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.     

Degradation of δ-crystallin mRNA in the lens fiber cells of the chicken

δ-Crystallin is the principal protein synthesized in the embryonic chicken lens. After hatching δ-crystallin synthesis decreases and eventually ceases. We have determined when the δ-crystallin messenger RNA (mRNA) disappears from the lens fiber cells during the first year of age by cell-free translation of lens RNA in a reticulocyte lysate, RNA blot (Northern) hybridization, and in situ hybridization. The hybridization was performed with a nick-translated, cloned δ-crystallin cDNA (pδCr2). δ-Crystallin mRNA was present in the lens until 3 months of age and disappeared between the third and fifth month after hatching. The in situ hybridization experiments indicated that the δ-crystallin mRNA was present throughout the lens fiber mass until 1 month after hatching and was greatly reduced in the cortical fiber cells thereafter. In contrast to earlier stages, then, the cortical fiber cells differentiating at the lens equator after about 1 month of age do not accumulate δ-crystallin mRNA. The data also indicate that the maximal half-life of functional δ-crystallin mRNA in the posthatched chicken lens is about 2 months.     

Xtr, a plural tudor domain-containing protein, is involved in the translational regulation of maternal mRNA during oocyte maturation in Xenopus laevis

Xtr in the fertilized eggs of Xenopus has been demonstrated to be a member of a messenger ribonucleoprotein (mRNP) complex that plays a crucial role in karyokinesis during cleavage. Since the Xtr is also present both in oocytes and spermatocytes and its amount increases immediately after spematogenic cells enter into the meiotic phase, this protein was also predicted to act during meiotic progression. Taking advantage of Xenopus oocytes' large size to microinject anti-Xtr antibody into them for inhibition of Xtr function, we examined the role of Xtr in meiotic progression of oocytes. Microinjection of anti-Xtr antibody into immature oocytes followed by reinitiation of oocyte maturation did not affect germinal vesicle break down and the oscillation of Cdc2/cyclin B activity during meiotic progression but caused abnormal spindle formation and chromosomal alignment at meiotic metaphase I and II. Immunoprecipitation of Xtr showed the association of Xtr with FRGY2 and mRNAs such as RCC1 and XL-INCENP mRNAs, which are involved in the progression of karyokinesis. When anti-Xtr antibody was injected into oocytes, translation of XL-INCENP mRNA, which is known to be repressed in immature oocytes and induced after reinitiation of oocyte maturation, was inhibited even if the oocytes were treated with progesterone. A similar translational regulation was observed in oocytes injected with a reporter mRNA, which was composed of an enhanced green fluorescent protein open reading frame followed by the 3' untranslational region (3'UTR) of XL-INCENP mRNA. These results indicate that Xtr regulates the translation of XL-INCENP mRNA through its 3'UTR during meiotic progression of oocyte.     

Eukaryotic initiation factor 4A stimulates translation in microinjected Xenopus oocytes

The injection of heterologous mRNA into fully grown Xenopus oocytes results not only in the synthesis of the heterologous protein but also in a reciprocal decrease in the synthesis of endogenous proteins. This indicates that injected and endogenous mRNAs compete for some component which is rate-limiting for translation in oocytes. We have attempted to identify this rate-limiting translational component. We find that heterologous and homologous polysomes compete with endogenous mRNAs as effectively as naked mRNA, indicating that polysomes do not contain detectable levels of the rate-limiting factor. In addition, we have used micrococcal nuclease digestion and a mRNA-specific oligonucleotide to destroy the mRNA component of polysomes. The remaining polysome factors, when injected into oocytes, failed to stimulate translation. When several eukaryotic translation initiation factors were injected into oocytes, initiation factor 4A consistently increased general oocyte protein synthesis by about twofold. It is possible that the availability of eIF-4A in oocytes is a key factor in limiting the overall rate of protein synthesis.     

Amounts and modulation of actin mRNAs in mouse oocytes and embryos

In order to measure the content of beta- and gamma-actin mRNA in mouse oocytes and ovulated eggs, Northern and slot blots were hybridized to complementary RNA probes transcribed from mouse isotype-specific cDNA sequences. The blots included samples of isotype-specific sense strand RNA standards prepared from the same cDNA sequences. Total actin mRNA content was estimated to be 40 fg per preovulatory full-grown oocyte or egg, consisting of one-third beta-actin mRNA and two-thirds gamma-actin mRNA. Ninety per cent of the actin mRNA is on polysomes in full-grown oocytes. The per cent of actin mRNA in polysomal mRNA is similar to the per cent of actin in newly synthesized proteins. Measurements on other developmental stages showed that, in mid-growth-phase oocytes, each actin mRNA reaches a level twofold higher than in full-grown oocytes. Thereafter, all modulations of the two isotypic mRNAs occur in parallel; that is, they are maintained at constant levels during the late growth phase (oocytes from females 8-14 days old); gradually degraded in oocytes that have completed their rapid growth phase (oocytes from females 15-18 days old), in maturing oocytes, and in 1- and 2-cell embryos; and deadenylated after about 7 h of progression into meiotic maturation.     

Two delta-crystallin polypeptides are derived from a cloned delta 1-crystallin cDNA

Previous studies have shown that there are 2 similar delta-crystallin genes (delta 1 and delta 2) and at least 2 delta-crystallin polypeptides in the chicken eye lens. We show here that both delta-crystallin polypeptides can be synthesized from mRNA transcribed in vitro from a cloned delta 1-crystallin cDNA. Both polypeptides co-migrate in SDS-urea-polyacrylamide electrophoresis with their authentic counterparts isolated from 15-day-old embryonic chicken lenses, and both react with sheep anti-chicken delta-crystallin serum. Screening nearly 900 delta-crystallin cDNA clones from a 15-day-old embryonic lens library with an oligonucleotide probe specific for exon 2 of the delta 2-crystallin gene failed to detect any delta 2 cDNA clones, indicating that the delta 2 gene produces little or no mRNA in the lens at this stage of development. Our results suggest that both of the observed delta-crystallin polypeptides are derived from mRNA transcribed from the delta 1 gene, with heterogeneity arising at the translational or co-translational level.     

The oligo(A) tail on histone mRNA plays an active role in translational silencing of histone mRNA during Xenopus oogenesis

Metazoan replication-dependent histone mRNAs end in a stem-loop sequence. The one known exception is the histone mRNA in amphibian oocytes, which has a short oligo(A) tail attached to the stem-loop sequence. Amphibian oocytes also contain two proteins that bind the 3' end of histone mRNA: xSLBP1, the homologue of the mammalian SLBP, and xSLBP2, which is present only in oocytes. xSLBP2 is an inhibitor of histone mRNA translation, while xSLBP1 activates translation. The short A tail on histone mRNAs appears at stage II to III of oogenesis and is present on histone mRNAs throughout the rest of oogenesis. At oocyte maturation, the oligo(A) tail is removed and the xSLBP2 is degraded, resulting in the activation of translation of histone mRNA. Both SLBPs bind to the stem-loop with the oligo(A) tail with similar affinities. Reporter mRNAs ending in the stem-loop with or without the oligo(A) tail are translated equally well in a reticulocyte lysate, and their translation is stimulated by the presence of xSLBP1. In contrast, translation of the reporter mRNA with an oligo(A) tail is not activated in frog oocytes in response to the presence of xSLBP1. These results suggest that the oligo(A) tail is an active part of the translation repression mechanism that silences histone mRNA during oogenesis and that its removal is part of the mechanism that activates translation.     

Cloning and sequencing of a carp βs-crystallin cDNA

The mRNAs were extracted from common carp (Cyprinus carpio) lenses, purified, reverse transcribed, dC tailed and cloned into Escherichia coli with pBR322 as vector. The cloning efficiency was around 1·10⁷ colonies per μg of mRNA. A clone (pC20) was found by hybrid-arrested translation to contain the cDNA related to carp crystallins. However, comparison of the derived amino-acid sequence with bovine γ-II and β_s-crystallins indicates that this carp crystallin sequence resembles closely the bovine β_s-crystallin and should be better classified as such except that this fish sequence does not contain the N-terminal ‘arm’ of four amino-acid residues present in bovine β_s-crystallin.     

In vivo uptake of mitochondrial malate dehydrogenase from watermelon by mitochondria of Xenopus laevis oocytes

The heterologous in vivo translation system of Xenopus laevis oocytes was used to translate messenger RNA isolated from water-melon cotyledons. Immunocytochemistry was used to localize the translation products in situ within the oocyte. In addition, the translation products were immunoprecipitated from homogenized oocytes, separated on SDS-polyacrylamide electrophoresis and visualized by fluorography. A variety of watermelon proteins encoded in the injected mRNA were translated within the oocytes. Among them was the mitochondrial isoenzyme of malate dehydrogenase (mtMDH). The mtMDH was correctly imported into the mitochondria of the oocytes, as detected by immunocytochemistry.     

The control of δ-crystallin gene expression during lens cell development: Dissociation of cell elongation,cell division, δ-crystallin synthesis,and δ-crystallin mRNA accumulation

Previous studies have shown that freshly explanted 6-day-old embryonic chick lens epithelial cells elongate, differentially increase their synthesis of δ-crystallin, and accumulate δ-crystallin mRNA when cultured with fetal calf serum; in contrast, precultured serum-starved 6-day-old and freshly explanted 19-day-old embryonic epithelial cells divide when treated with fetal calf serum. We have explored whether the stimulation of δ-crystallin gene expression (as measured by δ-crystallin synthesis and δ-crystallin mRNA accumulation) is affected by inhibiting lens cell elongation with colchicine, and whether δ-crystallin gene expression is increased in lens epithelial cells stimulated to divide by treatment with fetal calf serum, as it is in those stimulated to elongate by treatment with serum. Three new findings were made in this study. First, the stimulation of δ-crystallin gene expression does not require elongation of the cultured lens cells. Second, a decreased proportion of δ-crystallin synthesis is observed in lens epithelial cells during normal development and during serum starvation; in neither case is this decrease associated with a reduction in the number of δ-crystallin mRNA sequences per cell. Finally, serum stimulation of lens cell division does not increase the proportion of δ-crystallin synthesis, but can promote the accumulation of δ-crystallin mRNA. Thus, the relative proportion of δ-crystallin synthesized during chick lens development is not solely a function of the number of δ-crystallin mRNA sequences in the lens cells.     

Characteristics of the Na+/K+-ATPase from Torpedo californica expressed in Xenopus oocytes: a combination of tracer flux measurements with electrophysiological measurements

The Na+/K+-ATPase from electroplax of Torpedo californica was incorporated into the plasma membrane of Xenopus oocytes by microinjection of mRNA coding for the alpha- and beta-subunit of the enzyme; the mRNAs were obtained by in vitro translation of cloned cDNAs (Noguchi et al. (1988) FEBS Lett. 225, 27-32). (1) Measurements of ouabain-sensitive membrane current revealed that the Na+/K+-ATPase of Torpedo is less sensitive to ouabain than the endogenous enzyme. (2) The ouabain-sensitive membrane currents in mRNA-injected oocytes exhibit similar voltage dependence as the currents generated by the endogenous ATPase of Xenopus oocytes; in particular, the current-voltage relation exhibits a maximum and a negative slope at potentials more positive than +20 mV. (3) A maximum can also be detected if the rate of 22Na+ efflux is determined under different voltage-clamp conditions. If membrane current and rate of Na+2 efflux are determined simultaneously, a voltage-independent ratio between current and flux is obtained suggesting voltage-independent Na+-K+ stoichiometry. The data are compatible with a 3Na+-2K+ stoichiometry.     

The stem-loop binding protein is required for efficient translation of histone mRNA in vivo and in vitro

Metazoan replication-dependent histone mRNAs end in a conserved stem-loop rather than in the poly(A) tail found on all other mRNAs. The 3' end of histone mRNA binds a single class of proteins, the stem-loop binding proteins (SLBP). In Xenopus, there are two SLBPs: xSLBP1, the homologue of the mammalian SLBP, which is required for processing of histone pre-mRNA, and xSLBP2, which is expressed only during oogenesis and is bound to the stored histone mRNA in Xenopus oocytes. The stem-loop is required for efficient translation of histone mRNAs and substitutes for the poly(A) tail, which is required for efficient translation of other eucaryotic mRNAs. When a rabbit reticulocyte lysate is programmed with uncapped luciferase mRNA ending in the histone stem-loop, there is a three- to sixfold increase in translation in the presence of xSLBP1 while xSLBP2 has no effect on translation. Neither SLBP affected the translation of a luciferase mRNA ending in a mutant stem-loop that does not bind SLBP. Capped luciferase mRNAs ending in the stem-loop were injected into Xenopus oocytes after overexpression of either xSLBP1 or xSLBP2. Overexpression of xSLBP1 in the oocytes stimulated translation, while overexpression of xSLBP2 reduced translation of the luciferase mRNA ending in the histone stem-loop. A small region in the N-terminal portion of xSLBP1 is required to stimulate translation both in vivo and in vitro. An MS2-human SLBP1 fusion protein can activate translation of a reporter mRNA ending in an MS2 binding site, indicating that xSLBP1 only needs to be recruited to the 3' end of the mRNA but does not need to be directly bound to the histone stem-loop to activate translation.     

Comparative inhibition of rabbit globin mRNA translation by modified antisense oligodeoxynucleotides.

We have studied the translation of rabbit globin mRNA in cell free systems (reticulocyte lysate and wheat germ extract) and in microinjected Xenopus oocytes in the presence of anti-sense oligodeoxynucleotides. Results obtained with the unmodified all-oxygen compounds were compared with those obtained when phosphorothioate or alpha-DNA was used. In the wheat germ system a 17-mer sequence targeted to the coding region of beta-globin mRNA was specifically inhibitory when either the unmodified phosphodiester oligonucleotide or its phosphorothioate analogue were used. In contrast no effect was observed with the alpha-oligomer. These results were ascribed to the fact that phosphorothioate oligomers elicit an RNase-H activity comparable to the all-oxygen congeners, while alpha-DNA/mRNA hybrids were a poor substrate. Microinjected Xenopus oocytes followed a similar pattern. The phosphorothioate oligomer was more efficient to prevent translation than the unmodified 17-mer. Inhibition of beta-globin synthesis was observed in the nanomolar concentration range. This result can be ascribed to the nuclease resistance of phosphorothioates as compared to natural phosphodiester linkages, alpha-oligomers were devoid of any inhibitory effect up to 30 microM. Phosphorothioate oligodeoxyribonucleotides were shown to be non-specific inhibitors of protein translation, at concentrations in the micromolar range, in both cell-free systems and oocytes. Non-specific inhibition of translation was dependent on the length of the phosphorothioate oligomer. These non-specific effects were not observed with the unmodified or the alpha-oligonucleotides.     

Maturation of Xenopus laevis oocyte by progesterone requires poly(A) tail elongation of mRNA.

Meiotic maturation of Xenopus laevis oocytes by progesterone requires translation of stored maternal mRNAs. We investigated the role of poly(A) tail elongation of mRNAs during this process using cordycepin, which inhibits poly(A) tail elongation of mRNAs. When oocytes were treated with the buffer containing 10 mM cordycepin for 12 h, concentration of 3'-dATP in cytosol of oocytes increased to 0.7 mM, while that of ATP remained constant at around 1.2 mM. Incorporation of [32P]AMP into poly(A) mRNA was inhibited almost completely by this treatment. Progesterone-induced germinal vesicle breakdown (GVBD) was also abolished. Dose dependence of inhibition of progesterone-induced GVBD on cordycepin was similar to that of [32P]AMP incorporation into poly(A) mRNA. However, maturation-promoting factor-induced GVBD was unaffected by treatment of oocytes with cordycepin. Furthermore, the inhibition of GVBD by cordycepin was rescued by removal of cordycepin even in the presence of actinomycin D. Therefore, we concluded that poly(A) tail elongation of mRNA is required for induction of meiotic maturation of X. laevis oocytes. In addition, progesterone induced a 2.7-fold activation of [32P]AMP incorporation into the poly(A) tail of mRNA after a lag period of 3 h whereas GVBD was induced after 6-8 h from the progesterone treatment. Syntheses of most of the proteins were unaffected by treatment of oocytes with progesterone or cordycepin. However, syntheses of several proteins were increased or decreased by progesterone and cordycepin treatment.     

The asynchrony of gamma- and beta-chain synthesis during human erythroid cell maturation. III. gamma- and beta-mRNA in immature and mature erythroid clones

The synthesis of the β-crystallin polypeptides has been studied in different regions of the embryonic chicken lens. Seven β-crystallin polypeptides ranging in molecular weight from approximately 19,000 (19K) to 35,000 (35K) daltons were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Each polypeptide was synthesized in a rabbit reticulocyte cell-free system supplemented with RNA from the embryonic lens fiber cells suggesting that each is encoded by a separate mRNA. Analysis of the cell-free translation products of the RNAs from 6-, 15-, and 19-day-old embryonic chicken lens fibers demonstrated that all seven polypeptides are translated at each of the stages and that the proportion of β-crystallin mRNAs increases as the chicken embryo matures. Fingerprints of methionine-containing tryptic peptides indicated that the three predominant β-crystallin polypeptides synthesized in the reticulocyte lysate (20K, 26K, and 35K) have related but distinct primary structures. Surprisingly, both the 35K β-crystallin polypeptide and its mRNA were selectively absent from the cells in the central region of the epithelium. Synthesis of this polypeptide from extracted RNAs was detected in the elongating cells of the equatorial region of the epithelium and from the fiber cells. In contrast to the 35K polypeptide, the six lower-molecular-weight β-crystallin polypeptides were synthesized in a reticulocyte lysate directed by RNAs extracted from all three regions of the lens. These data indicate that lens cell elongation and fiber cell differentiation in the embryonic chicken are accompanied by the appearance of the mRNA for the 35K polypeptide.     

Expression of rat renal sodium/phosphate cotransporter in Xenopus laevis oocytes.

In an attempt to identify the renal Na+/Pi cotransporter, Xenopus laevis oocytes were used to express mRNA isolated from the renal cortex of rat kidney. Na(+)-dependent uptake of Pi in oocytes, injected with mRNA, resulted in an increase of 2-4-fold as compared to oocytes injected with water. Both the new expressed and endogenous Na(+)-dependent Pi uptake activity were inhibited with 2 mM phosphonoformic acid (PFA). Expression of Pi uptake into oocytes was dose-dependent with the amount of mRNA injected. When mRNA was fractionated on a sucrose gradient, a mRNA fraction of 2.5 kilobases expressed the Na+/Pi cotransport activity in oocytes. This fraction resulted in a 6-fold stimulation of Na(+)-dependent Pi transport when compared to oocytes injected with water. The Km and Vmax for Na(+)-dependent Pi uptake were 0.18 mM and 118 pmol/oocyte per 30 min, respectively.     

Quantitative hybridization-arrest of mRNA in Xenopus oocytes using single-stranded complementary DNA or oligonucleotide probes.

The expression of heterologous mRNA in Xenopus oocytes was quantitatively inhibited by coinjection of single-stranded complementary DNA or synthetic complementary oligonucleotides. The lymphokines Interleukin-2 (IL-2) and Interleukin-3 (IL-3) were used as model systems to test the effectiveness of this procedure. Messenger RNA samples were hybridized to single stranded complementary DNA or oligonucleotides, injected into oocytes and the oocyte incubation medium assayed for the presence or absence of specific translation products 48 hours later. When IL-2 mRNA was hybridized to a large excess of long (490 bases) single stranded complementary DNA, the expression of IL-2 was effectively blocked (greater than 98%). Complementary oligonucleotides (18-23 bases) were almost as effective as the polynucleotide in inhibiting IL-2 activity (greater than 95%). Oligonucleotides derived from the 5' end, middle or 3' end of the coding sequence were all effective in arresting IL-2 mRNA translation. Oligonucleotide hybrid-arrest was effective even when no NaCl was present in the hybridization buffer, indicating that the annealing reaction could occur within the oocyte after injection. Definite proof that hybrid-arrest could occur in vivo was shown by the fact that oligonucleotides injected before or after mRNA injection, while not as effective as co-injection, still showed substantial inhibition of specific mRNA translation. The oligonucleotide hybrid-arrest method was equally effective in the case of IL-3, demonstrating its general applicability.