RALY regulates alternative splicing through targeting poly-uridine sequences

RBP associated with lethal yellow mutation (RALY) is a member of the heterogeneous nuclear ribonucleoprotein family (hnRNP). Although the role of RALY in splicing has been studied in a few specific genes, its regulatory mechanisms partially remain unclear. Here, we applied high-throughput RNA sequencing (RNA-seq) to identify global alternative splicing targets of RALY. Our results showed that RALY significantly affected exon skipping, while it had less influence on other events. To investigate the cis-elements controlling RALY-mediated splicing regulation, we generated a reporter based on the Fas minigene and demonstrated that RALY regulates splicing by targeting poly-uridine regions on pre-mRNA molecules. Moreover, analysis of various structural domains present in RALY showed that all of the domains in RALY are required for its splicing functions, whereas only the variable domain and the basic domain are necessary for its nuclear localization. Finally, we investigated RALY expression across different tumor types and characterized alternative splicing alterations associated with RALY depletion in cell culture. Our reporter-based and in vitro results show RALY can mediate splicing regulation via poly-uridine-rich sequences in reconstituted experimental systems, and multiple RALY domains are indispensable for its splicing activity.