2007 — 2009 |
Voronina, Ekaterina |
F32Activity Code Description: To provide postdoctoral research training to individuals to broaden their scientific background and extend their potential for research in specified health-related areas. |
Components of C. Elegans P-Granules and Regulation of Their Function @ Johns Hopkins University
[unreadable] DESCRIPTION (provided by applicant): Germ granules are evolutionary conserved ribonucleoprotein complexes necessary for fertility present in animal germ cells. The full complement of their components is not known in any organism, but many of discovered components are conserved from C. elegans, to Drosophila, to mouse and human. This proposal focuses on identifying new components and regulators of germ granule function in a model system C. elegans. This will be carried out by a combination of genetic screening and proteomic approaches. First, I will use RNAi to functionally screen the germline-expressed genes and assess localization of fluorescently tagged P granules of C. elegans. For this visual screen, I will use two reagents already available in the Seydoux lab: a strain of C. elegans expressing a GFP fusion to the germ granule component PGL-1, and an RNAi library targeting -3,000 genes expressed preferentially in the female germline. In a second approach, I will use biochemical methods to isolate P granule complexes. I will tag P granules in select germ cell sub- types (adult gonadal stem cells and embryonic primordial germ cells) by generating transgenic worms expressing tagged P granule components PGL-1 and GLH-1 in the germline under the control of defined regulatory elements. This will permit biochemical purification of populations of P granules specific to mitotic germline of embryonic primordial germ cells. The protein components of the isolated complexes will be identified by mass-spectrometry. Validity of candidate interactors will be assessed by alternative methods, such as yeast two-hybrid assays or in-vitro GST-pulldown assays. Positives resulting from both RNAi and proteomic screens will be studied in further detail: protein localization survey will assess whether any of these contribute to P granules themselves, and their function will be analyzed by disrupting gene function in vivo, by RNAi, or by expression of dominant-interfering constructs. Conservation of known germ granule components from invertebrates to vertebrates suggests that the results of these studies will advance our understanding of germline development in a broad array of species. In a number of reported cases, disruption of germ cell function results not only in lack of fertility, but also leads to malignant transformations (cancers) in the affected individuals. Advanced knowledge of P granule members and regulators will bring forth improved understanding of both fertility as well as malignancies in humans. [unreadable] [unreadable] [unreadable]
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0.966 |
2014 — 2018 |
Voronina, Ekaterina |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
P Granules and Control of Germ Cells Development
Abstract Germ cells carry out the reproductive function of the multicellular organisms, and normal germ cell development ensures survival of the species. Germ granules are conserved cytoplasmic organells of the germ cells, essential for the survival, differentiation, and function of these cells. Mutations in germ granule components or loss of their expression lead to infertility in model organisms and are associated with infertility in humans. By contrast, inappropriate expression of germ granule components in somatic cells is linked to carcinogenesis in humans. Many RNA-binding proteins and developmentally regulated mRNAs are found enriched in the germ granules, leading to a hypothesis that these organelles function in regulation of mRNA stability or translational activity; yet, the molecular function of these organelles is still undefined. The nematode C. elegans has been instrumental for understanding translational regulation of germline development. Our previous studies demonstrated specific contribution of C. elegans germ granules (P granules) to the regulation exerted by an RNA-binding regulatory protein FBF-2 in germline stem cells, yet much remains to be learned about the mechanistic basics of this contribution. Our experimental system is poised to address this question in molecular detail. Our studies will focus on FBF-2 as a paradigm of germ granule contribution to regulating the activity of RNA-binding proteins in germline. By integrating biochemical, molecular, genetic, and imaging-based approaches, we will: 1) Define the specific components of the FBF-2 regulatory complex; 2) Identify the cofactors of FBF-2 that depend on P granule integrity for their assembly with FBF-2; 3) Determine the sequences of FBF-2 mediating P granule recruitment and FBF-2-specific regulatory activity. These studies will reveal general mechanisms of germ granule-dependent regulation. Since the germ granules are conserved organelles and FBF-2 is a member of conserved PUF protein family, studies in this model system will provide critical insight into the causes of infertility in humans.
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0.966 |
2019 — 2021 |
Voronina, Ekaterina |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
P Granules and Control of Germ Cell Development
Abstract Germ cells carry out the reproductive function of multicellular organisms, and normal germ cell development ensures survival of the species. Germ granules are conserved cytoplasmic organelles of germ cells, essential for survival, differentiation, and function of these cells. Mutations in germ granule components or loss of their expression lead to infertility in model organisms and are associated with infertility in humans. By contrast, inappropriate expression of germ granule components in somatic cells is linked to carcinogenesis in humans. Many RNA-binding proteins and developmentally regulated mRNAs are found enriched in germ granules, leading to a hypothesis that these organelles function in regulation of mRNA stability or translational activity; yet, the molecular function of these organelles is still undefined. The nematode C. elegans has been instrumental for analysis of mRNA translational control in germline development. Through focusing on cofactors that promote localization of RNA-binding regulatory protein FBF-2 to germ granules (called P granules in this species) in germline stem cells we have generated a set of innovative tools that will address key mechanisms through which P granules regulate FBF-2 activity. By integrating genetic, molecular, biochemical and imaging-based approaches, we will: 1) Determine how FBF-2 interaction with a cofactor protein DLC-1 important for P granule localization impacts translational repression exerted by FBF-2; 2) Define the mechanisms of P granule remodeling that specifically degrade FBF-2 at the onset of meiosis when stem cells transition from proliferation to differentiation; 3) Determine the role of DLC-1 binding to core P granule components in regulation of stability of embryonic P granules and in recruitment of transient P granule components such as FBF-2 to adult germline P granules. Our experimental system is poised to reveal specific molecular mechanisms mediating the interplay between germ granules and translational regulation. Since germ granules are conserved organelles and our research focuses on conserved regulatory proteins, studies in this model system will provide critical insight into the causes of infertility in humans.
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0.966 |