ABSTRACT
The Caenorhabditis elegans sex determination gene, tra-2, is translationally regulated by elements in the 3'-untranslated region called TGEs. TGEs govern the translation of mRNAs in both invertebrates and vertebrates, indicating that this is a highly conserved mechanism for controlling gene activity. A factor called DRF, found in worm extracts binds the TGEs and may be a repressor of translation. Using the yeast three-hybrid screen and RNA gel shift analysis, we have found that the protein GLD-1, a germline-specific protein and a member of the STAR family of RNA-binding proteins, specifically binds to the TGEs. GLD-1 is essential for oogenesis, and is also necessary for spermatogenesis and inhibition of germ cell proliferation. Several lines of evidence demonstrate that GLD-1 is a translational repressor acting through the TGEs to repress tra-2 translation. GLD-1 can repress the translation of reporter RNAs via the TGEs both in vitro and in vivo, and is required to maintain low TRA-2A protein levels in the germline. Genetic analysis indicates that GLD-1 acts upstream of the TGE control. Finally, we show that endogenous GLD-1 is a component of DRF. The conservation of the TGE control and the STAR family suggests that at least a subset of STAR proteins may work through the TGEs to control translation.
Subject(s)
Caenorhabditis elegans Proteins , Caenorhabditis elegans/growth & development , Caenorhabditis elegans/genetics , Helminth Proteins/genetics , Helminth Proteins/physiology , Sex Determination Processes , Animals , Animals, Genetically Modified , Base Sequence , Caenorhabditis elegans/physiology , Female , Genes, Helminth , Male , Membrane Proteins/genetics , Membrane Proteins/physiology , Molecular Sequence Data , Mutation , Phenotype , Protein Biosynthesis , RNA, Helminth/genetics , Saccharomyces cerevisiae/geneticsABSTRACT
The Drosophila segment polarity gene cubitus interruptus (ci) encodes a zinc finger protein that is required for the proper patterning of segments and imaginal discs. Epistasis analysis indicates that ci functions in the Hedgehog (Hh) signal transduction pathway and is required to maintain wingless expression in the embryo. In this paper, the role of the Ci protein in the Hh signaling pathway is examined in more detail. Our results show that ectopic expression of ci in imaginal discs and the embryo activates the expression of Hh target genes. One of these target genes, patched, forms a negative feedback loop with ci that is regulated by Hh signal transduction. Activation is also achieved using the Ci zinc finger domain fused to a heterologous transactivation domain. Conversely, repression of Hh target genes occurs in animals expressing the Ci zinc finger domain fused to a repression domain. To examine Ci function in more detail, regions of the Ci protein that are responsible for its ability to transactivate and its subcellular distribution have been identified.
Subject(s)
DNA-Binding Proteins/biosynthesis , Drosophila Proteins , Embryo, Nonmammalian/physiology , Gene Expression Regulation, Developmental , Insect Proteins/biosynthesis , Animals , Drosophila/embryology , Drosophila/genetics , Feedback , Hedgehog Proteins , Molecular Sequence Data , Recombinant Fusion Proteins/biosynthesis , Signal Transduction , Transcription Factors , Transcriptional Activation , Wings, Animal , Zinc FingersABSTRACT
The segment polarity gene cubitus interruptus (ci) is required to maintain expression of the wingless gene and to specify naked cuticle within each epidermal segment. Antibodies were generated against the ci protein and used to analyze its pattern of expression. By stage 11, post-transcriptional regulation of ci is observed. ci transcript levels are uniform across the anterior compartment, but protein levels are higher next to the compartment boundaries. The distribution of the ci protein is altered in fused, hedgehog and wingless mutants suggesting cell-cell signaling may regulate ci protein levels. The role of ci in cell-cell signaling and pattern formation was examined in double mutants of ci with patched and zeste-white3/shaggy.
Subject(s)
DNA-Binding Proteins/physiology , Drosophila Proteins , Drosophila/embryology , Proteins/physiology , Proto-Oncogene Proteins/physiology , Animals , Gene Expression Regulation, Developmental , Hedgehog Proteins , Morphogenesis , Mutation , Signal Transduction , Transcription Factors , Wnt1 ProteinABSTRACT
The cubitus interruptus (ci) gene is a member of the Drosophila segment polarity gene family and encodes a protein with a zinc finger domain homologous to the vertebrate Gli genes and the nematode tra-1 gene. Three classes of existing mutations in the ci locus alter the regulation of ci expression and can be used to examine ci function during development. The first class of ci mutations causes interruptions in wing veins four and five due to inappropriate expression of the ci product in the posterior compartment of imaginal discs. The second class of mutations eliminates ci protein early in embryogenesis and causes the deletion of structures that are derived from the region including and adjacent to the engrailed expressing cells. The third class of mutations eliminates ci protein later in embryogenesis and blocks the formation of the ventral naked cuticle. The loss of ci expression at these two different stages in embryonic development correlates with the subsequent elimination of wingless expression. Adults heterozygous for the unique ciCe mutation have deletions between wing veins three and four. A similar wing defect is present in animals mutant for the segment polarity gene fused that encodes a putative serine/threonine kinase. In ciCe/+ and fused mutants, the deletions between wing veins three and four correlate with increased ci protein levels in the anterior compartment. Thus, proper regulation of both the ci mRNA and protein appears to be critical for normal Drosophila development.
