Non-canonical splice junction processing increases the diversity of RBFOX2 splicing isoforms.

Abstract

The underlying mechanisms of splicing regulation through non-canonical splice junction processing remain largely unknown. Here, we identified two RBFOX2 splicing isoforms by alternative 3' splice site selection of exon 9; the non-canonical splice junction processed RBFOX2 transcript (RBFOX2-N.C.) was expressed by the selection of the 3' splice GG acceptor sequence. The cytoplasmic localization of RBFOX2-C., a canonical splice junction-processed RBFOX2 transcript, was different from that of RBFOX2-N.C., which showed nuclear localization. In addition, we confirmed that RBFOX2-C. showed a significantly stronger localization into stress granules than RBFOX2-N.C. upon sodium arsenite treatment. Next, we investigated the importance of non-canonical 3' splice GG sequence selection of specific cis-regulatory elements using minigene constructs of the RBFOX2 gene. We found that the non-canonical 3' splice GG sequence and suboptimal branch point site adjacent region were critical for RBFOX2-N.C. expression through a non-canonical 3' splice selection. Our results suggest a regulatory mechanism for the non-canonical 3' splice selection in the RBFOX2 gene, providing a basis for studies related to the regulation of alternative pre-mRNA splicing through non-canonical splice junction processing.