The phylogenetic functional conservation ofDrosophilaSeven-In-Absentia (SINA) E3 ligase and its two human paralogs, SIAH1 and SIAH2, inDrosophilaeye development

Abstract

AbstractSeven-IN-Absentia (SINA) is the most downstream signaling gatekeeper identified thus far in the RAS/EGFR pathway that controls photoreceptor cell fate determination inDrosophila. Underscoring the central importance of SINA is its phylogenetic conservation in metazoans, with over 83% amino acid identities shared betweenDrosophilaSINA 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 newsinamutant alleles were isolated via ethyl methanesulfonate (EMS) and X-ray mutagenesis. Among them, 26 of the newsinamutants are embryonic, larval, or pupal lethal, and stronger than the five publishedsinamutants (sina1,sina2,sina3,sina4, andsina5) which are early adult lethal. By sequencing the SINA-coding region ofsinaES10,sinaES26,sinaES79, andsinaES473homozygous mutant animals, we identified three invariable amino acid residues in SINA’s RING-domain whose single point mutation ablates SINA function. To demonstrate the functional conservation of this medically important family of RING domain E3 ligases inDrosophila, we established a collection of transgenic lines, expressing either wild type (WT) or proteolysis-deficient (PD) SINA/SIAH inhibitors ofDrosophilaSINAWT/PDand human SIAH1WT/PD/2WT/PDunder tissue-specific GAL4-drivers inDrosophilaeye, wing, and salary gland. Our results showed thatDrosophilaSINA and human SIAH1/2 are functionally conserved. Our bioengineered SINAPD/SIAHPDinhibitors are effective in blocking the RAS-dependent neuronal cell fate determination inDrosophila.