Objective
In major animal model organisms, the germ line is established by action of localized oocyte determinants named germ plasm, which induce the cells inheriting them to become primordial germ cells (PGCs). One of the important components of germ plasm is a set of germ plasm-associated RNAs. In Xenopus, these RNAs are localized to the vegetal pole by the so-called early pathway. Very little is known about the mechanisms controlling formation of oocytes, germ plasm and PGCs in vertebrates. Also, no proteins with established role in the early localization pathway have yet been described. We recently identified a novel RNA named Germes that belongs to a class of early localizing germ plasm-associated RNAs. Overexpression of certain Germes protein mutants results in abnormalities of PGC number and migration. Using yeast two-hybrid system, we identified dlc8 dynein light chains as prominent protein partners of Germes. This finding was confirmed by in vivo co-localization and co-immunoprecipitation studies. 3' untranslated region of Germes RNA was found to be responsible for correct localization in oocytes. UV crosslinking experiments identified four early oocyte-specific proteins, which interact with Germes RNA but not with late-localizing Vg1 RNA. A well established role of dlc8 proteins in Drosophila oogenesis, in particular in macromolecule transport and localization, control of oocyte fate and polarity, highlights a potential function of Germes as a dlc8 interacting partner during oocyte formation and embryogenesis. The proposed project is aimed at dissection of oocyte and germ plasm formation in Xenopus through identification and characterization of macromolecular complexes that contain Germes protein or RNA. For isolation of Germes-interacting proteins, we will use a one- or two-step purification protocol using Tandem Affinity Purification tags, a trusted strategy for analysis of complex molecular interactions. Transgenic frogs expressing TAP-tagged Germes protein will be created using bacterial artificial chromosomes constructs. After affinity isolation of the Germes complexes followed by electrophoretic separation, the individual proteins will be identified by mass-spectrometry. For characterization of proteins interacting with Germes 3' UTR, we will affinity isolate RNA-protein complexes on the Streptavidin matrix. Using electrophoretic comparison of protein sets binding to early localizing Germes RNA and late localizing Vg1 RNA, we expect to detect proteins specifically interacting with Germes RNA, and then perform their molecular identification. Further experiments will involve verification of existence of identified protein-protein and RNA-protein interactions in vivo. As a result of these studies, detailed information on macromolecular complexes containing Germes RNA or protein will be obtained, which is likely to substantially advance our knowledge of oocyte, germ plasm and PGC formation, and the mechanisms of RNA localization in oocytes.
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Keywords
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Project’s keywords as indicated by the project coordinator. Not to be confused with the EuroSciVoc taxonomy (Fields of science)
Programme(s)
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Multi-annual funding programmes that define the EU’s priorities for research and innovation.
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Calls for proposals are divided into topics. A topic defines a specific subject or area for which applicants can submit proposals. The description of a topic comprises its specific scope and the expected impact of the funded project.
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Coordinator
COVENTRY
United Kingdom
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