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.     

Control of mRNA translation in oocytes and developing embryos of giant moths. I. Function of the 5' terminal "Cap"in the tobacco hornworm, Manduca sexta

Injection of labeled leucine into oocytes and developing embryos of the tobacco hornworm, Manduca sexta, revealed that the rate of protein synthesis increases dramatically after fertilization and continues to rise until gastrulation. Cell-free preparations of oocytes and developing embryos show a similar pattern of in vitro incorporation. When messenger RNA extracted from unfertilized oocytes was examined by gradient density centrifugation under denaturing conditions, a broad peak was observed which centered around 15 S. In contrast to mRNA extracted from oocytes, that from embryos was found to be capped by 7-methylguanosine at the 5′ terminus. When translation of oocyte mRNA was compared with that of embryo mRNA in a cell-free translation system derived from wheat germ, oocyte RNA translated less efficiently. In the presence of an inhibitor of methylation, S-adenosylhomocysteine, the differences were further widened. In competition with a cap analog, 7-methylguanosine 5′-monophosphate, embryo mRNA translation was inhibited more than oocyte at low concentrations of analog. These results are taken to indicate that the lack of a cap at the 5′ terminus could be one mechanism to inhibit translation prior to fertilization.     

Conditions affecting protein synthesis in amphibian oocytes.

The endogenous protein synthesis of Xenopus laevis and Calyptocephalella caudiverbera oocytes was studied by measuring the incorporation into acid-precipitable material of radioactive amino acids placed in the extracellular medium. Large differences of incorporation into protein were observed by using different labeled amino acids. For example, it was found that radioactive aspartic acid or glutamic acid was very poorly incorporated at concentrations under 0.1 mm. These differences are due to differences in uptake constants and in the internal pools of free amino acids which are very large for the acidic amino acids. Both types of oocytes behaved similarly with respect to magnesium ion concentration, temperature optimum and inhibitors of protein synthesis. They differed however in sensitivity to pH since Xenopus laevis oocyte protein synthesis was twofold higher at pH 8.5 than at pH 7 while Calyptocephalella caudiverbera oocytes showed no difference. Isolation of oocyte germinal vesicles allowed a study of the entrance of newly synthesized protein into the cell nuclei.     

LOCALIZATION OF RIBOSOMAL DNA WITHIN OOCYTES OF THE HOUSE CRICKET, ACHETA DOMESTICUS (ORTHOPTERA: GRYLLIDAE)

A large DNA-containing body is present in addition to the chromosomes in oocytes of the house cricket Acheta domesticus. Large masses of nucleolar material accumulate at the periphery of the DNA body during the diplotene stage of meiotic prophase I. RNA-DNA hybridization analysis demonstrates that the genes which code for 18S and 28S ribosomal RNA are amplified in the ovary. In situ hybridization indicates that the amplified genes are localized within the DNA body of early prophase cells. As the cells proceed through diplotene the DNA which hybridizes with ribosomal RNA is gradually incorporated into the developing nucleolar mass.     

Ribonucleoprotein localization in mouse oocytes

RNA molecules rarely function alone in cells. For most RNAs, their function requires formation of various ribonucleoprotein (RNP) complexes. For example, mRNP composition can determine mRNA localization, translational repression, level of translation or mRNA stability. RNPs are usually studied by biochemical methods. However, biochemical approaches are unsuitable for some model systems, such as mammalian oocytes and early embryos, due to the small amounts that can be obtained for experimental analysis. In such cases, microscopic techniques are often used to learn about RNPs. Here, we present a review of immunostaining, fluorescence in situ hybridization with subcellular resolution and a combination of both, with emphasis on the mouse oocyte and early embryos models. Application of these techniques to whole-mount fixed oocytes and early embryos can provide information about RNP composition and localization with three-dimensional resolution.     

A simple real-time assay for in vitro translation

A high-throughput assay for real-time measurement of translation rates in cell-free protein synthesis (SNAP assay) is described. The SNAP assay enables quantitative, real-time measurement of overall translation rates in vitro via the synthesis of O⁶-alkylguanine DNA O⁶-alkyltransferase (SNAP). SNAP production is continuously detected by fluorescence produced by the reaction of SNAP with a range of quenched fluorogenic substrates. The capabilities of the assay are exemplified by measurements of the activities of Escherichia coli MRE600 ribosomes and fluorescently labeled E. coli mutant ribosomes in the PURExpress translation system and by determination of the 50% inhibitory concentrations (IC₅₀) of three common macrolide antibiotics.     

Partial purification and characterization of the maturation-promoting factor from eggs of Xenopus laevis

Maturation-promoting factor (MPF) was purified 20- to 30-fold from unfertilized eggs of Xenopus laevis, by ammonium sulfate precipitation and chromatography on pentyl-agarose and arginine-agarose. The final material induces maturation in 50% of the recipient oocytes when 5 ng of protein is injected in a volume of 20 ml. The maturation response includes precocious germinal vesical breakdown, elevated protein phosphorylation, amplification of cytoplasmic MPF, and formation of an activatable egg blocked at second meiotic metaphase. These eggs are capable of cleavage and, in some cases, of gastrulation. A quantitative in vivo assay of MPF is described and a unit of MPF activity is defined as that amount causing a 50% maturation frequency when oocytes are injected each with a 20-nl test volume. Maturation frequency has a very high-order dependence on MPF concentration. The purification procedure selects simultaneously for endogenous protein phosphorylation systems containing kinases, protein substrates, and phosphatases. This fact, as well as the finding that ATP enhances MPF activity at least twofold when included in the dilution medium for assay, is discussed in terms of the possible involvement of protein phosphorylation in MPF activation and inactivation.     

Insect protein synthesis in frog cells: the translation of honey bee promelittin messenger RNA in Xenopus oocytes

Venom glands of young queen bees (Apis mellifera) synthesize the toxic peptide melittin as their main product. Melittin is formed by proteolytic cleavage of a precursor, promelittin. Unfractionated RNA prepared from venom glands was injected into Xenopus oocytes and was shown to direct the synthesis of a promelittin-like substance. About half of the peptide chain made in oocytes has been sequenced; the 17 amino acid residues identified correspond exactly with sequences found in promelittin from venom gland cells. These results yield final proof that injected messenger RNAs can be read with great fidelity. The translation of a messenger from an insect gland shows that at least some of the translational systems within the oocyte are neither cell-type nor phylum specific. It seems likely that the oocyte can be used to assay any kind of eukaryotic mRNA.The conversion of promelittin to melittin could not be detected in oocytes. Moreover, the promelittin synthesized in oocytes differs at the carboxyl end from the product made in gland cells, for the latter terminates with glutamine amide while the oocyte material probably ends with an amino acid with a free α-carboxyl group. Some of the post-translational modifications characteristic of gland cells thus do not seem to take place in oocytes.     

Baciphelacin: a new eukaryotic translation inhibitor

Baciphelacin an antibiotic produced by Bacillus thiaminolyticus was a potent inhibitor of protein synthesis in HeLa cells and other mammalian cell lines. It had no effect on DNA or RNA synthesis. Concentrations of baciphelacin around 10⁻⁷ M inhibited protein synthesis by 50% in intact cells. The antibiotic had no effect on protein synthesis in Saccharomyces cerevisiae or Escherichia coli, but inhibited the protozoan Trypanosoma brucei. In vitro protein synthesis in a rabbit reticulocyte cell-free system was blocked by baciphelacin. However, translation of globin mRNA in a wheat cell-free system was not affected by this antibiotic. Baciphelacin had no activity against a number of cell-free systems used to measure different steps of translation, including binding of substrates to the ribosome, peptide bond formation and polyphenylalanine synthesis. Therefore, it is assumed that it affects the initiation of translation or the charging of tRNA. Finally, the inhibition of protein synthesis by compounds structurally related to baciphelacin was tested and their effects compared to baciphelacin.     

Apoptotic and developmental effects of bovine Herpesvirus type-5 infection on in vitro-produced bovine embryos

Bovine Herpesvirus type-5 (BoHV-5), which is potentially neuropathogenic, was recently described to be related with reproductive disorders in cows. The objective was to elucidate mechanisms involved in propagation of BoHV-5 in embryonic cells. For this purpose, bovine embryos produced in vitro were assayed for apoptotic markers after experimental infection of oocytes, in vitro fertilization, and development. Host DNA fragmentation was detected with a TUNEL assay, expression of annexin-V was measured with indirect immunofluorescence, and viral DNA was detected with in situ hybridization. Infective BoHV-5 virus was recovered from embryos derived from exposed oocytes after two consecutive passages on Madin-Darby bovine kidney (MDBK) cells. The viral DNA corresponding to US9 gene, localized between nucleotides 126243 to 126493, was detected in situ and amplified. There was no significant difference between the ratio of TUNEL stained nuclei and total cells in good quality blastocysts (0.87 ± 0.05, mean ± SD), but there were differences (P < 0.05) between infected (0.18 ± 0.05) and uninfected blastocysts (0.73 ± 0.07). The Annexin-V label was more intense in uninfected embryos (0.79 ± 0.04; P < 0.05). The quality of infected and uninfected embryos was considered equal, with no significant effect on embryonic development. In conclusion, we inferred that BoHV-5 infected bovine oocytes, replicated, and suppressed some apoptotic pathways, without significantly affecting embryonic development.     

Quantitative and qualitative analysis of RNA synthesis in stage 6 and stage 4 oocytes of Xenopus laevis

RNA synthesis has been studied in oocytes taken from Xenopus laevis females which have not recently ovulated. Such females contain a population of large (stage 6) oocytes which exhibit white equatorial bands and which are considered to represent the terminal stage of oocyte development. Rates of RNA synthesis in these “banded” oocytes were measured by analyzing the kinetics of incorporation of ³H-guanosine into acid-precipitable, alkaline-labile material, and changes in precursor pool (GTP) specific activity during incubations. In additional experiments, rates of RNA synthesis were measured after ³H-GTP was injected directly into stage 6 oocytes. For comparison, rates of RNA synthesis were measured in lampbrush chromosome stage oocytes (stage 4; 0.5–0.6 mm diameter). The results show that, under the in vitro conditions employed, stage 6 oocytes are not metabolically dormant, but synthesize total RNA at a rate at least as great as the stage 4 oocytes.Qualitative studies on newly synthesized RNA in the two oocyte classes have been performed using sucrose density gradient centrifugation and acrylamide gel electrophoresis. Both stage 4 and stage 6 oocytes exhibited similar patterns, and the bulk of the RNA synthesized and accumulated during 12-hr pulses appears to be ribosomal. These observations are discussed in terms of existing concepts concerning synthetic activity in stage 6 oocytes.     

A tale of two functions: enzymatic activity and translational repression by carboxyltransferase

Acetyl-CoA Carboxylase catalyzes the first committed step in fatty acid synthesis. Escherichia coli acetyl-CoA carboxylase is composed of biotin carboxylase, carboxyltransferase and biotin carboxyl carrier protein functions. The accA and accD genes that code for the α- and β-subunits, respectively, are not in an operon, yet yield an α₂β₂ carboxyltransferase. Here, we report that carboxyltransferase regulates its own translation by binding the mRNA encoding its subunits. This interaction is mediated by a zinc finger on the β-subunit; mutation of the four cysteines to alanine diminished nucleic acid binding and catalytic activity. Carboxyltransferase binds the coding regions of both subunit mRNAs and inhibits translation, an inhibition that is relieved by the substrate acetyl-CoA. mRNA binding reciprocally inhibits catalytic activity. Preferential binding of carboxyltransferase to RNA in situ was shown using fluorescence resonance energy transfer. We propose an unusual regulatory mechanism by which carboxyltransferase acts as a ‘dimmer switch’ to regulate protein production and catalytic activity, while sensing the metabolic state of the cell through acetyl-CoA concentration.     

Cell-free translation analysis of messenger RNA in echinoderm and amphibian early development

A wheat germ cell-free translation system has been used to analyze populations of abundant messenger RNA from sea urchin eggs and embryos and from amphibian oocytes and ovaries. We show directly that sea urchin eggs and embryos contain translatable mRNA of three general classes: poly(A)⁺ mRNA, poly(A)^? histone mRNA, and poly(A)^? nonhistone mRNA. Additionally, some histone synthesis appears to be promoted by poly(A)⁺ RNA. Sea urchin eggs seem to contain a higher proportion of prevalent poly(A)^? nonhistone mRNAS than do embryos. Some differences in the proteins encoded by poly(A)⁺ and poly(A)^? RNAs are detectable. Many coding sequences in the egg appear to be represented in both poly(A)⁺ and poly(A)^? RNAs, since the translation products of the two RNA classes exhibit many common bands when run on one-dimensional polyacrylamide gels. However, some of this overlap is probably due to fortuitous comigration of nonidentical proteins. Distinct stage-specific changes in the spectra of prevalent translatable mRNAs of all three classes occur, although many mRNAs are detectable throughout early development. Particularly striking is the presence of an egg poly(A)^? mRNA, encoding a 70,000–80,000 molecular weight protein, which is not detected in morula or later-stage embryos. In amphibian (Xenopus laevis and Triturus viridescens) ovary RNA, the translation assay detects the following three mRNA classes: poly(A)⁺ nonhistone mRNA, poly(A)^? histone mRNA, and poly(A)⁺ histone mRNA. Amphibian ovary RNA appearently lacks an abundant poly(A)^? nonhistone mRNA component of the magnitude detectable in sea urchin eggs. mRNA encoding histone-like proteins is found in the very earliest (small stage 1) oocytes of Xenopus as well as in later stage oocytes. During oogenesis there appear to be no striking qualitative changes in the spectra of prevalent translatable mRNAs which are detected by the cell-free translation assay.     

Protein synthesis during meiotic maturation of mouse oocytes in vitro: Synthesis and phosphorylation of a protein localized in the germinal vesicle

Isolated fully grown mouse oocytes, arrested in dictyate of the first meiotic prophase, synthesize a protein with an apparent molecular weight of 28,000 which is localized in the germinal vesicle of the oocyte (germinal vesicle-associated protein; GVAP). Analyses of the distribution of GVAP have been carried out on SDS-polyacrylamide gels using oocytes cultured in vitro in the presence of [³⁵S]methionine or [³H]lysine and germinal vesicles isolated individually from these cultured oocytes. The results of such analyses show that GVAP contains only about 2% of the total radiolabel incorporated into mouse oocyte proteins, but as much as 40% of the total radiolabel incorporated into proteins associated with isolated germinal vesicles. These measurements indicate that GVAP is at least 1000-fold more concentrated in the germinal vesicle than in the cytoplasm of the oocyte. Furthermore, the synthesis and phosphorylation of GVAP are apparently terminated at a time which coincides with germinal vesicle breakdown during spontaneous meiotic maturation of mouse oocytes in vitro. Although the exact nature of GVAP is not known as yet, it appears to be an example of a protein that is selectively sequestered in the germinal vesicle of the oocyte during oogenesis and whose synthesis and modification are dependent upon the presence of an intact germinal vesicle.     

Modifications of the 5′ Cap of mRNAs during Xenopus Oocyte Maturation: Independence from Changes in Poly(A) Length and Impact on Translation

The translation of specific maternal mRNAs is regulated during early development. For some mRNAs, an increase in translational activity is correlated with cytoplasmic extension of their poly(A) tails; for others, translational inactivation is correlated with removal of their poly(A) tails. Recent results in several systems suggest that events at the 3′ end of the mRNA can affect the state of the 5′ cap structure, m⁷G(5′)ppp(5′)G. We focus here on the potential role of cap modifications on translation during early development and on the question of whether any such modifications are dependent on cytoplasmic poly(A) addition or removal. To do so, we injected synthetic RNAs into Xenopus oocytes and examined their cap structures and translational activities during meiotic maturation. We draw four main conclusions. First, the activity of a cytoplasmic guanine-7-methyltransferase increases during oocyte maturation and stimulates translation of an injected mRNA bearing a nonmethylated GpppG cap. The importance of the cap for translation in oocytes is corroborated by the sensitivity of protein synthesis to cap analogs and by the inefficient translation of mRNAs bearing nonphysiologically capped 5′ termini. Second, deadenylation during oocyte maturation does not cause decapping, in contrast to deadenylation-triggered decapping in Saccharomyces cerevisiae. Third, the poly(A) tail and the N-7 methyl group of the cap stimulate translation synergistically during oocyte maturation. Fourth, cap ribose methylation of certain mRNAs is very inefficient and is not required for their translational recruitment by poly(A). These results demonstrate that polyadenylation can cause translational recruitment independent of ribose methylation. We propose that polyadenylation enhances translation through at least two mechanisms that are distinguished by their dependence on ribose modification.