| dc.description.abstracteng | Reproduction is an essential feature of life that ensures the perpetuation and survival in many multicellular organisms while passing the genetic information to the next generation. The zebrafish germline is specified during early embryogenesis by maternal RNP granules collectively called germ plasm. It has been discovered that the zebrafish bucky ball gene, which is a key gene responsible for germ plasm formation and encodes a novel vertebrate-specific protein with unknown biochemical function. Overexpression experiments revealed that Buc induces ectopic primordial germ cells (PGCs) in vivo. So far, only the Oskar protein in Drosophila shows equivalent activities. Remarkably, we recently revealed that Oskar induces ectopic PGCs in zebrafish, albeit Oskar does not share sequence homology with Buc. Moreover, zebrafish Vasa is also sufficient to induce PGCs in vivo suggesting that Buc and Vasa act in the same molecular process involving in germ cell specification. This conserved activity between Oskar and Buc suggests that both proteins share a similar biochemical interactome, whose molecular identity is unknown.
In this study, I characterize the interaction of Buc and the RNA helicase Vasa investigating their binding motifs. I identified a peptide encoding amino acids 372-394 in Buc as Vasa Binding Motif (VBM) and a peptide encoding amino acids 600-625 in Vasa as Buc Binding Motif (BBM) using bimolecular fluorescence complementation (BiFC). Further, I show that the Buc-VBM is highly conserved in vertebrates. Moreover, Buc and Vasa directly interact in vitro and independent of RNA. The Buc-VBM encodes an intrinsically disordered peptide, but CD spectroscopy reveals that a crowding agent induces formation of an alpha-helix. Fascinatingly, I discovered that Buc activates Vasa ATPase activity annotating the first biochemical function of Buc as a helicase activator. Collectively, these results propose a model in which the activity of Vasa helicase is a central regulator of PGC formation, which is tightly controlled by the germ plasm organizer Buc. | de |