Maturation-specific deadenylation in Xenopus oocytes requires nuclear and cytoplasmic factors.

During the meiotic maturation of Xenopus oocytes, maternal mRNAs that lack a cytoplasmic polyadenylation element are deadenylated and translationally inactivated. In this report, we have characterized the regulation of poly(A) removal during maturation. Deadenylation in vivo is detected only after germinal vesicle breakdown and does not require de novo protein synthesis. Enucleated oocytes do not deadenylate either endogenous or microinjected RNAs upon maturation, indicating that a nuclear component is required for poly(A) removal. Whole cell extracts prepared from both immature and mature oocytes deadenylate exogenous RNA substrates in vitro. Deadenylation activity is not detected in isolated nuclear or cytoplasmic extracts obtained from immature oocytes, but is reconstituted when these fractions are combined in vitro. These results indicate that the factors required for deadenylation activity are present in immature oocytes, but that poly(A) removal is prevented by the sequestration of one or more of these components within the nucleus. Maturation-specific deadenylation of maternal mRNAs occurs upon the release of nuclear factors into the cytoplasm at germinal vesicle breakdown.

Deadenylation of maternal mRNAs during Xenopus oocyte maturation does not require specific cis-sequences: a default mechanism for translational control.

The meiotic maturation of Xenopus oocytes initiates significant changes in the translation of a number of maternal mRNAs that coincide with alterations in their polyadenylation states. A considerable number of maternal mRNAs are deadenylated in mature oocytes, thereby reducing their translational efficiencies. In this report we demonstrate that deadenylation does not require specific cis-acting sequences. Polyadenylated RNAs derived from either ribosomal protein or beta-globin mRNAs, or that contain non-mRNA-derived sequences, are deadenylated in mature oocytes. Translation of a substrate RNA is not required for its deadenylation. G10 mRNA is representative of a class of mRNAs that is translationally activated at maturation and contains the cytoplasmic polyadenylation element (U)6AU. A deletion mutant G10 transcript that lacks the (U)6AU element is not polyadenylated in mature oocytes but is deadenylated instead. Insertion of the (U)6AU element into the 3'-untranslated region of the ribosomal protein L1 mRNA is sufficient to prevent both its deadenylation and polysomal release in mature oocytes. These results indicate that the deadenylation and translational inactivation of maternal mRNAs during Xenopus oocyte maturation occur by a default pathway in which transcripts lacking a cytoplasmic polyadenylation element undergo poly(A) removal.

Nucleotide sequence and 40 S subunit assembly of Xenopus laevis ribosomal protein S22.

We have isolated and determined the nucleotide sequence of a cDNA encoding Xenopus laevis ribosomal protein S22. A synthetic S22 mRNA derived from this cDNA directs the synthesis of an in vitro translation product that is indistinguishable from S22 purified from Xenopus ovarian ribosomes. In vitro translated S22 is assembled into 40 S subunits when microinjected into the cytoplasm of oocytes. Analysis of the derived amino acid sequence indicates that Xenopus S22 is homologous to Escherichia coli ribosomal protein S10.

Developmental expression and 5S rRNA-binding activity of Xenopus laevis ribosomal protein L5.

Ribosomal protein L5 binds specifically to 5S rRNA to form a complex that is a precursor to 60S subunit assembly in vivo. Analyses in yeast cells, mammalian cells, and Xenopus embryos have shown that the accumulation of L5 is not coordinated with the expression of other ribosomal proteins. In this study, the primary structure and developmental expression of Xenopus ribosomal protein L5 were examined to determine the basis for its distinct regulation. These analyses showed that L5 expression could either coincide with 5S rRNA synthesis and ribosome assembly or be controlled independently of these events at different stages of Xenopus development. L5 synthesis during oogenesis was uncoupled from the accumulation of 5S rRNa but coincided with subunit assembly. In early embryos, the inefficient translation of L5 mRNA resulted in the accumulation of a stable L5-5S rRNA complex before ribosome assembly at later stages of development. Additional results demonstrated that L5 protein synthesized in vitro bound specifically to 5S rRNA.

The mRNA encoding elongation factor 1-alpha (EF-1 alpha) is a major transcript at the midblastula transition in Xenopus.

A Xenopus laevis gastrula cDNA library has been screened in order to identify sequences that are expressed early in development. We find that the mRNA encoding translation elongation factor 1-alpha (EF-1 alpha) is synthesized in very large amounts in the early embryo. Transcription of EF-1 alpha mRNA commences at the midblastula transition (MBT), and new EF-1 alpha protein is synthesized very soon after this, as determined by the association of EF-1 alpha mRNA with polysomes. The nucleotide sequence of a full-length EF-1 alpha cDNA clone and the deduced amino acid sequence of Xenopus EF-1 alpha protein are presented.

Selective enhancement of bovine papillomavirus type 1 DNA replication in Xenopus laevis eggs by the E6 gene product.

Genetic analyses of bovine papillomavirus type 1 (BPV-1) DNA in transformed mammalian cells have indicated that the E6 gene product is essential for the establishment and maintenance of a high plasmid copy number. In order to analyze the direct effect of the E6 protein on the replication of a BPV-1-derived plasmid, a cDNA containing the BPV-1 E6 open reading frame was subcloned into an SP6 vector for the in vitro synthesis of the corresponding mRNA. The SP6 E6 mRNA was injected into Xenopus laevis oocytes to determine the subcellular localization of the E6 gene product and to analyze the effect of the protein on BPV-1 DNA replication. SP6 E6 mRNA microinjected into stage VI oocytes was translated into a 15.5-kilodalton protein that was specifically immunoprecipitated by antibodies directed against the E6 gene product. The E6 protein preferentially accumulated in oocyte nuclei, a localization which is consistent with the replicative functions in which it has been implicated. The expression of E6 in replication-competent mature oocytes selectively enhanced the replication of a BPV-derived plasmid, indicating a direct role for this gene product in the control of BPV-1 DNA replication.

Translational inactivation of ribosomal protein mRNAs during Xenopus oocyte maturation.

Ribosomal protein synthesis ceases upon maturation of Xenopus oocytes. We find that this cessation results from the dissociation of ribosomal protein mRNAs from polysomes and is accompanied by the deadenylation of these transcripts. A synthetic mRNA encoding ribosomal protein L1, microinjected into stage VI oocytes, is deadenylated and released from polysomes upon maturation. Our results indicate that sequences located within 387 bp of the 3' terminus of L1 mRNA direct both the deadenylation and polysomal release of this ribosomal protein mRNA. The proper translational regulation of an exogenous ribosomal protein mRNA in microinjected oocytes provides a basis for determining the sequence specificity for the differential utilization of maternal mRNAs during oocyte maturation.

Post-translational control of ribosomal protein L1 accumulation in Xenopus oocytes.

A functional ribosomal protein mRNA, encoding the 60 S subunit protein L1, has been synthesized in vitro using bacteriophage SP6 RNA polymerase. This mRNA directs the synthesis of a product indistinguishable from L1 protein purified from Xenopus ovarian ribosomes. Our results show that L1 synthesis in stage VI oocytes increases in response to microinjection of exogenous SP6-L1 mRNA, but excess L1 protein is not stably accumulated. These results indicate that dosage compensation does not occur at the translational level for this ribosomal protein mRNA and that the abundance of this protein in fully grown oocytes is subject to post-translational regulation.

Expression of ribosomal protein genes during Xenopus development.

The Xenopus ribosomal protein genes provide an excellent system to elucidate the complex regulation encompassing 60 functionally related proteins present in equimolar amounts in ribosomal subunits. Oogenesis and embryogenesis provide unique opportunities to investigate ribosome biosynthesis in situations wherein gene activation of individual components is uncoupled from assembly of the ribosomal subunits. This chapter has focused on the basic parameters that control ribosomal protein gene expression during development. Translational control is clearly a major level for coordinating the regulation of these genes during development, as is posttranslational stability of the ribosomal proteins and RNA splicing of the L1 gene. In addition to these levels of control under active investigation, a number of intriguing problems remain to be addressed in any detail. For example, the mechanisms that balance ribosomal protein production with subunit assembly in oocytes remain to be determined. Resolution of these events must also define the processes by which ribosomal proteins, upon synthesis in the cytoplasm, are first translocated to the nucleus and subsequently to the nucleolus for subunit assembly. Functional approaches in which these genes are assayed for accurate developmental control in microinjected oocytes and fertilized eggs will undoubtedly provide information on the synthesis of this eukaryotic organelle and the signals responsible for altering these processes at different developmental stages.

Stable repression of ribosomal protein L1 synthesis in Xenopus oocytes by microinjection of antisense RNA.

The synthesis of an endogenous ribosomal protein, L1, is selectively and efficiently inhibited by microinjection of antisense L1 RNAs into Xenopus oocytes. Repression of L1 synthesis is achieved within 12 hr and is maintained for 48 hr. RNase-protection assays reveal the formation of RNA X RNA duplexes in vivo between the endogenous L1 mRNA and injected antisense transcripts. Partial-length antisense RNAs, complementary to only the 3'-terminal region of L1 mRNA, repress translation as effectively as a full-length antisense RNA, indicating that complementarity to the 5' region of L1 mRNA is not required for efficient inhibition. The use of antisense RNA to repress synthesis of an endogenous ribosomal protein provides a functional basis for determining mechanisms that integrate ribosomal protein synthesis with ribosome assembly during oogenesis.

Coordinate expression of ribosomal protein genes during Xenopus development.

The expression of ribosomal protein and rRNA genes during Xenopus oogenesis results in the synthesis of sufficient ribosomes to support development of the swimming tadpole. cDNA clones for ribosomal proteins L13, L15, L23, and S22 have been isolated and used as probes to examine ribosomal protein gene transcripts during oogenesis and embryogenesis. Our results show that ribosomal protein mRNAs attain maximal steady-state levels in stage II oocytes concomitant with the onset of vitellogenesis. Approximately 50% of ribosomal protein mRNAs are associated with polysomes throughout oogenesis, resulting in a constant rate of ribosomal protein synthesis in stage III through stage VI oocytes. In contrast, the polysomal to nonpolysomal distribution of bulk poly(A)+ RNA increases during oogenesis, resulting in a five- to eightfold stimulation in the rate of overall protein synthesis. Following fertilization, maternal ribosomal protein mRNAs are degraded. Accumulation of de novo ribosomal protein transcripts is first detectable during gastrulation, but ribosomal protein mRNAs do not enter polysomes until stage 30 tailbud embryos. We find no discernible structural or functional differences between ribosomal protein transcripts in the polysomal and the nonpolysomal fractions for the observed stages of oocytes and embryos. These results are consistent with a model in which control of ribosomal protein synthesis is regulated at the translational level during Xenopus development.

Onset of 5 S RNA gene regulation during Xenopus embryogenesis.

The transcription of 5 S RNA genes during oogenesis results in the storage of sufficient 5 S RNA in ribosomes to support subsequent embryogenesis. Xenopus oocytes of all stages synthesize oocyte-type 5 S RNA. A generalized repression of transcription occurs at meiosis and is maintained throughout early cleavage. The onset of 5 S RNA synthesis is detected at approximately the 4000-cell blastula stage (stage 9), concomitant with de novo synthesis of other species of RNA. At this developmental stage the level of 5 S RNA synthesis is low relative to the synthesis of tRNA and small nuclear RNAs. Analysis of this newly synthesized 5 S RNA reveals it to be a nearly equal mixture of oocyte and somatic 5 S RNA derived from both maternal and paternal genes. Given the 50:1 ratio of oocyte to somatic 5 S RNA genes in X. laevis, these results indicate that the majority of the oocyte 5 S RNA genes are inactivated at this time. This reflects differential transcription of the two families of 5 S RNA genes rather than post-transcriptional stability as demonstrated by the ability of a chromatin template isolated from stage 9 embryos to direct the same ratio of oocyte to somatic 5 S RNA synthesis in vitro as that observed in vivo. By completion of gastrulation, 5 S RNA synthesized in vivo and directed from chromatin in vitro is at least 90% somatic 5 S RNA. These results are consistent with a model in which the decrease in concentration of the 5 S-specific transcription factor relative to the number of 5 S RNA genes during embryogenesis contributes to the inactivation of the oocyte 5 S RNA genes.

Stable transcription complexes of Xenopus 5S RNA genes: a means to maintain the differentiated state.

Cloned 5S RNA genes added to Xenopus oocyte nuclear extract assemble into stable active transcription complexes that persist for many rounds of 5S RNA synthesis. This stability of the complex has been demonstrated by its resistance to dilution and to competitor DNA. A stable complex is formed within minutes and lasts for at least 40 rounds of transcription per template over several hours. Stable, transcriptionally inactive complexes can be formed by incubation of cloned 5S RNA genes in an oocyte nuclear extract depleted of a 5S-specific transcription factor and supplemented with histones. The stable, transcriptionally active and inactive states of 5S RNA gene complexes that can be formed in vitro are analogous to the states of the somatic and oocyte 5S RNA genes as they exist in somatic cell chromatin. Oocyte 5S RNa genes remain repressed in chromatin isolated from somatic cells, but can be activated by washing chromatin with high salt. Maintenance of the differentiated state of cell requires that selected genes remain stably active while others are stably repressed for long periods of time. We propose that stable transcription complexes may play an important role in the maintenance of the differentiated state in eucaryotic cells.

A quantitative assay for Xenopus 5S RNA gene transcription in vitro.

The in vitro transcription of Xenopus 5S RNA genes and of deletion mutants of these genes has been quantitated by assays that measure the efficiency of transcription and the ability to compete with the transcription of a "wild-type" 5S RNA gene. The difference between the competition strength of one repeating unit of X. borealis somatic 5S DNA and its plasmid vector is fifteenfold. tRNA genes and the adenovirus VA RNA genes are weak competitors of 5S RNA synthesis (and vice versa). Deletion of the 5' flanking region reduces the competition strength of somatic but not oocyte 5S DNA. Except for the influence of the flanking sequence, the regions within the 5S RNA gene that determine competition strength are those that have been shown to interact with a specific transcription factor that is required for accurate initiation of 5S RNA transcription. The major oocyte (Xlo) and trace oocyte (Xlt) 5S RNA genes from X. laevis are transcribed as efficiently as somatic 5S DNAs but compete only one fourth as well. This fourfold difference in the competition strength is due to oocyte-specific base changes within the intragenic control region.

Related 5S RNA transcription factors in Xenopus oocytes and somatic cells.

Xenopus oocytes contain an abundant protein that binds specifically to the center of 5S RNA genes and directs their transcription by RNA polymerase III. This protein also binds to 5S RNA. We show here that transcription of cloned 5S RNA genes in extracts derived from Xenopus tissue culture cells is dependent on the intragenic control region and is inhibited by 5S RNA and by antibodies raised against the previously characterized oocyte transcription factor. Somatic cells contain a protein that is similar to the oocyte factor in charge, affinity for heparin-agarose, and antigenicity but has an apparent molecular mass about 2000 daltons greater than that of the oocyte protein. Our experiments strongly suggest that this larger protein is the transcription factor for 5S RNA genes in somatic cells. The 5S RNA may regulate its own synthesis in somatic cells by binding to this protein, which is present at a low concentration. The presence of two different proteins responsible for 5S RNA synthesis in oocytes and in somatic cells cannot by itself explain the developmental control of oocyte and somatic 5S RNA genes, because somatic cell extracts transcribe both types of gene.

Photoreceptor pigment that induces differentiation in the slime mold Physarum polycephalum.

An extract of small molecules (molecular weight less than 500) of the slime mold Physarum polycephalum undergoes a shift in ultraviolet-visible absorption spectrum upon illumination. This illumination also confers on the extract the ability to induce sporulation when injected into a starved, unilluminated slime mold. The spectral shift and appearance of the sporulation-inducing activity both occur regardless of whether the illumination is carried out on an intact slime mold or on the plasmodium-free extract itself. Thin-layer chromatography resolves the slime mold extract into four major visible fractions. One of these has high sporulation-inducing activity after illumination in vitro.