Elizabeth R. Gavis - US grants

9506828 Gavis Establishment of anterior-posterior polarity of the Drosophila embryo requires the asymmetric distribution of determinant molecules. Restriction of the posterior determinant, encoded by the nanos gene, to the posterior of the embryo is essential for normal development. High concentrations of Nanos in the anterior of the embryo suppresses head and thorax development. Spatial restriction of nanos expression occurs by a novel mechanism; translational repression of unlocalized nanos RNA and derepression by localization of nanos RNA to the posterior pole. Independent but overlapping cis-acting sequence elements have been identified within the nanos 3' untranslated region (3' UTR) that mediate RNA localization and translational repression of unlocalized nanos RNA. The proposed research aims to elucidate the molecular mechanism that effects translational regulation of nanos RNA and its linkage to RNA localization by identification and characterization of components of the cellular machinery that act through the defined nanos 3'UTR sequences. The experiments described will be directed toward: 1) further analysis of the cis-acting sequences that mediate localization dependent translational control guided by evolutionary sequence and secondary structure comparisons and identification of binding sites for trans acting factors and 2) isolation and characterization of factors that interact with the translational control element by both biochemical methods and genetic screens. ***

DESCRIPTION (Applicant's Abstract): Control of mRNA translation is an important mechanism for the temporal and spatial regulation of gene expression that underlies the development of an organism. Translational control plays a large role in coordinating early developmental events that rely on proteins synthesized from maternally provided mRNAs. While numerous developmentally important mRNAs have been shown to be translationally regulated by sequences within their 3' untranslated regions (3UTRs), little is known about the mechanisms by regulation is achieved. Translational repression of nanos RNA plays an essential role in generating the restricted distribution of Nanos protein that is necessary for proper patterning of the anterior-posterior body axis of the Drosophila embryo. Translational repression of nanos RNA is mediated by a 90 nucleotide translational control element (TCE) within the nanos 3'UTR. The proposed work focuses on translational repression of nanos RNA in Drosophila since it affords biochemical, molecular, and genetic approaches to investigation of 3'UTR-dependent translational regulatory mechanisms. Determination of the precise step at which translation is blocked by the nanos TCE, using biochemical approaches, will provide an important framework for analysis of components of the regulatory machinery. Since TCEprotein recognition underlies TCE function, sequence and structural features of the TCE necessary for recognition as well as proteins that recognize these features will be characterized. Proteins that interact with the TCE will be identified using in vitro biochemical assays and a yeast 3-hybrid assay. The roles that these proteins play in TCE-mediated translational repression will be investigated using both biochemical and genetic assays. In a complementary approach, sensitized genetic screens will permit isolation of genes encoding components of the regulatory machinery that do not necessarily interact directly with the TCE as well as those that have more pleiotropic roles in translational regulation. These studies will lead to an understanding of how 3'UTR sequences regulate translation during development. In addition, they will shed light on how RNA-protein interactions regulate basic cellular processes to provide the highly selective control needed for development, growth, and differentiation.

Control of mRNA translation is an important mechanism for temporal and spatial regulation of gene expression during the development of an organism. In the early Drosophila embryo, translational repression of nanos mRNA is essential for formation of the anterior-posterior body axis. Repression is mediated by a translational control element (TCE) within the nanos 3' untranslated region (3'UTR). Ectopic expression studies have shown that the nanos TCE can repress translation in several different somatic cell types at later stages of development as well. These include subsets of cells in the nervous system and the dorsal pouch epithelium. Analysis of the effect of TCE mutations on translational repression has revealed that the TCE acts through a different recognition mechanism in the dorsal pouch than is required for its function in the early embryo. Complimentarity of sequences required for TCE function in the dorsal pouch to a Drosophila microRNA suggests that the TCE may be a microRNA target in somatic tissues.
The proposed work combines genetic and molecular approaches to investigate this second mode of TCE-mediated translational regulation. Specific Aim 1 addresses the mechanistic differences between TCE-mediated repression in the early embryo and TCE-mediated repression in the central and peripheral nervous systems. Aim 2 encompasses genetic and cell culture approaches to test the hypothesis that TCE-mediated regulation in somatic tissues is microRNA-dependent. This portion of the proposal will be carried out time and resource permitting. Finally, a novel genetic screen proposed in Aim 3 will permit isolation of mutations in regulatory factors required for TCE function.
Intellectual Merit: Post-transcriptional mechanisms that act through 3'UTR sequences play critical roles in regulation of gene expression in developmental processes like cell cycle regulation, embryonic patterning and cell fate determination. The mechanisms underlying the function of many 3'UTR regulatory elements is poorly understood, however. Based on the paradigm for 3'UTR-mediated translational regulation by small noncoding RNAs in C. elegans, the recent identification of hundreds of noncoding microRNAs in flies, worms, mammals, and plants suggests that translational control is more widespread than previously anticipated. The proposed investigation of a newly identified mode of TCE recognition will provide an important foundation for understanding how different mechanisms of 3'UTR recognition result in control of protein synthesis and may identify a new microRNA target. In addition, the nanos TCE provides a new paradigm to investigate how multiple modes of regulation can be combined within a single element.
Broader Impacts: Increasing the number of women entering careers in the biological sciences and remaining in these career paths is of critical importance to both research and education. Participation of female undergraduate and graduate students in the proposed research will provide appropriate training and mentoring to enable them to make optimal, sustainable career choices. A second priority, education outreach, is addressed through the use of portable laboratory modules to introduce fundamental concepts in modern molecular biology and genetics to New Jersey public elementary and middle school students. Participation of graduate students in the development and execution of these modules will provide them with valuable teaching experience. Additional efforts will focus on the development of a curriculum for a new, multi-institutional MD/PhD program and the implementation of that program.

[unreadable] DESCRIPTION (provided by applicant): Intracellular mRNA localization is an important mechanism for the spatial regulation of gene expression necessary for animal development. By restricting the distributions of developmental regulatory proteins to particular regions of oocytes and embryos, mRNA localization plays a critical role in patterning of body axes and specification of cell fates during embryonic development in a variety of organisms. In addition, RNA localization generates protein asymmetries necessary for the subsequent differentiation and function of many specialized cell types. Cis-acting signals that mediate localization have now been identified in a number of localized mRNAs. Little is known, however, about the mechanisms by which these signals are recognized specifically by cellular localization machinery and how they target their RNAs to unique intracellular locations. [unreadable] [unreadable] Restriction of Nanos protein to the posterior of the Drosophila embryo is essential for proper patterning of the anterior-posterior body axis. Nanos synthesis is limited to the posterior pole of the embryo by a combination of RNA localization and translational control. Localization of nanos mRNA to the posterior pole of the embryo generates the critical concentration Nanos protein in the posterior for abdominal development and is essential to activate nanos translation. [unreadable] [unreadable] Localization of nanos is mediated by a complex a cis-acting localization signal within its 3' untranslated region (3'UTR). Results from previous and preliminary studies indicate that cytoplasmic localization factors recognize different sequence or structural motifs within this localization signal. Using nanos as a model, the proposed work will provide insight into how complex RNA localization signals are recognized by the cellular localization machinery and how these RNA-protein interactions mediate transport and anchoring by localization pathways. More generally, these studies will shed light on mechanisms by which RNA-protein interactions provide the highly selective control of basic cellular processes needed for development, growth, and differentiation. Specific Aim 1 encompasses mutational analysis of the nanos localization signal to determine sequence and structural requirements for localization signal recognition and function. This work will be facilitated by phylogenetic analysis ofnanos 3'UTRs from ten different drosophilid species. Preliminary work has led to purification of one candidate nanos localization factor and biochemical identification of a second. Aim 2 focuses on biochemical and genetic characterization of these factors to determine their function in nanos localization. In addition, a new strategy for biochemical isolation of localization complexes is proposed. In Aim 3, a genetic screen for nanos localization factors will complement the biochemical approaches of Aim 2. Aim 4 takes advantage of a new system for GFP labeling of nanos RNA in vivo to investigate the dynamic pathway of nanos localization during oogenesis. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Intracellular mRNA localization is an important mechanism for the spatial regulation of gene expression necessary for animal development. By restricting the distributions of developmental regulatory proteins to particular regions of oocytes and embryos, mRNA localization plays a critical role in patterning of body axes and specification of cell fates during embryonic development in a variety of organisms. In addition, RNA localization generates protein asymmetries necessary for the subsequent differentiation and function of many specialized cell types. Cis-acting signals that mediate localization have now been identified in a number of localized mRNAs. Little is known, however, about the mechanisms by which these signals are recognized specifically by cellular localization machinery and how they target their RNAs to unique intracellular locations. [unreadable] [unreadable] Restriction of Nanos protein to the posterior of the Drosophila embryo is essential for proper patterning of the anterior-posterior body axis. Nanos synthesis is limited to the posterior pole of the embryo by a combination of RNA localization and translational control. Localization of nanos mRNA to the posterior pole of the embryo generates the critical concentration Nanos protein in the posterior for abdominal development and is essential to activate nanos translation. [unreadable] [unreadable] Localization of nanos is mediated by a complex a cis-acting localization signal within its 3' untranslated region (3'UTR). Results from previous and preliminary studies indicate that cytoplasmic localization factors recognize different sequence or structural motifs within this localization signal. Using nanos as a model, the proposed work will provide insight into how complex RNA localization signals are recognized by the cellular localization machinery and how these RNA-protein interactions mediate transport and anchoring by localization pathways. More generally, these studies will shed light on mechanisms by which RNA-protein interactions provide the highly selective control of basic cellular processes needed for development, growth, and differentiation. Specific Aim 1 encompasses mutational analysis of the nanos localization signal to determine sequence and structural requirements for localization signal recognition and function. This work will be facilitated by phylogenetic analysis ofnanos 3'UTRs from ten different drosophilid species. Preliminary work has led to purification of one candidate nanos localization factor and biochemical identification of a second. Aim 2 focuses on biochemical and genetic characterization of these factors to determine their function in nanos localization. In addition, a new strategy for biochemical isolation of localization complexes is proposed. In Aim 3, a genetic screen for nanos localization factors will complement the biochemical approaches of Aim 2. Aim 4 takes advantage of a new system for GFP labeling of nanos RNA in vivo to investigate the dynamic pathway of nanos localization during oogenesis. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): 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.

PROJECT SUMMARY Our long-term research goal is to understand post-transcriptional mechanisms that control gene activity during early animal development. We focus on intracellular mRNA localization and translational control, which play crucial roles in regulating the production of proteins from maternally supplied transcripts. Because these transcripts, which control the initial developmental program of nearly all animals, are pre-loaded in the egg, the spatial and temporal expression of the proteins they encode must be exerted post-transcriptionally. In animals as diverse as flies and frogs, mRNA localization and local control of translation produce asymmetric protein distributions required for axis formation, patterning, and germline development. Often many different transcripts must be localized concurrently to various subcellular locations. Additionally, translational control must be superimposed to repress unlocalized transcripts and activate properly localized transcripts. How specificity is conferred on these processes, so that each transcript is targeted to its correct destination and translated appropriately, is poorly understood. Our research has capitalized on the Drosophila egg, which relies heavily on maternal transcripts, to investigate mechanisms of mRNA localization and its coupling to translational control. Our early studies focusing on nanos mRNA led to the discovery of a diffusion-and-entrapment mechanism used by numerous transcripts for localization to the specialized germ plasm at the posterior of the oocyte. Produced by the ovarian nurse cells and then transferred to the oocyte, these transcripts are co- packaged at the posterior end into ribonucleoprotein complexes (RNPs) called germ granules. Later during embryogenesis, germ granule mRNAs are segregated as a cohort to the primordial germ cells, where they are required for germline development. Despite their shared dependence on germ granule localization tor translational activation, different transcripts have distinct temporal demands. Our recent studies have led to a stepwise model for germ granule assembly that provides a framework for understanding the composition, structure, and translational properties of RNPs and their functions. Determining the specific roles of shared and RNA-specific proteins in controlling RNP assembly and translation will, in turn, be fundamental to a deeper understanding of mRNA localization as a mechanism for generating protein ? and consequently cellular ? asymmetries. To elucidate how localized assembly and function of complex RNA granules is controlled, we will take advantage of quantitative high resolution imaging, in vivo fluorescent RNA labeling, and new biochemical strategies to identify cis-acting regulatory elements and interacting proteins that mediate both individualistic and coordinate RNA behaviors. Ribosome footprinting, a genome-level approach for monitoring translation, will be employed to investigate mechanisms that impose translational arrest on unlocalized transcripts. Finally, we will use high resolution imaging of protein synthesis in vivo to decipher the relationship between germ granule association and translational activity.