Vg1RBP phosphorylation by Erk2 MAP kinase correlates with the cortical release of Vg1 mRNA during meiotic maturation of Xenopus oocytes

Xenopus Vg1RBP is a member of the highly conserved IMP family of four KH-domain RNA binding proteins, with roles in RNA localization, translational control, RNA stability, and cell motility. Vg1RBP has been implicated in localizing Vg1 mRNAs to the vegetal cortex during oogenesis, in a process mediated by microtubules and microfilaments, and in migration of neural crest cells in embryos. Using c-mos morpholino, kinase inhibitors, and constitutely active recombinant kinases we show that Vg1RBP undergoes regulated phosphorylation by Erk2 MAPK during meiotic maturation, on a single residue, S402, located between the KH2 and KH3 domains. Phosphorylation temporally correlates with the release of Vg1 mRNA from its tight cortical association, assayed in lysates in physiological salt buffers, but does not affect RNA binding, nor self-association of Vg1RBP. U0126, a MAP kinase inhibitor, prevents Vg1RBP cortical release and Vg1 mRNA solubilization in meiotically maturing eggs, while injection of MKK6-DD, a constitutively activated MAP kinase kinase, promotes the release of both Vg1RBP and Vg1 mRNA from insoluble cortical structures. We propose that Erk2 MAP kinase phosphorylation of Vg1RBP regulates the protein:protein-mediated association of Vg1 mRNP with the cytoskeleton and/or ER. Since the MAP kinase site in Vg1RBP is conserved in several IMP homologs, this modification also has important implications for the regulation of IMP proteins in somatic cells.     

PTB/hnRNP I is required for RNP remodeling during RNA localization in Xenopus oocytes

Transport of specific mRNAs to defined regions within the cell cytoplasm is a fundamental mechanism for regulating cell and developmental polarity. In the Xenopus oocyte, Vg1 RNA is transported to the vegetal cytoplasm, where localized expression of the encoded protein is critical for embryonic polarity. The Vg1 localization pathway is directed by interactions between key motifs within Vg1 RNA and protein factors recognizing those RNA sequences. We have investigated how RNA-protein interactions could be modulated to trigger distinct steps in the localization pathway and found that the Vg1 RNP is remodeled during cytoplasmic RNA transport. Our results implicate two RNA-binding proteins with key roles in Vg1 RNA localization, PTB/hnRNP I and Vg1RBP/vera, in this process. We show that PTB/hnRNP I is required for remodeling of the interaction between Vg1 RNA and Vg1RBP/vera. Critically, mutations that block this remodeling event also eliminate vegetal localization of the RNA, suggesting that RNP remodeling is required for localization.     

Vg1 RNA binding protein mediates the association of Vg1 RNA with microtubules in Xenopus oocytes.

Localized RNAs are found in a variety of somatic and developing cell types. In many cases, microtubules have been implicated as playing a role in facilitating transport of these RNAs. Here we report that Vg1 RNA, which is localized to the vegetal cortex of Xenopus laevis oocytes, is associated with microtubules in vivo. Because of the ubiquitous nature of tubulin, the association of specific RNAs with microtubules is likely to involve factors that recognize both RNA and microtubules. Vg1 RNA binding protein (Vg1 RBP), previously shown to bind with high affinity to the vegetal localization site in Vg1 RNA, appears to function in this capacity. Vg1 RBP is associated with microtubules: it is enriched in microtubule extracts of oocytes and is also co-precipitated by heterologous, polymerized tubulin. Furthermore, Vg1 RBP binding activity is required for the specific association of Vg1 RNA to microtubules in vitro. These data suggest a general model for how specific RNAs can be localized to particular sites via common cytoskeletal elements.     

The KH domains of Xenopus Vg1RBP mediate RNA binding and self-association

Xenopus Vg1 mRNA is localized to the vegetal cortex during oogenesis in a process involving microtubules and microfilaments and proteins that specifically recognize the vegetal localization element (VLE) within the 3' untranslated region. One of the best characterized VLE-binding proteins is Vg1RBP or Vera. Primary sequence analysis of Vg1RBP and its homologs suggests that most of its open reading frame is occupied by RNA-binding modules, including two RRMs and four KH domains, arranged as three pairs of didomains. In the first detailed domain analysis of Vg1RBP, we show that the interaction of Vg1RBP with the VLE requires both KH didomains, but not the RRM didomain, and moreover that the KH didomains contribute cooperatively to RNA binding. In the full-length protein, individual KH domains display significant redundancy, and their relative importance appears to vary with the RNA target. We also demonstrate that the KH34 didomain mediates Vg1RBP self-association, which is stabilized by RNA, and occurs in vivo as well as in vitro. Altogether, our findings highlight the importance of multiple KH domains in mediating RNA-protein and protein-protein interactions in the formation of a stable complex of Vg1RBP and Vg1 mRNA.     

A ubiquitous and conserved signal for RNA localization in chordates

During oogenesis in Xenopus laevis, several RNAs that localize to the vegetal cortex via one of three temporally defined pathways have been identified. Although individual mRNAs utilize only one pathway, there is functional overlap and apparent continuity between them, suggesting that common cis-acting sequences may exist. Because previous work with the Vg1 mRNA revealed that short nontandem repeats are important for localization, we developed a new computer program, called REPFIND, to expedite the identification of repeated motifs in other localized RNAs. Here we show that clusters of short CAC-containing motifs characterize the localization elements (LEs) of virtually all mRNAs localized to the vegetal cortex of Xenopus oocytes. A search for this signal in GenBank [9] resulted in the identification of new localized mRNAs, demonstrating the applicability of REPFIND to predict localized RNAs. CAC-rich LEs are also found in ascidians and other vertebrates, indicating that these cis regulatory elements are conserved in chordates. Interestingly, biochemical evidence shows that distinct CAC-containing motifs have different functions in the localization process. Thus, clusters of CAC-containing motifs are a ubiquitous signal for RNA localization and can signal localization in a variety of pathways through slight variations in sequence composition.     

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.     

Xvelo1 uses a novel 75-nucleotide signal sequence that drives vegetal localization along the late pathway in Xenopus oocytes

Vegetally localized RNAs in Xenopus laevis oocytes are involved in the patterning of the early embryo as well as in cell fate specification. Here we report on the isolation and characterization of a novel, vegetally localized RNA in Xenopus oocytes termed Xvelo1. It encodes a protein of unknown biological function and it represents an antisense RNA for XPc1 over a length of more than 1.8 kb. Xvelo1 exhibits a localization pattern reminiscent of the late pathway RNAs Vg1 and VegT; it contains RNA localization elements (LE) which do not match with the consensus structural features as deduced from Vg1 and VegT LEs. Nevertheless, the protein binding pattern as observed for Xvelo1-LE in UV cross-linking experiments and coimmunoprecipitation assays is largely overlapping with the one obtained for Vg1-LE. These observations suggest that the structural features recognized by the protein machinery that drives localization of maternal mRNAs along the late pathway in Xenopus oocytes must be redefined.     

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.     

40LoVe interacts with Vg1RBP/Vera and hnRNP I in binding the Vg1-localization element

Localizing mRNAs within the cytoplasm gives cells the ability to spatially restrict protein production, a powerful means to regulate gene expression. Localized mRNA is often visible in microscopically observable particles or granules, and the association of mRNA localization with these structures is an indication that particles or granules may be essential to the localization process. Understanding how such structures form will therefore be important for understanding the function of localization RNPs (L-RNPs). We previously identified a novel component of an L-RNP from the Vg1 mRNA from Xenopus oocytes called 40LoVe. 40LoVe interaction with the Vg1-localization element (Vg1LE) was previously shown to be dependent on the VM1 and E2 sequence motifs within the Vg1LE that cross-link to hnRNP I and Vg1RBP/Vera, respectively. We report interaction of these motif-binding proteins with 40LoVe and identify a 40LoVe-Xenopus hnRNP D/AUF1 interaction. We further demonstrate that titration of VM1 and E2 motif binding activity in vivo surprisingly suggests that the motif binding proteins have differing roles during Vg1LE-dependent mRNA localization.     

The mRNA coding for Xenopus glutamate receptor interacting protein 2 (XGRIP2) is maternally transcribed, transported through the late pathway and localized to the germ plasm

Using a large-scale in situ hybridization screening, we found that the mRNA coding for Xenopus glutamate receptor interacting protein 2 (XGRIP2) was localized to the germ plasm of Xenopus laevis. The mRNA is maternally transcribed in oocytes and, during maturation, transported to the vegetal germ plasm through the late pathway where VegT and Vg1 mRNAs are transported. In the 3'-untranslated region (UTR) of the mRNA, there are clusters of E2 and VM1 localization motifs that were reported to exist in the mRNAs classified as the late pathway group. With in situ hybridization to the sections of embryos, the signal could be detected in the cytoplasm of migrating presumptive primordial germ cells (pPGCs) until stage 35. At stage 40, when the cells cease to migrate and reach the dorsal mesentery, the signal disappeared. A possible role of XGRIP2 in pPGCs of Xenopus will be discussed.     

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.     

Embryonic expression of Drosophila IMP in the developing CNS and PNS

Drosophila IMP (dIMP) is related to the vertebrate RNA-binding proteins IMP1-3, ZBP1, Vg1RBP and CRD-BP, which are involved in RNA regulatory processes such as translational repression, localization and stabilization. The proteins are expressed in many fetal tissues, including the developing nervous system, and IMP up-regulation in solid tumors correlates with a high metastatic potential and poor prognosis. In this study, we used immunohistochemistry and live-imaging of an endogenous promoter-driven GFP-dIMP fusion protein to reveal the expression pattern of dIMP protein throughout embryogenesis. In the cellular blastoderm, immunoreactivity was seen in the entire cell-layer, where it was localized apically to the nucleus, and in the pole cells. Later, the GFP-dIMP fusion protein appeared in the developing central nervous system, both in the brain and in the ventral nerve cord. In the peripheral nervous system, immunoreactivity was detected in both neurons and accessory cells of chordotonal and external sensory organs.     

Distinct roles of two conserved Staufen domains in oskar mRNA localization and translation

Drosophila Staufen protein is required for the localization of oskar mRNA to the posterior of the oocyte, the anterior anchoring of bicoid mRNA and the basal localization of prospero mRNA in dividing neuroblasts. The only regions of Staufen that have been conserved throughout animal evolution are five double-stranded (ds)RNA-binding domains (dsRBDs) and a short region within an insertion that splits dsRBD2 into two halves. dsRBDs 1, 3 and 4 bind dsRNA in vitro, but dsRBDs 2 and 5 do not, although dsRBD2 does bind dsRNA when the insertion is removed. Full-length Staufen protein lacking this insertion is able to associate with oskar mRNA and activate its translation, but fails to localize the RNA to the posterior. In contrast, Staufen lacking dsRBD5 localizes oskar mRNA normally, but does not activate its translation. Thus, dsRBD2 is required for the microtubule-dependent localization of osk mRNA, and dsRBD5 for the derepression of oskar mRNA translation, once localized. Since dsRBD5 has been shown to direct the actin-dependent localization of prospero mRNA, distinct domains of Staufen mediate microtubule- and actin-based mRNA 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.     

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.     

PKA-R1 spatially restricts Oskar expression for Drosophila embryonic patterning

Targeting proteins to specific domains within the cell is central to the generation of polarity, which underlies many processes including cell fate specification and pattern formation during development. The anteroposterior and dorsoventral axes of the Drosophila melanogaster embryo are determined by the activities of localized maternal gene products. At the posterior pole of the oocyte, Oskar directs the assembly of the pole plasm, and is thus responsible for formation of abdomen and germline in the embryo. Tight restriction of oskar activity is achieved by mRNA localization, localization-dependent translation, anchoring of the RNA and protein, and stabilization of Oskar at the posterior pole. Here we report that the type 1 regulatory subunit of cAMP-dependent protein kinase (Pka-R1) is crucial for the restriction of Oskar protein to the oocyte posterior. Mutations in PKA-R1 cause premature and ectopic accumulation of Oskar protein throughout the oocyte. This phenotype is due to misregulation of PKA catalytic subunit activity and is suppressed by reducing catalytic subunit gene dosage. These data demonstrate that PKA mediates the spatial restriction of Oskar for anteroposterior patterning of the Drosophila embryo and that control of PKA activity by PKA-R1 is crucial in this process.     

A novel mode of RBD-protein recognition in the Y14-Mago complex

Y14 and Mago are conserved eukaryotic proteins that associate with spliced mRNAs in the nucleus and remain associated at exon junctions during and after nuclear export. In the cytoplasm, Y14 is involved in mRNA quality control via the nonsense-mediated mRNA decay (NMD) pathway and, together with Mago, is involved in localization of osk (oskar) mRNA. We have determined the crystal structure of the complex between Drosophila melanogaster Y14 and Mago at a resolution of 2.5 A. The structure reveals an atypical mode of protein-protein recognition mediated by an RNA-binding domain (RBD). Instead of binding RNA, the RBD of Y14 engages its RNP1 and RNP2 motifs to bind Mago. Using structure-guided mutagenesis, we show that Mago is also a component of the NMD pathway, and that its association with Y14 is essential for function. Heterodimerization creates a single structural platform that interacts with the NMD machinery via phylogenetically conserved residues.