Elizabeth R. Gavis
Department of Molecular Biology
Princeton, NJ 08544
telephone: (609) 258-3857
fax: (609) 258-1343
presenter: Elizabeth Gavis
Sherri Evans Bergsten, Seema Chatterjee, Ira E. Clark, Susan Crucs, Tao Huang,
Bingyi Yao, and Elizabeth R. Gavis
Department of Molecular Biology, Princeton University
In multi-cellular organisms, localization of proteins to particular domains within a cell is fundamental to the generation of asymmetry during development and to the polarization of differentiated cells. Apart from protein targeting, many cell types employ localized translation of mRNAs to ensure restricted accumulation of proteins. As a model system, my laboratory studies the Drosophila nanos (nos) mRNA, whose localized translation is essential for patterning of the anterior-posterior body axis during embryonic development. Remarkably, two post-transcriptional mechanisms, subcellular mRNA localization and translational control, are coupled to restrict Nos protein synthesis to the posterior of the embryo. Nos protein is required in the posterior of the embryo for abdominal development but must be excluded from the anterior to permit head and thorax development. Localization of nos RNA to the posterior pole of the embryo is required to activate nos translation; when localization is abolished, nos mRNA remains translationally repressed. Localization is not sufficient to restrict nos to the posterior, however; more than 95% of nos mRNA in the embryo is unlocalized. Translational repression of this unlocalized mRNA is essential for anterior development. We have shown that translational repression of unlocalized nos mRNA is mediated by a 90 nucleotide translational control element (TCE) within the nos 3' untranslated region (3'UTR).
Both the primary sequence and predicted secondary structure of the TCE is conserved between D. melanogaster and D. virilis and TCE function is conserved as well. Through a systematic analysis of TCE mutations, we have shown that TCE function in vivo requires formation of a bipartite structure consisting of two stem-loops. One RNA binding protein, Smg, has been identified that interacts with TCE stem-loop II; TCE function requires at least two additional, as yet unidentified factors, however.
Posterior localization is mediated by a 540 nucleotide cis-acting localization signal that can be subdivided into partially redundant localization elements, one of which is coincident with the TCE. Each element contains a domain whose sequence is conserved between the D. melanogaster and D. virilis nos 3'UTRs. Results from our analysis of localization element function indicate that wild-type nos RNA localization requires recognition of multiple sequence or structural motifs. Our demonstration that the D. virilis nos 3'UTR confers wild-type localization in a D. melanogaster embryo suggests that the conserved domains define functionally relevant motifs that are the targets for binding by putative localization factors. Through biochemical assays, we have identified several candidate localization factors that interact specifically with these sequences.
Our results indicate that the coincidence of cis-acting translational regulatory sequences and localization signal sequences is functionally significant. Our data suggest that interaction of the nos 3'UTR with translational repressors and localization factors is mutually exclusive; consequently interaction with localization factors is sufficient to activate nos translation. Recent results suggest that the nos 3'UTR exists in alternate structural conformations depending on whether nos RNA is localized or unlocalized. Formation of alternate 3'UTR structures may underlie the switch between translational repression and localization-dependent translational activation.
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