Control of mRNA translation plays a key role in temporal and spatial regulation of gene expression during development. Translational regulation of mRNAs involved in a variety of developmental processes, including oocyte polarization, cell cycle regulation, embryonic patterning, and neuronal morphogenesis often depends on sequences found within their 3'untranslated regions (3'UTRs). Identification of proteins that interact with these 3'UTR regulatory elements has begun to shed light on the diversity of mechanisms that control translation. Recent studies indicate that multiple modes of translational regulation can be imposed on a transcript by different 3'UTR-binding factors, but how RNA-protein interactions affect such layers of control is poorly understood. The proposed research aims to elucidate translational control mechanisms underlying developmental processes, using the Drosophila nanos (nos) gene as a model. nos is ideal these studies, as nos encodes a translational repressor whose own synthesis must be highly regulated for proper embryonic development. During late oogenesis and early embryogenesis, nos translation must be repressed within the bulk cytoplasm for patterning of the anterior-posterior body axis. This repression is mediated by a translational control element (TCE) in the nos 3'UTR comprising two stem-loops that function in oogenesis and embryogenesis, respectively, through their interaction with the repressor proteins Glorund (Glo) and Smaug (Smg).
In Aim 1, biochemical experiments that take advantage of a robust in vitro translation system based on ovary extract and a new method for ribosome footprinting are proposed to investigate the molecular mechanisms by which the TCE-Glo interaction inhibits translation by targeting both initiation and post-initiation events. This effort is supported by the identification and characterization of natie nos RNA-protein complexes and Glo interacting proteins proposed in Aim 2. In the early embryo, Nos protein forms a translational repressor complex with its partner Pumilio (Pum) to silence genes that inhibit abdominal and germline development. Identification of roles for Nos, Pum, and numerous additional RNA-binding proteins in neuronal morphogenesis during the previous grant period motivate biochemical and genetic studies proposed in Aim 3 to investigate the neuronal functions of these regulators and how post-transcriptional mechanisms modulate cellular processes that underlie morphogenesis and function of highly polarized cells. Mutations in translational regulatory proteins have been associated with a variety of cancers and diseases with neurological dysfunction. The proposed work will provide fundamental insight into how these factors control development, growth and differentiation, and how the disruption of translational control can result in disease.

Public Health Relevance

Control of mRNA translation enables cells to rapidly start or stop making proteins and to restrict where in the cell particular proteins are produced. This flexibility is particularly important in animal development, from the formation of eggs to the differentiation, growth, and function of specialized cells like neurons. Mutations in proteins that control mRNA translation have been associated with a variety of cancers and diseases with neurological dysfunction;thus, by investigating translational regulatory factors and the mechanisms by which they function, the proposed work will provide fundamental insight into how these factors control development, growth and differentiation, and how the disruption of translational control can result in disease.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
5R01GM061107-14
Application #
8643240
Study Section
Development - 1 Study Section (DEV1)
Program Officer
Hoodbhoy, Tanya
Project Start
Project End
Budget Start
Budget End
Support Year
14
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Princeton University
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
City
Princeton
State
NJ
Country
United States
Zip Code
08543
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