A high-throughput screening strategy identifies regulators of alternative splicing via interaction with RNA G-quadruplexes

RNA secondary structures have been increasingly reported to serve critical regulatory roles in post-transcriptional gene regulation. RNA G-quadruplex secondary structures can serve as cis-elements to recruit splicing factors and regulate alternative RNA splicing. We recently showed that RNA G-quadruplexes play a critical regulatory role in regulating alternative splicing during the epithelial mesenchymal transition. Due to the critical role alternative splicing plays in human health and disease, an unmet need exists to identify small molecule modulators of alternative splicing. In this study, we performed high-throughput screening using a dual-output splicing reporter to identify small molecules capable of regulating alternative splicing by interacting with RNA secondary structure G-quadruplexes. We identify emetine and its analog cephaeline as small molecules that denature RNA G-quadruplexes in a sequence and location independent manner to modify alternative splicing. Transcriptome analysis reveals that treatment with emetine globally regulates alternative splicing, including events associated with exon-proximal G-quadruplexes. These data suggest a critical role for emetine and cephealine as splicing regulators with the selective ability to disrupt RNA G-quadruplex-associated alternative splicing in vivo. PolyA-RNA-sequencing of control and emetine treated cell lines with two biological replicates each