Establishment of polarity in the egg can be viewed as the earliest step in embryonic patterning. Thus, differences in cell fate among the early cleavage cells are a consequence of asymmetric distributions of informational molecules in the egg cytoplasm before fertilization. In a wide variety of organisms, the basis for such polarity can be provided by localized maternal determinants in the form of mRNA. Among vertebrates, Vg1 mRNA is a prominent example of a localized mRNA that plays a role in embryonic patterning. Restricted expression of Vg1 protein in the vegetal hemisphere of the egg is critical for correct patterning of the embryo, making localization of Vg1 mRNA an important model for understanding how maternal molecules are localized to influence pattern and polarity. The goal of this research project is to investigate the mechanisms that direct targeting of mRNAs to specific regions of the cell cytoplasm to generate cell and developmental polarity. The foundation for this investigation has been laid by our progress studying the molecular pathway that directs maternal mRNA molecules to the vegetal cortex of the Xenopus oocyte. Through development of approaches to track mRNA transport in live oocytes and biochemical strategies to isolate transport complexes, our experiments have uncovered new steps in the pathway and revealed new components of the cellular transport machinery. Our current proposal seeks to capitalize on these findings in order to delineate the molecular pathway that orchestrates delivery of maternal mRNAs to their cytoplasmic destinations.
Three specific aims are proposed:
In Aim 1, we will analyze the molecular interactions that drive vegetal RNP transport granule assembly, in Aim 2, we will determine the mechanisms that regulate directionality during RNA transport and in Aim 3 we will investigate the mechanisms that control retention of localized RNAs at the oocyte cortex. The proposed research is designed to reveal the mechanisms by which mRNA molecules are transported within cells to generate spatially restricted protein expression, and will provide insight into how developmental signals are spatially distributed in the vertebrate embryo. This work will impact issues related to human health such as birth defects, as well as neurological diseases that have been linked to defective RNA transport and localized protein synthesis.

Public Health Relevance

The goal of this research project is to determine the molecular mechanisms responsible for localization of RNA in the cytoplasm; RNA localization and local protein synthesis is critical for both embryonic development and neurological function. In particular, certain human diseases, including fragile X mental retardation syndrome and spinal muscular atrophy, have been linked to defects in RNA localization and localized protein synthesis. Our proposed studies are particularly relevant to NIH's mission because in addition to investigating the molecular mechanisms directing mRNA trafficking, we will investigate roles for human disease genes in this process, potentially providing new insight into the disease pathologies.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM071049-21
Application #
9431214
Study Section
Development - 2 Study Section (DEV2)
Program Officer
Hoodbhoy, Tanya
Project Start
1993-09-01
Project End
2019-02-28
Budget Start
2018-03-01
Budget End
2019-02-28
Support Year
21
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Brown University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
001785542
City
Providence
State
RI
Country
United States
Zip Code
Jeschonek, Samantha P; Mowry, Kimberly L (2018) Whole-Mount Immunofluorescence for Visualizing Endogenous Protein and Injected RNA in Xenopus Oocytes. Cold Spring Harb Protoc 2018:pdb.prot097022
Neil, Christopher R; Mowry, Kimberly (2018) Fluorescence In Situ Hybridization of Cryosectioned Xenopus Oocytes. Cold Spring Harb Protoc 2018:pdb.prot097030
Ciocanel, Maria-Veronica; Kreiling, Jill A; Gagnon, James A et al. (2017) Analysis of Active Transport by Fluorescence Recovery after Photobleaching. Biophys J 112:1714-1725
Powrie, Erin A; Ciocanel, Veronica; Kreiling, Jill A et al. (2016) Using in vivo imaging to measure RNA mobility in Xenopus laevis oocytes. Methods 98:60-65
Gagnon, James A; Kreiling, Jill A; Powrie, Erin A et al. (2013) Directional transport is mediated by a Dynein-dependent step in an RNA localization pathway. PLoS Biol 11:e1001551
Pratt, Catherine A; Mowry, Kimberly L (2013) Taking a cellular road-trip: mRNA transport and anchoring. Curr Opin Cell Biol 25:99-106
Medioni, Caroline; Mowry, Kimberly; Besse, Florence (2012) Principles and roles of mRNA localization in animal development. Development 139:3263-76
Gagnon, James A; Mowry, Kimberly L (2011) Visualization of mRNA localization in Xenopus oocytes. Methods Mol Biol 714:71-82
Gagnon, James A; Mowry, Kimberly L (2011) Molecular motors: directing traffic during RNA localization. Crit Rev Biochem Mol Biol 46:229-39
Pratt, Catherine A; Mowry, Kimberly L (2010) Preparation of a highly active cell-free translation system from immature Xenopus laevis oocytes. Methods 51:101-5

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