Bruno: a double turn-off for Oskar

In Drosophila, the posterior localization of oskar mRNA and its translational regulation are essential for axis specification and germline formation. Recently in Cell, demonstrated that Bruno inhibits cap-dependent translation of oskar mRNA and uncovered a novel Bruno-dependent assembly of oskar mRNA into multimeric RNP particles, which are inaccessible to the translational machinery. This work provides a novel link between mRNA localization, particle formation, and translational regulation.     

Control of oskar mRNA translation by Bruno in a novel cell-free system from Drosophila ovaries

The coupled regulation of oskar mRNA localization and translation in time and space is critical for correct anteroposterior patterning of the Drosophila embryo. Localization-dependent translation of oskar mRNA, a mechanism whereby oskar RNA localized at the posterior of the oocyte is selectively translated and the unlocalized RNA remains in a translationally repressed state, ensures that Oskar activity is present exclusively at the posterior pole. Genetic experiments indicate that translational repression involves the binding of Bruno protein to multiple sites, the Bruno Response Elements (BRE), in the 3' untranslated region (UTR) of oskar mRNA. We have established a cell-free translation system derived from Drosophila ovaries, which faithfully reproduces critical features of mRNA translation in vivo, namely cap structure and poly(A) tail dependence. We show that this ovary extract, containing endogenous Bruno, is able to recapitulate oskar mRNA regulation in a BRE-dependent way. Thus, the assembly of a ribonucleoprotein (RNP) complex leading to the translationally repressed state occurs in vitro. Moreover, we show that a Drosophila embryo extract lacking Bruno efficiently translates oskar mRNA. Addition of recombinant Bruno to this extract establishes the repressed state in a BRE-dependent manner, providing a direct biochemical demonstration of the critical role of Bruno in oskar mRNA translation. The approach that we describe opens new avenues to investigate translational regulation in Drosophila oogenesis at a biochemical level.     

Drosophila cup is an eIF4E binding protein that associates with Bruno and regulates oskar mRNA translation in oogenesis

Translational control is a critical process in the spatio-temporal restriction of protein production. In Drosophila oogenesis, translational repression of oskar (osk) RNA during its localization to the posterior pole of the oocyte is essential for embryonic patterning and germ cell formation. This repression is mediated by the osk 3' UTR binding protein Bruno (Bru), but the underlying mechanism has remained elusive. Here, we report that an ovarian protein, Cup, is required to repress precocious osk translation. Cup binds the 5'-cap binding translation initiation factor eIF4E through a sequence conserved among eIF4E binding proteins. A mutant Cup protein lacking this sequence fails to repress osk translation in vivo. Furthermore, Cup interacts with Bru in a yeast two-hybrid assay, and the Cup-eIF4E complex associates with Bru in an RNA-independent manner. These results suggest that translational repression of osk RNA is achieved through a 5'/3' interaction mediated by an eIF4E-Cup-Bru complex.     

In vivo imaging of oskar mRNA transport reveals the mechanism of posterior localization

oskar mRNA localization to the posterior of the Drosophila oocyte defines where the abdomen and germ cells form in the embryo. Although this localization requires microtubules and the plus end-directed motor, kinesin, its mechanism is controversial and has been proposed to involve active transport to the posterior, diffusion and trapping, or exclusion from the anterior and lateral cortex. By following oskar mRNA particles in living oocytes, we show that the mRNA is actively transported along microtubules in all directions, with a slight bias toward the posterior. This bias is sufficient to localize the mRNA and is reversed in mago, barentsz, and Tropomyosin II mutants, which mislocalize the mRNA anteriorly. Since almost all transport is mediated by kinesin, oskar mRNA localizes by a biased random walk along a weakly polarized cytoskeleton. We also show that each component of the oskar mRNA complex plays a distinct role in particle formation and transport.     

A late phase of Oskar accumulation is crucial for posterior patterning of the Drosophila embryo, and is blocked by ectopic expression of Bruno

In Drosophila, posterior embryonic body patterning and germ cell formation rely on Oskar, a protein that is concentrated at the posterior pole of the oocyte. A program of mRNA localization and translational regulation ensures that Oskar is only expressed at the proper location. One key regulatory factor is Bruno, which represses translation of oskar mRNA before its localization. Ectopic expression of a bruno cDNA prolongs repression, even after oskar mRNA is localized, and posterior body patterning is efficiently and selectively blocked. Surprisingly, the initial accumulation of Oskar, while frequently reduced, is not eliminated, arguing that levels of Oskar previously thought to be sufficient for patterning do not suffice, or that Bruno acts at a downstream step in patterning. Expression of the bruno cDNA does not inhibit posterior patterning when Oskar is expressed independent of Bruno-mediated regulation, ruling out a downstream requirement for Bruno. Notably, an Oskar::GFP reporter protein reveals continual accumulation during the late phases of oogenesis. Taken together, these results strongly argue that a late phase in accumulation of Osk protein, typically not monitored because of imperviousness of late stage oocytes to antibodies, is crucial for body patterning.     

An RNA‐binding atypical tropomyosin recruits kinesin‐1 dynamically to oskar mRNPs

Localization and local translation of oskar mRNA at the posterior pole of the Drosophila oocyte directs abdominal patterning and germline formation in the embryo. The process requires recruitment and precise regulation of motor proteins to form transport‐competent mRNPs. We show that the posterior‐targeting kinesin‐1 is loaded upon nuclear export of oskar mRNPs, prior to their dynein‐dependent transport from the nurse cells into the oocyte. We demonstrate that kinesin‐1 recruitment requires the DmTropomyosin1‐I/C isoform, an atypical RNA‐binding tropomyosin that binds directly to dimerizing oskar 3′UTRs. Finally, we show that a small but dynamically changing subset of oskar mRNPs gets loaded with inactive kinesin‐1 and that the motor is activated during mid‐oogenesis by the functionalized spliced oskar RNA localization element. This inefficient, dynamic recruitment of Khc decoupled from cargo‐dependent motor activation constitutes an optimized, coordinated mechanism of mRNP transport, by minimizing interference with other cargo‐transport processes and between the cargo‐associated dynein and kinesin‐1.     

An oskar-dependent positive feedback loop maintains the polarity of the Drosophila oocyte

The localization of oskar mRNA to the posterior of the Drosophila oocyte defines the site of assembly of the pole plasm, which contains the abdominal and germline determinants. oskar mRNA localization requires the polarization of the microtubule cytoskeleton, which depends on the recruitment of PAR-1 to the posterior cortex in response to a signal from the follicle cells, where it induces an enrichment of microtubule plus ends. Here, we show that overexpressed oskar mRNA localizes to the middle of the oocyte, as well as the posterior. This ectopic localization depends on the premature translation of Oskar protein, which recruits PAR-1 and microtubule-plus-end markers to the oocyte center instead of the posterior pole, indicating that Oskar regulates the polarity of the cytoskeleton. Oskar also plays a role in the normal polarization of the oocyte; mutants that disrupt oskar mRNA localization or translation strongly reduce the posterior recruitment of microtubule plus ends. Thus, oskar mRNA localization is required to stabilize and amplify microtubule polarity, generating a positive feedback loop in which Oskar recruits PAR-1 to the posterior to increase the microtubule cytoskeleton's polarization, which in turn directs the localization of more oskar mRNA.     

Hrp48, a Drosophila hnRNPA/B homolog, binds and regulates translation of oskar mRNA

Establishment of the Drosophila embryonic axes provides a striking example of RNA localization as an efficient mechanism for protein targeting within a cell. oskar mRNA encodes the posterior determinant and is essential for germline and abdominal development in the embryo. Tight restriction of Oskar activity to the posterior is achieved by mRNA localization-dependent translational control, whereby unlocalized mRNA is translationally repressed and repression is overcome upon mRNA localization. Here we identify the previously reported oskar RNA binding protein p50 as Hrp48, an abundant Drosophila hnRNP. Analysis of three hrp48 mutant alleles reveals that Hrp48 levels are crucial for polarization of the oocyte during mid-oogenesis. Our data also show that Hrp48, which binds to the 5' and 3' regions of oskar mRNA, plays an important role in restricting Oskar activity to the posterior of the oocyte, by repressing oskar mRNA translation during transport.     

Drosophila Y14 shuttles to the posterior of the oocyte and is required for oskar mRNA transport.

BACKGROUND: mRNA localization is a powerful and widely employed mechanism for generating cell asymmetry. In Drosophila, localization of mRNAs in the oocyte determines the axes of the future embryo. oskar mRNA localization at the posterior pole is essential and sufficient for the specification of the germline and the abdomen. Its posterior transport along the microtubules is mediated by Kinesin I and several proteins, such as Mago-nashi, which, together with oskar mRNA, form a posterior localization complex. It was recently shown that human Y14, a nuclear protein that associates with mRNAs upon splicing and shuttles to the cytoplasm, interacts with MAGOH, the human homolog of Mago-nashi. RESULTS: Here, we show that Drosophila Y14 interacts with Mago-nashi in vivo. Immunohistochemistry reveals that Y14 is predominantly nuclear and colocalizes with oskar mRNA at the posterior pole. We show that, in y14 mutant oocytes, oskar mRNA localization to the posterior pole is specifically affected, while the cytoskeleton appears to be intact. CONCLUSIONS: Our findings indicate that Y14 is part of the oskar mRNA localization complex and that the nuclear shuttling protein Y14 has a specific and direct role in oskar mRNA cytoplasmic localization.     

Miranda couples oskar mRNA/Staufen complexes to the bicoid mRNA localization pathway

The double-stranded RNA binding protein Staufen is required for the microtubule-dependent localization of bicoid and oskar mRNAs to opposite poles of the Drosophila oocyte and also mediates the actin-dependent localization of prospero mRNA during the asymmetric neuroblast divisions. The posterior localization of oskar mRNA requires Staufen RNA binding domain 2, whereas prospero mRNA localization mediated the binding of Miranda to RNA binding domain 5, suggesting that different Staufen domains couple mRNAs to distinct localization pathways. Here, we show that the expression of Miranda during mid-oogenesis targets Staufen/oskar mRNA complexes to the anterior of the oocyte, resulting in bicaudal embryos that develop an abdomen and pole cells instead of the head and thorax. Anterior Miranda localization requires microtubules, rather than actin, and depends on the function of Exuperantia and Swallow, indicating that Miranda links Staufen/oskar mRNA complexes to the bicoid mRNA localization pathway. Since Miranda is expressed in late oocytes and bicoid mRNA localization requires the Miranda-binding domain of Staufen, Miranda may play a redundant role in the final step of bicoid mRNA localization. Our results demonstrate that different Staufen-interacting proteins couple Staufen/mRNA complexes to distinct localization pathways and reveal that Miranda mediates both actin- and microtubule-dependent mRNA localization.     

The Drosophila hnRNPA/B homolog, Hrp48, is specifically required for a distinct step in osk mRNA localization

The Staufen-dependent localization of oskar mRNA to the posterior of the Drosophila oocyte induces the formation of the pole plasm, which contains the abdominal and germline determinants. In a germline clone screen for mutations that disrupt the posterior localization of GFP-Staufen, we isolated three missense alleles in the hnRNPA/B homolog, Hrp48. These mutants specifically abolish osk mRNA localization, without affecting its translational control or splicing, or the localization of bicoid and gurken mRNAs and the organization of the microtubule cytoskeleton. Hrp48 colocalizes with osk mRNA throughout oogenesis, and interacts with its 5' and 3' regulatory regions, suggesting that it binds directly to oskar mRNA to mediate its posterior transport. The hrp48 alleles cause a different oskar mRNA localization defect from other mutants, and disrupt the formation of GFP-Staufen particles. This suggests a new step in the localization pathway, which may correspond to the assembly of Staufen/oskar mRNA transport particles.     

Barentsz is essential for the posterior localization of oskar mRNA and colocalizes with it to the posterior pole

The localization of Oskar at the posterior pole of the Drosophila oocyte induces the assembly of the pole plasm and therefore defines where the abdomen and germ cells form in the embryo. This localization is achieved by the targeting of oskar mRNA to the posterior and the localized activation of its translation. oskar mRNA seems likely to be actively transported along microtubules, since its localization requires both an intact microtubule cytoskeleton and the plus end-directed motor kinesin I, but nothing is known about how the RNA is coupled to the motor. Here, we describe barentsz, a novel gene required for the localization of oskar mRNA. In contrast to all other mutations that disrupt this process, barentsz-null mutants completely block the posterior localization of oskar mRNA without affecting bicoid and gurken mRNA localization, the organization of the microtubules, or subsequent steps in pole plasm assembly. Surprisingly, most mutant embryos still form an abdomen, indicating that oskar mRNA localization is partially redundant with the translational control. Barentsz protein colocalizes to the posterior with oskar mRNA, and this localization is oskar mRNA dependent. Thus, Barentsz is essential for the posterior localization of oskar mRNA and behaves as a specific component of the oskar RNA transport complex.     

A repeated IMP-binding motif controls oskar mRNA translation and anchoring independently of Drosophila melanogaster IMP

Zip code-binding protein 1 (ZBP-1) and its Xenopus laevis homologue, Vg1 RNA and endoplasmic reticulum-associated protein (VERA)/Vg1 RNA-binding protein (RBP), bind repeated motifs in the 3' untranslated regions (UTRs) of localized mRNAs. Although these motifs are required for RNA localization, the necessity of ZBP-1/VERA remains unresolved. We address the role of ZBP-1/VERA through analysis of the Drosophila melanogaster homologue insulin growth factor II mRNA-binding protein (IMP). Using systematic evolution of ligands by exponential enrichment, we identified the IMP-binding element (IBE) UUUAY, a motif that occurs 13 times in the oskar 3'UTR. IMP colocalizes with oskar mRNA at the oocyte posterior, and this depends on the IBEs. Furthermore, mutation of all, or subsets of, the IBEs prevents oskar mRNA translation and anchoring at the posterior. However, oocytes lacking IMP localize and translate oskar mRNA normally, illustrating that one cannot necessarily infer the function of an RBP from mutations in its binding sites. Thus, the translational activation of oskar mRNA must depend on the binding of another factor to the IBEs, and IMP may serve a different purpose, such as masking IBEs in RNAs where they occur by chance. Our findings establish a parallel requirement for IBEs in the regulation of localized maternal mRNAs in D. melanogaster and X. laevis.