteomes. However, it remains unclear how RNA structures contribute to AS regulation. Here, we systematically search transcriptomes for secondary structures with concealed branch-sites (BSs) in the alternatively spliced introns and predict thousands from six organisms, of which many are evolutionarily conserved. Intriguingly, a highly conserved stem-loop structure with concealed BSs is found in animal SF3B3 genes and colocalizes with a downstream poison exon (PE). Destabilization of this structure allows increased usage of the branch sites and results in enhanced PE inclusion in both human and Drosophila cells, leading to decreased expression of SF3B3. Through an RNAi screen of 28 RNA-binding proteins, we find that this stem-loop structure is sensitive to U2 and U2-associated proteins. Furthermore, we find that SF3B3 also facilitates DNA repair and protects genome stability by enhancing interaction between ERCC6/CSB and arrested RNA polymerase II. Importantly, both Drosophila and human cells with the secondary structure mutated by genome editing exhibit altered DNA repair in vivo. This study provides a novel and common mechanism for AS regulation of PEs and reveals a physiological function of SF3B3 in DNA repair.