Squid, Cup, and PABP55B function together to regulate gurken translation in Drosophila

During Drosophila melanogaster oogenesis, the proper localization of gurken (grk) mRNA and protein is required for the establishment of the dorsal-ventral axis of the egg and future embryo. Squid (Sqd) is an RNA-binding protein that is required for the correct localization and translational regulation of the grk message. We show that Cup and polyA-binding protein (PABP) interact physically with Sqd and with each other in ovaries. We show that cup mutants lay dorsalized eggs, enhance dorsalization of weak sqd alleles, and display defects in grk mRNA localization and Grk protein accumulation. In contrast, pAbp mutants lay ventralized eggs and enhance grk haploinsufficiency. PABP also interacts genetically and biochemically with Encore. These data predict a model in which Cup and Sqd mediate translational repression of unlocalized grk mRNA, and PABP and Enc facilitate translational activation of the message once it is fully localized to the dorsal-anterior region of the oocyte. These data also provide the first evidence of a link between the complex of commonly used trans-acting factors and Enc, a factor that is required for grk translation.     

Hrb27C, Sqd and Otu cooperatively regulate gurken RNA localization and mediate nurse cell chromosome dispersion in Drosophila oogenesis

Heterogeneous nuclear ribonucleoproteins, hnRNPs, are RNA-binding proteins that play crucial roles in controlling gene expression. In Drosophila oogenesis, the hnRNP Squid (Sqd) functions in the localization and translational regulation of gurken (grk) mRNA. We show that Sqd interacts with Hrb27C, an hnRNP previously implicated in splicing. Like sqd, hrb27C mutants lay eggs with dorsoventral defects and Hrb27C can directly bind to grk RNA. Our data demonstrate a novel role for Hrb27C in promoting grk localization. We also observe a direct physical interaction between Hrb27C and Ovarian tumor (Otu), a cytoplasmic protein implicated in RNA localization. We find that some otu alleles produce dorsalized eggs and it appears that Otu cooperates with Hrb27C and Sqd in the oocyte to mediate proper grk localization. All three mutants share another phenotype, persistent polytene nurse cell chromosomes. Our analyses support dual cooperative roles for Sqd, Hrb27C and Otu during Drosophila oogenesis.     

Drosophila Dynein light chain (DDLC1) binds to gurken mRNA and is required for its localization

During oogenesis in Drosophila, mRNAs encoding determinants required for the polarization of egg and embryo become localized in the oocyte in a spatially restricted manner. The TGF-alpha like signaling molecule Gurken has a central role in the polarization of both body axes and the corresponding mRNA displays a unique localization pattern, accumulating initially at the posterior and later at the anterior-dorsal of the oocyte. Correct localization of gurken RNA requires a number of cis-acting sequence elements, a complex of trans-acting proteins, of which only several have been identified, and the motor proteins Dynein and Kinesin, traveling along polarized microtubules. Here we report that the cytoplasmic Dynein-light-chain (DDLC1) which is the cargo-binding subunit of the Dynein motor protein, directly bound with high specificity and affinity to a 230-nucleotide region within the 3'UTR of gurken, making it the first Drosophila mRNA-cargo to directly bind to the DLC. Although DDLC1 lacks known RNA-binding motifs, comparison to double-stranded RNA-binding proteins suggested structural resemblance. Phenotypic analysis of ddlc1 mutants supports a role for DDLC1 in gurken RNA localization and anchoring as well as in correct positioning of the oocyte nucleus.     

Functioning of the Drosophila orb gene in gurken mRNA localization and translation.

The orb gene encodes an RNA recognition motif (RRM)-type RNA-binding protein that is a member of the cytoplasmic polyadenylation element binding protein (CPEB) family of translational regulators. Early in oogenesis, orb is required for the formation and initial differentiation of the egg chamber, while later in oogenesis it functions in the determination of the dorsoventral (DV) and anteroposterior axes of egg and embryo. In the studies reported here, we have examined the role of the orb gene in the gurken (grk)-Drosophila epidermal growth factor receptor (DER) signaling pathway. During the previtellogenic stages of oogenesis, the grk-DER signaling pathway defines the posterior pole of the oocyte by specifying posterior follicle cell identity. This is accomplished through the localized expression of Grk at the very posterior of the oocyte. Later in oogenesis, the grk-DER pathway is used to establish the DV axis. Grk protein synthesized at the dorsal anterior corner of the oocyte signals dorsal fate to the overlying follicle cell epithelium. We show that orb functions in both the early and late grk-DER signaling pathways, and in each case is required for the localized expression of Grk protein. We have found that orb is also required to promote the synthesis of a key component of the DV polarity pathway, K(10). Finally, we present evidence that Orb protein expression during the mid- to late stages of oogenesis is, in turn, negatively regulated by K(10).     

gurken and the I factor retrotransposon RNAs share common localization signals and machinery

Drosophila gurken mRNA is localized by dynein-mediated transport to a crescent near the oocyte nucleus, thus targeting the TGFalpha signal and forming the primary embryonic axes. Here, we show that gurken and the I factor, a non-LTR retrotransposon, share a small consensus RNA stem loop of defined secondary structure, which forms a conserved signal for dynein-mediated RNA transport to the oocyte nucleus. Furthermore, gurken and the I factor compete in vivo for the same localization machinery. I factor transposition leads to its mRNA accumulating near and within the oocyte nucleus, thus causing perturbations in gurken and bicoid mRNA localization and axis specification. These observations further our understanding of the close association of transposable elements with their host and provide an explanation for how I factor transposition causes female sterility. We propose that the transposition of other elements may exploit the host's RNA transport signals and machinery.     

An autoregulatory feedback loop directs the localized expression of the Drosophila CPEB protein Orb in the developing oocyte

The RRM-type RNA binding protein Orb plays a central role in the establishment of polarity in the Drosophila egg and embryo. In addition to its role in the formation and initial differentiation of the egg chamber, orb is required later in oogenesis for the determination of the dorsoventral (DV) and anteroposterior (AP) axes. In DV axis formation, Orb protein is required to localize and translate gurken mRNA at the dorsoanterior part of the oocyte. In AP axis formation, Orb is required for the translation of oskar mRNA. In each case, Orb protein is already localized at the appropriate sites within the oocyte before the arrival of the mRNAs encoding axis determinants. We present evidence that an autoregulatory mechanism is responsible for directing the on site accumulation of Orb protein in the Drosophila oocyte. This orb autoregulatory activity ensures the accumulation of high levels of Orb protein at sites in the oocyte that contain localized orb message.     

The transmembrane region of Gurken is not required for biological activity, but is necessary for transport to the oocyte membrane in Drosophila

During Drosophila oogenesis, localization of the transforming growth factor alpha (TGFalpha)-like signaling molecule Gurken to the oocyte membrane is required for polarity establishment of the egg and embryo. To test Gurken domain functions, full-length and truncated forms of Gurken were expressed ectopically using the UAS/Gal4 expression system, or in the germline using the endogenous promoter. GrkDeltaC, a deletion of the cytoplasmic domain, localizes to the oocyte membrane and can signal. GrkDeltaTC, which lacks the transmembrane and cytoplasmic domains, retains signaling ability when ectopically expressed in somatic cells. However, in the germline, the GrkDeltaTC protein accumulates throughout the oocyte cytoplasm and cannot signal. In addition, we found that several strong gurken alleles contain point mutations in the transmembrane region. We conclude that secretion of Gurken requires its transmembrane region, and propose a model in which the gene cornichon mediates this process.     

Staufen, a gene required to localize maternal RNAs in the Drosophila egg.

The posterior group gene staufen is required both for the localization of maternal determinants to the posterior pole of the Drosophila egg and for bicoid RNA to localize correctly to the anterior pole. We report the cloning and sequencing of staufen and show that staufen protein is one of the first molecules to localize to the posterior pole of the oocyte, perhaps in association with oskar RNA. Once localized, staufen is found in the polar granules and is required to hold other polar granule components at the posterior pole. By the time the egg is laid, staufen protein is also concentrated at the anterior pole, in the same region as bicoid RNA.     

Encore is a member of a novel family of proteins and affects multiple processes in Drosophila oogenesis

Mutations in the encore (enc) gene of Drosophila melanogaster cause one extra round of mitosis in the germline, resulting in the formation of egg chambers with extra nurse cells. In addition, enc mutations affect the accumulation of Gurken protein within the oocyte, leading to the production of ventralized eggs. Here we show that enc mutants also exhibit abnormalities in karyosome morphology, similar to other ventralizing mutants such as okra and spindle B. Unlike these mutants, however, the defects in Gurken accumulation and karyosome formation do not result from activation of a meiotic checkpoint. Furthermore, we demonstrate that the requirement for enc in these processes is temporally distinct from its role in germline mitosis. Cloning of the enc locus and generation of anti-Enc antibodies reveal that enc encodes a large novel protein that accumulates within the oocyte cytoplasm and colocalizes with grk mRNA. We argue that the enc mutant phenotypes reflect a role for Enc in the regulation of several RNA targets.     

An interaction type of genetic screen reveals a role of the Rab11 gene in oskar mRNA localization in the developing Drosophila melanogaster oocyte.

Abdomen and germ cell development of Drosophila melanogaster embryo requires proper localization of oskar mRNA to the posterior pole of the developing oocyte. oskar mRNA localization depends on complex cell biological events like cell-cell communication, dynamic rearrangement of the microtubule network, and function of the actin cytoskeleton of the oocyte. To investigate the cellular mechanisms involved, we developed a novel interaction type of genetic screen by which we isolated 14 dominant enhancers of a sensitized genetic background composed of mutations in oskar and in TropomyosinII, an actin binding protein. Here we describe the detailed analysis of two allelic modifiers that identify Drosophila Rab11, a gene encoding small monomeric GTPase. We demonstrate that mutation of the Rab11 gene, involved in various vesicle transport processes, results in ectopic localization of oskar mRNA, whereas localization of gurken and bicoid mRNAs and signaling between the oocyte and the somatic follicle cells are unaffected. We show that the ectopic oskar mRNA localization in the Rab11 mutants is a consequence of an abnormally polarized oocyte microtubule cytoskeleton. Our results indicate that the internal membranous structures play an important role in the microtubule organization in the Drosophila oocyte and, thus, in oskar RNA localization.