Novel functions of Sex-lethal interactor (Sin) and minisomes

Abstract

Part 1.Sol-narae (Sona) is a Drosophila ADAMTS (a disintegrin and metalloproteinase with thrombospondin domains) that promotes Wg signaling by positively regulating secretion of Wg and processing extracellular Wg. A genetic screen for suppressors of Sona-induced lethality identified a suppressor that has a mutation in the Sex-lethal interactor (Sin) gene. Nothing has been known for Sin except its physical interaction with Sex-lethal protein. Drosophila Sin is quite homologous with RPC5, a mammalian RNA polymerase III subunit. I found that Sin mutants show apoptosis and defects in PCP signaling. Similar to Sona, Sin was identified in the exosome fraction and was secreted in wing discs under stress conditions such as gamma-ray irradiation. Human RPC5 was also present in the exosome fraction and its secretion was increased by UV irradiation and heat-shock treatment. Hence, I propose that Sin is involved in cell survival and secrets exosomes to cope against environmental stresses in order to promote cell survival.Part II.Extracellular vesicles play important roles in intercellular communication by delivering RNA, lipid, and proteins to neighboring or distant cells. Identification and classification of extracellular vesicles secreted from diverse cell types are essential for understanding their signaling properties. Here, I identified a new group of small extracellular vesicles that I named minisomes. Minisomes were isolated by a subsequent ultracentrifugation of the supernatant fraction after the conventional exosome sepaation. Electron microscopy and nanoparticle tracking analyses showed that minisomes are smaller than exosomes. Typical exosome markers such as Hsp70, TSG101, and CD63 were found in both exosomes and minisomes. However, CD81 was highly enriched in exosomes but not in minisomes, suggesting that minisomes belong to a distinguished group of extracellular vesicles. Furthermore, chemicals that inhibit major exosome production pathways did not decrease minisome release, indicating that the biogenesis pathways are also distinct. Exosomes and minisomes were found in conditioned media of human cell lines as well as fetal bovine serum. Addition of exosomes and minisomes to serum-free media enhanced exosome production and cell proliferation of tissue culture cells, respectively. Our study for the first time identifies minisomes and their specific cellular functions.