The phylogenetic functional conservation of Drosophila Seven-In-Absentia (SINA) E3 ligase and its two human paralogs, SIAH1 and SIAH2, in Drosophila eye development

Seven-IN-Absentia (SINA) is the most downstream signaling gatekeeper identified thus far in the RAS/EGFR pathway that controls photoreceptor cell fate determination in Drosophila. Underscoring the central importance of SINA is its phylogenetic conservation in metazoans, with over 83% amino acid identities shared between Drosophila SINA and human SINA homologs (SIAHs). SIAH is a major tumor vulnerability in multidrug-resistant and incurable cancer. SIAH inhibition is an effective strategy to shut down the tumor-driving K-RAS/EGFR/HER2 pathway activation that promotes malignant tumor growth and metastatic dissemination. To further delineate the SINA function in the RAS/EGFR pathway, a genetic modifier screen was conducted, and 28 new sina mutant alleles were isolated via ethyl methanesulfonate (EMS) and X-ray mutagenesis. Among them, 26 of the new sina mutants are embryonic, larval, or pupal lethal, and stronger than the five published sina mutants (sina1, sina2, sina3, sina4, and sina5) which are early adult lethal. By sequencing the SINA-coding region of sinaES10, sinaES26, sinaES79, and sinaES473 homozygous mutant animals, we identified three invariable amino acid residues in SINAs RING-domain whose single point mutation ablates SINA function. To demonstrate the functional conservation of this medically important family of RING domain E3 ligases in Drosophila, we established a collection of transgenic lines, expressing either wild type (WT) or proteolysis-deficient (PD) SINA/SIAH inhibitors of Drosophila SINAWT/PD and human SIAH1WT/PD/2WT/PD under tissue-specific GAL4-drivers in Drosophila eye, wing, and salary gland. Our results showed that Drosophila SINA and human SIAH1/2 are functionally conserved. Our bioengineered SINAPD/SIAHPD inhibitors are effective in blocking the RAS-dependent neuronal cell fate determination in Drosophila.