Xenopus cold-inducible RNA-binding protein 2 interacts with ElrA,the Xenopus homolog of HuR,and inhibits deadenylation of specific mRNAs

Xenopus cold-inducible RNA-binding protein 2 (xCIRP2) is a major cytoplasmic RNA-binding protein in oocytes. In this study, we identify another RNA-binding protein ElrA, the Xenopus homolog of HuR, as an interacting protein of xCIRP2 by yeast two-hybrid screening. As ElrA stabilizes the RNA body in the in vitro mRNA stability system, we examine the role of xCIRP2 in the stabilization of mRNA and find that xCIRP2 inhibits deadenylation of AU-rich element-containing mRNA. These results suggest that xCIRP2 and ElrA may be involved in the regulation of mRNA stability at different steps. By immunoprecipitation with anti-xCIRP2 antibody, we find that xCIRP2 interacts with several mRNAs including mRNA encoding the centrosomal kinase Nek2B in oocytes. xCIRP2 also inhibits deadenylation of the mRNA substrate containing the 3'-untranslated region of Nek2B mRNA in the in vitro system. Our results suggest that xCIRP2 associates with specific mRNAs and can regulate the length of poly(A) tail in Xenopus oocytes.     

Visualization of the reconstituted FRGY2-mRNA complexes by electron microscopy

Xenopus oocytes store large quantities of translationally dormant mRNA in the cytoplasm as storage messenger ribonucleoprotein particles (mRNPs). The Y-box proteins, mRNP3 and FRGY2/mRNP4, are major RNA binding components of maternal storage mRNPs in oocytes. In this study, we show that the FRGY2 proteins form complexes with mRNA, which leads to mRNA stabilization and translational repression. Visualization of the FRGY2-mRNA complexes by electron microscopy reveals that FRGY2 packages mRNA into a compact RNP. Our results are consistent with a model that the Y-box proteins function in packaging of mRNAs to store them stably for a long time in the oocyte cytoplasm.     

Different size of the product of the 14S light chain mRNA translated in vitro and in amphibian oocytes

When purified 14S mRNA for light chain of immunoglobulin is translated in a reticulocyte lysate and in Xenopus oocytes, two major differences are observed: (1) In the lysate 14S RNA competes efficiently with endogenous mRNA whereas in the oocyte it is translated without reducing the synthesis of endogenous proteins. (2) The translation product of 14S light chain mRNA in the lysate is a protein about 20 amino acids longer than light chain whereas in the oocyte it is a chain of the exact size of authentic secreted light chain. This difference can be explained if 14S mRNA codes for a precursor protein, which is not cleaved in the lysate but can be efficiently converted into light chain in the oocytes.     

Possible involvement of insulin-like growth factor 2 mRNA-binding protein 3 in zebrafish oocyte maturation as a novel cyclin B1 mRNA-binding protein that represses the translation in immature oocytes

In immature zebrafish oocytes, dormant cyclin B1 mRNAs localize to the animal polar cytoplasm as aggregates. After hormonal stimulation, cyclin B1 mRNAs are dispersed and translationally activated, which are necessary and sufficient for the induction of zebrafish oocyte maturation. Besides cytoplasmic polyadenylation element-binding protein (CPEB) and cis-acting elements in the 3′ untranslated region (UTR), Pumilio1 and a cis-acting element in the coding region of cyclin B1 mRNA are important for the subcellular localization and timing of translational activation of the mRNA. However, mechanisms underlying the spatio-temporal control of cyclin B1 mRNA translation during oocyte maturation are not fully understood. We report that insulin-like growth factor 2 mRNA-binding protein 3 (IMP3), which was initially described as a protein bound to Vg1 mRNA localized to the vegetal pole of Xenopus oocytes, binds to the 3′ UTR of cyclin B1 mRNA that localizes to the animal pole of zebrafish oocytes. IMP3 and cyclin B1 mRNA co-localize to the animal polar cytoplasm of immature oocytes, but in mature oocytes, IMP3 dissociates from the mRNA despite the fact that its protein content and phosphorylation state are unchanged during oocyte maturation. IMP3 interacts with Pumilio1 and CPEB in an mRNA-dependent manner in immature oocytes but not in mature oocytes. Overexpression of IMP3 and injection of anti-IMP3 antibody delayed the progression of oocyte maturation. On the basis of these results, we propose that IMP3 represses the translation of cyclin B1 mRNA in immature zebrafish oocytes and that its release from the mRNA triggers the translational activation.     

RNA-binding strategies common to cold-shock domain- and RNA recognition motif-containing proteins

Numerous RNA-binding proteins have modular structures, comprising one or several copies of a selective RNA-binding domain generally coupled to an auxiliary domain that binds RNA non-specifically. We have built and compared homology-based models of the cold-shock domain (CSD) of the Xenopus protein, FRGY2, and of the third RNA recognition motif (RRM) of the ubiquitous nucleolar protein, nucleolin. Our model of the CSD_FRG–RNA complex constitutes the first prediction of the three-dimensional structure of a CSD–RNA complex and is consistent with the hypothesis of a convergent evolution of CSD and RRM towards a related single-stranded RNA-binding surface. Circular dichroism spectroscopy studies have revealed that these RNA-binding domains are capable of orchestrating similar types of RNA conformational change. Our results further show that the respective auxiliary domains, despite their lack of sequence homology, are functionally equivalent and indispensable for modulating the properties of the specific RNA-binding domains. A comparative analysis of FRGY2 and nucleolin C-terminal domains has revealed common structural features representing the signature of a particular type of auxiliary domain, which has co-evolved with the CSD and the RRM.     

Translational repression by the oocyte-specific protein P100 in Xenopus

The translational regulation of maternal mRNAs is one of the most important steps in the control of temporal-spatial gene expression during oocyte maturation and early embryogenesis in various species. Recently, it has become clear that protein components of mRNPs play essential roles in the translational regulation of maternal mRNAs. In the present study, we investigated the function of P100 in Xenopus oocytes. P100 exhibits sequence conservation with budding yeast Pat1 and is likely the orthologue of human Pat1a (also called PatL2). P100 is maternally expressed in immature oocytes, but disappears during oocyte maturation. In oocytes, P100 is an RNA binding component of ribosome-free mRNPs, associating with other mRNP components such as Xp54, xRAP55 and CPEB. Translational repression by overexpression of P100 occurred when reporter mRNAs were injected into oocytes. Intriguingly, we found that when P100 was overexpressed in the oocytes, the kinetics of oocyte maturation was considerably retarded. In addition, overexpression of P100 in oocytes significantly affected the accumulation of c-Mos and cyclin B1 during oocyte maturation. These results suggest that P100 plays a role in regulating the translation of specific maternal mRNAs required for the progression of Xenopus oocyte maturation.     

Recruitment of Orc6l, a dormant maternal mRNA in mouse oocytes, is essential for DNA replication in 1-cell embryos

Mouse oocytes acquire the ability to replicate DNA during meiotic maturation, presumably to ensure that DNA replication does not occur precociously between MI and MII and only after fertilization. Acquisition of DNA replication competence requires protein synthesis, but the identity of the proteins required for DNA replication is poorly described. In Xenopus, the only component missing for DNA replication competence is CDC6, which is synthesized from a dormant maternal mRNA recruited during oocyte maturation, and a similar situation also occurs during mouse oocyte maturation. We report that ORC6L is another component required for acquisition of DNA replication competence that is absent in mouse oocytes. The dormant maternal Orc6l mRNA is recruited during maturation via a CPE present in its 3′ UTR. RNAi-mediated ablation of maternal Orc6l mRNA prevents the maturation-associated increase in ORC6L protein and inhibits DNA replication in 1-cell embryos. These results suggest that mammalian oocytes have more complex mechanisms to establish DNA replication competence when compared to their Xenopus counterparts.     

The stability and translation of sea urchin histone messenger RNA molecules injected into Xenopus laevis eggs and developing embryos

Embryonic sea urchin histone mRNA was injected into eggs and developing zygotes of Xenopus. The functional stability of the mRNA was monitored by separating newly synthesized sea urchin histones from those of Xenopus. Just as when injected into Xenopus oocytes, sea urchin H1, H2A, and H2B mRNA molecules have a functional half-life of about 3 hr in the developing embryo. This suggests that the endogenous Xenopus histone mRNA is also unstable and has a number of implications for the amount of histone mRNA that is stored in the oocyte and the time at which histone genes should become active in development. The injected mRNA is translated with little, if any, greater efficiency in the egg than in the oocyte. However, Xenopus histone synthesis increases about 20- to 50-fold during the transition from oocyte to egg. The injection experiments therefore suggest that this increase is brought about primarily by the mobilization of stored mRNA, rather than an increase in the efficiency of histone synthesis.     

Large P body-like RNPs form in C. elegans oocytes in response to arrested ovulation, heat shock, osmotic stress, and anoxia and are regulated by the major sperm protein pathway

As Caenorhabditis elegans hermaphrodites age, sperm become depleted, ovulation arrests, and oocytes accumulate in the gonad arm. Large ribonucleoprotein (RNP) foci form in these arrested oocytes that contain RNA-binding proteins and translationally masked maternal mRNAs. Within 65 min of mating, the RNP foci dissociate and fertilization proceeds. The majority of arrested oocytes with foci result in viable embryos upon fertilization, suggesting that foci are not deleterious to oocyte function. We have determined that foci formation is not strictly a function of aging, and the somatic, ceh-18, branch of the major sperm protein pathway regulates the formation and dissociation of oocyte foci. Our hypothesis for the function of oocyte RNP foci is similar to the RNA-related functions of processing bodies (P bodies) and stress granules; here, we show three orthologs of P body proteins, DCP-2, CAR-1 and CGH-1, and two markers of stress granules, poly (A) binding protein (PABP) and TIA-1, appear to be present in the oocyte RNP foci. Our results are the first in vivo demonstration linking components of P bodies and stress granules in the germ line of a metazoan. Furthermore, our data demonstrate that formation of oocyte RNP foci is inducible in non-arrested oocytes by heat shock, osmotic stress, or anoxia, similar to the induction of stress granules in mammalian cells and P bodies in yeast. These data suggest commonalities between oocytes undergoing delayed fertilization and cells that are stressed environmentally, as to how they modulate mRNAs and regulate translation.     

Distinct functions of maternal and somatic Pat1 protein paralogs

We previously identified Xenopus Pat1a (P100) as a member of the maternal CPEB RNP complex, whose components resemble those of P-(rocessing) bodies, and which is implicated in translational control in Xenopus oocytes. Database searches have identified Pat1a proteins in other vertebrates, as well as paralogous Pat1b proteins. Here we characterize Pat1 proteins, which have no readily discernable sequence features, in Xenopus oocytes, eggs, and early embryos and in human tissue culture cells. xPat1a and 1b have essentially mutually exclusive expression patterns in oogenesis and embryogenesis. xPat1a is degraded during meiotic maturation, via PEST-like regions, while xPat1b mRNA is translationally activated at GVBD by cytoplasmic polyadenylation. Pat1 proteins bind RNA in vitro, via a central domain, with a preference for G-rich sequences, including the NRAS 5′ UTR G-quadruplex-forming sequence. When tethered to reporter mRNA, both Pat proteins repress translation in oocytes. Indeed, both epitope-tagged proteins interact with the same components of the CPEB RNP complex, including CPEB, Xp54, eIF4E1b, Rap55B, and ePAB. However, examining endogenous protein interactions, we find that in oocytes only xPat1a is a bona fide component of the CPEB RNP, and that xPat1b resides in a separate large complex. In tissue culture cells, hPat1b localizes to P-bodies, while mPat1a-GFP is either found weakly in P-bodies or disperses P-bodies in a dominant-negative fashion. Altogether we conclude that Pat1a and Pat1b proteins have distinct functions, mediated in separate complexes. Pat1a is a translational repressor in oocytes in a CPEB-containing complex, and Pat1b is a component of P-bodies in somatic cells.     

A cis-acting element in the coding region of cyclin B1 mRNA couples subcellular localization to translational timing

Subcellular localization of messenger RNAs (mRNAs) to correct sites and translational activation at appropriate timings are crucial for normal progression of various biological events. However, a molecular link between the spatial regulation and temporal regulation remains unresolved. In immature zebrafish oocytes, translationally repressed cyclin B1 mRNA is localized to the animal polar cytoplasm and its temporally regulated translational activation in response to a maturation-inducing hormone is essential to promote oocyte maturation. We previously reported that the coding region of cyclin B1 mRNA is required for the spatio-temporal regulation. Here, we report that a sequence, CAGGAGACC, that is conserved in the coding region of vertebrate cyclin B1 mRNA is involved in the regulation. Like endogenous cyclin B1 mRNA, reporter mRNAs harboring the sequence CAGGAGACC were localized to the animal polar cytoplasm of oocytes, while those carrying mutations in the sequence (with no change in the coding amino acids) were dispersed in the animal hemisphere of oocytes. Furthermore, translational activation of the mutant mRNAs was initiated at a timing earlier than that of endogenous and wild-type reporter mRNAs during oocyte maturation. Interaction of CAGGAGACC with proteins in vitro suggests that this sequence functions in collaboration with a trans-acting protein factor(s) in oocytes. These findings reveal that the sequence in the coding region of cyclin B1 mRNA plays an important role as a cis-acting element in both subcellular localization and translational timing of mRNA, providing a direct molecular link between the spatial and temporal regulation of mRNA translation.     

Analyses of zebrafish and Xenopus oocyte maturation reveal conserved and diverged features of translational regulation of maternal cyclin B1 mRNA

Background  

Vertebrate development relies on the regulated translation of stored maternal mRNAs, but how these regulatory mechanisms may have evolved to control translational efficiency of individual mRNAs is poorly understood. We compared the translational regulation and polyadenylation of the cyclin B1 mRNA during zebrafish and Xenopus oocyte maturation. Polyadenylation and translational activation of cyclin B1 mRNA is well characterized during Xenopus oocyte maturation. Specifically, Xenopus cyclin B1 mRNA is polyadenylated and translationally activated during oocyte maturation by proteins that recognize the conserved AAUAAA hexanucleotide and U-rich Cytoplasmic Polyadenylation Elements (CPEs) within cyclin B1 mRNA's 3'UnTranslated Region (3'UTR).     

Translation of mouse interferon mRNA in Xenopus laevis oocytes and in rabbit reticulocyte lysates

Mouse interferon mRNA, extracted from NDV (Newcastle disease virus)-induced L-929 cells has been translated with high efficiency in $\text{Xenopus laevis}$ oocytes and rabbit reticulocyte lysates. The translational efficiency of a crude RNA extract was 10 640 interferon units/mg RNA/hour for the $\text{Xenopus}$ oocytes and 4 012 interferon units/mg RNA/hour for the reticulocyte lysates. The translation product fulfilled the usual criteria for mouse interferon, $\text{viz.}$ species specificity and neutralization by specific anti-mouse interferon antiserum. Upon injection of crude interferon mRNA into $\text{Xenopus}$ oocytes, interferon activity appeared both in the oocyte homogenates and the oocyte incubation medium. When analyzed by velocity sedimentation in formamidesucrose, the mouse interferon mRNA showed a rather sharp peak halfway between the 4 S and 18 S RNA markers, as could be expected from a mRNA which codes for a 20,000 dalton protein.     

Direct injection of cell-free Kir1.1 protein into Xenopus oocytes replicates single-channel currents derived from Kir1.1 mRNA

The development of integral membrane protein cell-free synthesis permits in-vitro labeling of accessible cysteines for real-time FRET and LRET measurements. The functional integrity of these synthetic ion channel proteins has been verified at the whole oocyte level by direct injection into, and recording from, Xenopus oocytes. However, the microscopic single-channel properties of cell-free translated protein have not been systematically examined. In the present study, we compare patch-clamp currents originating from cell-free protein with currents derived from mRNA injection, using the same (single-Cys) inward rectifier DNA template (C189-Kir1.1b). Results indicate that cell-free Kir protein, incorporated into liposomes and injected into oocytes, is trafficked to the plasma membrane where it inserts in an outside-out orientation and exhibits single-channel characteristics identical to that derived from a corresponding mRNA.     

Levels of free PABP are limited by newly polyadenylated mRNA in early Spisula embryogenesis

The poly(A) tail of eukaryotic mRNAs regulates translation and RNA stability through an association with the poly(A)-binding protein (PABP). The role of PABP in selective polyadenylation/deadenylation and translational recruitment/repression of maternal mRNAs that occurs in early development is not fully understood. Here, we report studies including UV-crosslinking and immunoblotting assays to characterise PABP in the early developmental stages of the clam Spisula solidissima. A single, 70 kDa PABP, whose sequence is highly homologous to vertebrate, yeast and plant PABPs, is detected in oocytes. The levels of clam PABP are constant in early embryogenesis, although its ability to crosslink labelled poly(A) is ‘masked’ shortly after fertilisation and remains so until the larval stage. Full RNA-binding potential of PABP in embryo lysates was achieved by brief denaturation with guanidinium hydrochloride followed by dilution for binding and crosslinking or by controlled treatment of lysates with Ca²⁺-dependent micrococcal nuclease. Masking of PABP, which accompanies cytoplasmic polyadenylation in maturing oocytes and in in vitro activated oocyte lysates, is very likely due to an association with mRNAs that bear new PABP target binding sites and thus prevent protein binding to the labelled A-rich probe. Functional implications of these findings as well as the potential application of this unmasking method to other RNA-binding proteins is discussed.     

Glycosaminoglycans in the basal lamina and extracellular matrix of the developing mouse mammary duct

When meiotic maturation of primary oocytes of the starfish Asterias forbesi is induced by 1-methyladenine, rapid and striking changes in the pattern of protein synthesis detectable by electrophoresis occur after germinal vesicle breakdown. These include a decline in relative labeling with [³⁵S]methionine of several polypeptides synthesized in the oocyte, and increased labeling and new appearance of several polypeptides. Fertilization does not result in other detectable changes. The population of total mRNA translatable in a rabbit reticulocyte lysate cell-free system does not change, but the distribution of mRNAs between polysomes and the postribosomal supernatant reflects the changes observed in vivo. Thus these changes are regulated at the translational level. A review of the literature indicates that translationally mediated changes in patterns of protein synthesis during maturation of oocytes may be a widespread phenomenon.