The Drosophila maternal effect gene oskar encodes the posterior determinant responsible for the formation of the posterior pole plasm in the egg, and thus of the abdomen and germline of the future fly. Previously identified oskar mutants give rise to offspring that lack both abdominal segments and a germline, thus defining the 'posterior group phenotype'. Common to these classical oskar alleles is that they all produce significant amounts of oskar mRNA. By contrast, two new oskar mutants in which oskar RNA levels are strongly reduced or undetectable are sterile, because of an early arrest of oogenesis. This egg-less phenotype is complemented by oskar nonsense mutant alleles, as well as by oskar transgenes, the protein-coding capacities of which have been annulled. Moreover, we show that expression of the oskar 3Ј untranslated region (3ЈUTR) is sufficient to rescue the egg-less defect of the RNA null mutant. Our analysis thus reveals an unexpected role for oskar RNA during early oogenesis, independent of Oskar protein.These findings indicate that oskar RNA acts as a scaffold or regulatory RNA essential for development of the oocyte.
Kinesin-1 carries cargos including proteins, RNAs, vesicles, and pathogens over long distances within cells. The mechanochemical cycle of kinesins is well described, but how they establish cargo specificity is not fully understood. Transport of oskar mRNA to the posterior pole of the Drosophila oocyte is mediated by Drosophila kinesin-1, also called kinesin heavy chain (Khc), and a putative cargo adaptor, the atypical tropomyosin, aTm1. How the proteins cooperate in mRNA transport is unknown. Here, we present the high-resolution crystal structure of a Khc-aTm1 complex. The proteins form a tripartite coiled coil comprising two in-register Khc chains and one aTm1 chain, in antiparallel orientation. We show that aTm1 binds to an evolutionarily conserved cargo binding site on Khc, and mutational analysis confirms the importance of this interaction for mRNA transport in vivo. Furthermore, we demonstrate that Khc binds RNA directly and that it does so via its alternative cargo binding domain, which forms a positively charged joint surface with aTm1, as well as through its adjacent auxiliary microtubule binding domain. Finally, we show that aTm1 plays a stabilizing role in the interaction of Khc with RNA, which distinguishes aTm1 from classical motor adaptors.
Iron-regulatory protein-1 (IRP-1) plays a dual role as a regulatory RNA-binding protein and as a cytoplasmic aconitase. When bound to iron-responsive elements (IRE), IRP-1 post-transcriptionally regulates the expression of mRNAs involved in iron metabolism. IRP have been cloned from several vertebrate species. Using a degenerate-primer PCR strategy and the screening of data bases, we now identify the homologues of IRP-1 in two invertebrate species, Drosophila melanogaster and Caenorhabditis elegans. Comparative sequence analysis shows that these invertebrate IRP are closely related to vertebrate IRP, and that the amino acid residues that have been implicated in aconitase function are particularly highly conserved, suggesting that invertebrate IRP may function as cytoplasmic aconitases. Antibodies raised against recombinant human IRP-1 immunoprecipitate the Drosophila homologue expressed from the cloned cDNA. In contrast to vertebrates, two IRP-1 homologues (Drosophila IRP-1A and Drosophila IRP-1B), displaying 86% identity to each other, are expressed in D. melanogaster. Both of these homologues are distinct from vertebrate IRP-2. In contrast to the mammalian system where the two IRP (IRP-1 and IRP-2) are differentially expressed, Drosophila IRP-1A and Drosophila IRP-1B are not preferentially expressed in specific organs. The localization of Drosophila IRP-1A to position 94C1-8 and of Drosophila IRP-1B to position 86B3-6 on the right arm of chromosome 3 and the availability of an IRP-1 cDNA from C. elegans will facilitate a genetic analysis of the IRE/IRP system, thus opening a new avenue to explore this regulatory network.Keywords : Drosophila melanogaster; Caenorhabditis elegans ; iron-regulatory protein; RNA binding; iron regulation.The post-transcriptional regulation of mRNAs involved in tion of IRP-1 and IRP-2 occurs post-translationally by distinct mechanisms. Striking similarities were discovered between cellular iron metabolism by iron-regulatory proteins (IRP) and iron-responsive elements (IRE) is widely used in the animal IRP-1 and mitochondrial [4, 5] and bacterial aconitases [6, 7], which are Fe-S proteins that reversibly convert citrate to isokingdom. In mammals, where this system is best characterized, IRP control the translation of mRNAs for the iron-storage pro-citrate. This finding led to experiments showing that IRP-1 is converted in iron-replete cultured cells into a cytoplasmic aconitein ferritin, the erythroid 5-aminolevulinate synthase a rate-limiting enzyme for the main iron-utilization pathway, and the mito-tase by insertion of a [4Fe-4S] cluster liganded to three highly conserved cysteine residues [8Ϫ10]. The [4Fe-4S] IRP-1 is inchondrial aconitase, by binding to a single IRE located in the 5′ active in IRE binding. Removal or loss of the Fe-S cluster conuntranslated region (UTR) of the respective messages. IRP verts the aconitase form into an RNA-binding protein. In conbound to multiple IRE in the 3′ UTR of the transferrin receptor trast to IRP-1, IRP-2 does not exhibit aconitase activi...
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