Some somatic sequences are absent or exceedingly rare in Xenopus oocyte RNA

A variety of Xenopus laevis cDNA clones derived from somatic cell RNAs were hybridized to oocyte pA⁺ RNA separated on Northern gels. We were unable to detect oocyte pA⁺ sequences complementary to three undefined tadpole cDNA clones. With one of these clones, a complex pattern of bands appears during embryogenesis. With the other two clones, a single band appears. Two additional tadpole clones hybridize to both oocyte and tadpole RNA, but yield a more complex RNA pattern from embryos than from oocytes. One of these additional tadpole clones has complementarity to actin DNA, suggesting that the additional RNA band which appears during embryogenesis is α-actin mRNA (E. A. Sturgess, J. E. M. Ballantine, H. R. Woodland, P. R. Mohun, C. D. Lane, and G. J. Dimitriadis, 1980, J. Embryol. Exp. Morphol.58, 303–320). We have also failed to detect hybridization to oocyte pA⁺ RNA with one vitellogenin and three adult globin cDNA clones. Reconstruction experiments with purified globin mRNA from anemic adult blood cells set the lower level of sensitivity for globin mRNA at one part in 10⁶. The data suggest that some Xenopus mRNA sequences are absent or very rare in the oocyte pA⁺ RNA population.     

Differential inhibition with partially purified and endogenous rabbit reticulocyte globin mRNA by 7-methylguanosine 5'-monophosphate.

The effect of 7-methylguanosine 5′-monophosphate (m⁷G^5′ p) on translation of partially purified globin mRNA and of polysome-associated endogenous globin mRNA has been studied. Under identical experimental conditions, with 0.4 mM m⁷G^5′ p, translation with partially purified globin mRNA is inhibited 50%; translation with endogenous globin mRNA is inhibited 10%. The inhibition of protein synthesis by m⁷G^5′ p occurs at a step before the first peptide bond formation as evidenced by studies with pactamycin; 0.4 mM m⁷G^5′ p inhibited the first dipeptide synthesis 43% when the partially purified globin mRNA was used whereas 15% inhibition was observed with the endogenous mRNA. The inhibition of m⁷G^5′ p appears to be related to the structural integrity of globin mRNA.     

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).     

Effects of estradiol-17β in the male xenopus laevis: Isolation and translation of cytoplasmic messenger rna populations

Summary Total Xenopus liver cytoplasmic RNA isolated following long-term estrogen administration (14 days) was fractioned using Sepharose 4B chromatography. One of the Sepharose 4B peaks was shown to contain RNA with a molecular weight reported for vitellogenin mRNA (34S). The presence of estrogeninduced vitellogenin mRNA in the peak 5 RNA was determined by translation of the RNA in the oocyte and analysis of the oocyte translational products by immunoprecipitation with anti-vitellogenin.Sepharose 4B peaks 2 and 3 were also observed to contain estrogen induced mRNA populations sedimenting between 9-18S. These findings suggest that Sepharose 4B chromatography might prove useful in separating different mRNA populations following estrogen-induced gene activation.     

Isolation of globin messenger RNA of Xenopus laevis

The polypeptide chains of Xenopus laevis hemoglobin have been analyzed by sodium dodecyl sulfate (SDS) and acid-urea gel electrophoresis. Four components can be distinguished, each having an approximate molecular weight of 13,000 daltons. Messenger RNA coding for the globin chains has been isolated and characterized. In a denaturing acrylamide gel the mRNA has an approximate molecular weight of 250,000 daltons. The complexity of the RNA is consistent with the presence of four different mRNA molecules, each of this molecular weight. When the mRNA is assayed in a wheat germ in vitro translation system, four polypeptides are synthesized corresponding to the four globin subunits. The relative proportion of the four synthesized polypeptides appears to vary according to the developmental stage of the red blood cells used for mRNA isolation. Hybridization of a complementary DNA (cDNA) copy of the globin mRNA to Xenopus laevis DNA in DNA excess indicates that each of the globin genes is present in one to three copies per haploid genome.     

Structural messenger RNA contains cytokeratin polymerization and depolymerization signals

We have previously shown that VegT mRNA plays a structural (translation-independent) role in the organization of the cytokeratin cytoskeleton in Xenopus oocytes. The depletion of VegT mRNA causes the fragmentation of the cytokeratin network in the vegetal cortex of Xenopus oocytes. This effect can be rescued by the injection of synthetic VegT RNA into the oocyte. Here, we show that the structural function of VegT mRNA in Xenopus oocyte depends on its combinatory signals for the induction or facilitation and for the maintenance of the depolymerization vs. polymerization status of cytokeratin filaments and that the 300-nucleotide fragment of VegT RNA isolated from the context of the entire molecule induces and maintains the depolymerization of cytokeratin filaments when injected into Xenopus oocytes. A computational analysis of three homologous Xenopus VegT mRNAs has revealed the presence, within this 300-nucleotide region, of a conserved base-pairing (hairpin) configuration that might function in RNA/protein interactions.     

Protein synthesis in oocytes of Xenopus laevis is not regulated by the supply of messenger RNA

The control of protein synthesis in oocytes of Xenopus laevis has been investigated by injecting oocytes with mRNA and polysomes followed by labeling with ¹⁴C-amino acid mixtures. Contrary to previous reports in which injected oocytes were labeled with ³H-histidine, injected globin mRNA is found to decrease amino acid incorporation into endogenous proteins competitively at all concentrations tested. No increase in overall amino acid incorporation is detected when more mRNA is supplied. Similar results are obtained after labeling injected oocytes with leucine, methionine, proline or valine individually. An explanation is presented for the conflicting results obtained when histidine is used as a label.When reticulocyte polysomes are injected, rather than purified globin mRNA, incorporation of amino acids into endogenous proteins remains roughly constant and overall incorporation increases. Similarly, when encephalomyocarditis viral RNA is injected together with either globin mRNA or reticulocyte polysomes, the globin mRNA causes decreased amino acid incorporation into encephalomyocarditis proteins, but the polysomes do not do so. The results demonstrate that different types of mRNA compete for a strictly limited translational capacity which is saturated in the normal oocyte. The limiting component is present in polysomes and is not message-specific. The constraint on protein synthesis in the amphibian oocyte cannot be fully explained by masked mRNA.     

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.     

Developmental timing of mRNA translation—integration of distinct regulatory elements

Targeted mRNA translation is emerging as a critical mechanism to control gene expression during developmental processes. Exciting new findings have revealed a critical role for regulatory elements within the mRNA untranslated regions to direct the timing of mRNA translation. Regulatory elements can be targeted by sequence‐specific binding proteins to direct either repression or activation of mRNA translation in response to developmental signals. As new regulatory elements continue to be identified it has become clear that targeted mRNAs can contain multiple regulatory elements, directing apparently contradictory translational patterns. How is this complex regulatory input integrated? In this review, we focus on a new challenge area—how sequence‐specific RNA binding proteins respond to developmental signals and functionally integrate to regulate the extent and timing of target mRNA translation. We discuss current understanding with a particular emphasis on the control of cell cycle progression that is mediated through a complex interplay of distinct mRNA regulatory elements during Xenopus oocyte maturation. Mol. Reprod. Dev. 77: 662–669, 2010. © 2010 Wiley‐Liss, Inc.     

Messenger RNA Nuclear Particles and their Attachment to Cytoplasmic Membranes in Krebs Tumour Cells

THE observation that some nuclear particles contain DNA-like RNA (D-RNA) supports the idea that messenger RNA (mRNA) species bind to specific proteins to form ribonucleoprotein complexes^1–4. The mechanism by which the nuclear particles are transported and eventually bind to ribosomes in a translation complex is still uncertain^5–9. We now present evidence that D-RNA nuclear particles of about 40–50S contain mRNA species which are transported into the cytoplasm and assemble on the network of the endoplasmic reticulum (ER). Ribosomes then bind to this membrane-bound mRNA to form a translation complex. The concept of membrane-bound mRNA has been suggested before¹⁰ but has not been conclusively demonstrated.     

Role of calcium in the localization of maternal poly(A)+RNA and tubulin mRNA in Xenopus oocytes

Summary Poly(A)⁺RNA and tubulin mRNA are localized in the periphery of Xenopus oocytes and become delocalized during meiotic maturation. Delocalization of this RNA can be triggered by incubation in agents which reduce entry of calcium ions into the cell (e.g. lanthanum chloride and verapamil). Although these agents ordinarily promote meiotic maturation, addition of theophylline to the medium will inhibit maturation but not delocalization. Manipulations which prevent calcium entry without inducing meiotic maturation (e.g. calcium-free buffer) are also shown to trigger disruption of the RNA localization. In addition, manipulations which reduce chloride efflux from the cell (e.g. increasing the external chloride ion concentration with choline chloride) result in disruption of the localization of poly (A)⁺ RNA and tubulin mRNA without inducing meiotic maturation. The calcium-dependent chloride efflux present in Xenopus oocytes disappears after the oocyte has been stimulated to proceed through meiotic maturation. We show that reduction of the influx of calcium ions or efflux of chloride ions induces the delocalization of poly (A)⁺RNA and tubulin mRNA without inducing meiotic maturation. We suggest, therefore, that reducing the transmembrane movement of these ions is likely to be the natural trigger for the delocalization of poly(A)⁺RNA and tubulin mRNA.     

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.     

Partial sequence analysis of Xenopus alpha- and beta-globin mRNA as determined from recombinant DNA plasmids

Recombinant plasmids containing Xenopus globin mRNA sequences have been constructed using the mRNA:cDNA hybrid conditions of Zain et al. (1979, Cell16, 851–861). The partial nucleotide sequence of two of these recombinants has been determined. They have been identified as containing α- and β-globin-like sequences by homology to other amphibian globin proteins. The nucleotide sequence of these recombinants permits the comparison of conserved regions in both the coding and 3′ nontranslated regions of Xenopus globin mRNAs with the known sequences of other eukaryotic globin proteins and mRNAs. Among the features which have been conserved though evolution is the sequence AAUAAA close to the 3′ terminus of the nontranslated region. Extensive regions of homology occur between the 3′ nontranslated regions of Xenopus α- and β-globin mRNA.     

mRNA translation during oocyte maturation plays a key role in development of primordial germ cells in<Emphasis Type="Italic">Xenopus</Emphasis> embryos

It is believed that cytoplasmic localization in the egg is necessary for development of primordial germ cells (PGCs) inXenopus embryos. In this study, we sought to determine if translation of maternal mRNA during oocyte maturation is involved in the development of PGCs. Donor oocytes were collected from both stimulated (those who receive gonadotropin) and unstimulated females, artificially matured and fertilized using a host transfer technique. Using chloramphenicol (50 μM and 500 μM RNA), RNA translation was inhibited during oocyte maturation. Our results showed that in unstimulated embryos treated with 50 μM chloramphenicol, there was a significant reduction in the number of PGCs reaching genital ridges. In stimulated embryos, however, the number of PGCs was unchanged unless a higher concentration (500 (μM) of chloramphenicol was used. From these results it is suggested that maternal mRNA translation during oocyte maturation plays a key role in development of PGCs.