Developmentally regulated RNA binding proteins during oogenesis in Xenopus laevis

During oogenesis, Xenopus oocytes synthesize and accumulate all types of RNA. In particular, they store poly(A+) RNA to such an extent that only about 5% is actually translated in the oocyte. Using a protein blotting and in vitro binding assay, we have identified proteins which are associated with poly(A+) RNA and perhaps other RNAs as well. Two groups of binding proteins were identified. The first group accumulates during oogenesis, generally is less than 50,000 molecular weight, and sediments in the 80 S and polysome regions of a gradient. These proteins most likely include ribosomal proteins. A second group of proteins is oocyte-specific, sediments less than 80 S as well 80 S and slightly heavier, generally has molecular weights greater than 50,000, and diminishes in amount as oogenesis progresses. In addition, these proteins are retained by oligo(dT)-cellulose when ribonucleoproteins are analyzed by chromatography and, when challenged with several different types of RNA in vitro, bind poly(A+) RNA preferentially. The possibility that some of these proteins might regulate the stability or translatability of mRNAs during oogenesis is discussed.     

Poly(A)+ RNA metabolism during oogenesis in Xenopus laevis.

In this study, we have measured the synthesis and turnover of oligo(dT)cellulose-bound RNA [poly(A)⁺ RNA] in Xenopus laevis oocytes at the maximal lampbrush chromosome stage (stage 3) and at the completion of oocyte growth (stage 6). Oocytes at both stages are shown to be active in the synthesis of poly(A)⁺ RNA. In stage 6 oocytes, the mean rate of synthesis of stable poly(A)⁺ RNA is 15% the instantaneous rate of synthesis, while the mean half-life of the unstable component is 1.6 hr. In contrast, the instantaneous rate of synthesis in stage 3 oocytes is about one-third that seen in stage 6, and most of it is devoted to the production of unstable species with an average half-life of 5 hr. Studies on the nuclear versus the cytoplasmic distribution of the newly synthesized poly(A)⁺ RNA demonstrated that by the end of a 12-hr labeling period for stage 3 oocytes and a 24-hr labeling period for stage 6 oocytes, approximately half of the material was cytoplasmic. This cytoplasmic material had the same electrophoretic mobility as bulk poly(A)⁺ RNA. Similarly, as with bulk poly(A)⁺ RNA, little, if any, of the newly synthesized material was found to be polysomal. Also, poly(A) labeling studies indicated that the newly synthesized poly(A)⁺ RNA was associated with the synthesis of poly(A) of the same length as that appearing on bulk poly(A)⁺ RNA. Studies on the content of bulk oligo(dT)cellulose-bound RNA indicated that about 86 ng is present in both stage 3 and stage 6 oocytes. The continual synthesis of poly(A)⁺ RNA throughout oogenesis in the absence of its accumulation led to the conclusion that it must be turning over. These data are discussed in relation to the hypothesis that bulk levels of poly(A)⁺ RNA are maintained by continually changing rates of synthesis and degradation.     

Xenopus borealis and Xenopus laevis 28S ribosomal DNA and the complete 40S ribosomal precursor RNA coding units of both species.

We have determined the nucleotide sequence of Xenopus borealis 28S ribosomal DNA (rDNA) and have revised the sequence of Xenopus laevis 28S rDNA (Ware et al., Nucl. Acids Res. 11, 7795-7817 (1983)). In the regions encoding the conserved structural core of 28S rRNA (2490 nucleotides) there are only four differences between the two species, each difference being a base substitution. In the variable regions, also called eukaryotic expansion segments (ca. 1630 nucleotides) there are some 61 differences, due to substitutions, mini-insertions and mini-deletions. Thus, evolutionary divergence in the variable regions has been at least 20-fold more rapid than in the conserved core. A search for intraspecies sequence variation has revealed minimal heterogeneity in X. laevis and none in X. borealis. At three out of four sites where heterogeneity was found in X. laevis (all in variable regions) the minority variant corresponded to the standard form in X. borealis. Intraspecies heterogeneity and interspecies divergence in the 28S variable regions are much less extensive than in the transcribed spacers. The 28S sequences are from the same clones that were used previously for sequencing the 18S genes and transcribed spacers. The complete sequences of the 40S precursor regions of the two reference clones are given.     

Accumulation of mitochondrial DNA during oogenesis in Xenopus laevis

The mitochondrial DNA (mtDNA) content of Xenopus laevis oocytes at various stages of oogenesis has been determined by molecular hybridization with ³H-labeled complementary RNA (cRNA). The previtellogenic oocyte less than 250 μm in diameter (stage 1) contains 0.95 ± 0.47 ng of mtDNA. Accumulation of mtDNA proceeds until stage 4 (500–750 μm diameter oocyte), by which time a steady-state level of 4.28 ± 0.40 ng/oocyte is attained. Using the hybridization assay, the stage 6 (full-grown) Xenopus oocyte contains 4.51 ± 0.69 ng of mtDNA, compared to the previously reported value of 3.8 ng determined by direct measurement on the unfertilized egg. There appears to be a reasonable correlation, therefore, between the termination of mtDNA accumulation and the dispersal of the juxtanuclear, mitochondrial aggregate (Balbiani body) at the onset of vitellogenesis in Xenopus. It is concluded that the enormous complement of oocyte mitochondria is accumulated well before the end of oocyte growth and is maintained at a constant level during the remainder of oogenesis, through maturation, fertilization, and on into early development.     

Regulation of protein synthesis and accumulation during oogenesis in Xenopus laevis

The tissue and developmental distribution of the various myosin subunits has been examined in bovine cardiac muscle. Electrophoretic analysis shows that a myosin light chain found in fetal but not in adult ventricular myosin is very similar and possibly identical to the light chain found in fetal or adult atrial and adult Purkinje fiber myosins. This light chain comigrates on two-dimensional gels with the bovine skeletal muscle embryonic light chain. Thus, this protein appears to be expressed only at early developmental stages in some tissues (cardiac ventricles, skeletal muscle) but at all stages in others (cardiac atria). The heavy chains of these myosins have been examined by one- and two-dimensional polypeptide mapping. The ventricular and Purkinje fiber heavy chains are indistinguishable. They are, however, different from the heavy chain found in cultured skeletal muscle myotubes, in contrast to the situation concerning the embryonic/atrial light chain.     

Quantitative changes in protein synthesis during oogenesis in Xenopus laevis

Protein synthesis rates in Xenopus laevis oocytes from stage 1 through stage 6 were measured. In addition, the translational efficiencies, total RNA contents, and percentages of ribosomes in polysomes in growing oocytes at several stages were determined. Stage 1 oocytes synthesize protein at a mean rate of 0.18 ng hr⁻¹ while stage 6 oocytes make protein at a rate of 22.8 ng hr⁻¹. Polysomes from growing and full-grown oocytes sedimented in a sucrose gradient with a peak value of 300 S, corresponding to a weight-average packing density of 10 ribosomes per mRNA. Ribosome transit times of endogenous mRNAs were essentially unchanged at all stages examined. While the oocyte's total ribosomal RNA content was observed to increase about 115-fold during oogenesis, the percentage of ribosomes in polysomes remained constant at approximately 2%. Taken together, the data suggest that the 127-fold increase in protein synthesis which occurs during Xenopus oogenesis involves the progressive recruitment onto polysomes of mRNA from the maternal stockpile.     

The nucleotide sequences of 5.8-S ribosomal RNA from Xenopus laevis and Xenopus borealis

1. The nucleotide sequence of 5.8-S rRNA from Xenopus laevis is given; it differs by a C in equilibrium U transition at position 140 from the 5.8-S rRNA of Xenopus borealis. 2. The sequence contains two completely modified and two partially modified residues. 3. Three different 5' nucleotides are found: pU-C-G (0.4) pC-G (0.2) and pG (0.4). 4. The 3' terminus is C not U as in all other 5.8-S sequences so far determined. 5. The X. laevis sequence differs from the mammalian and turtle sequences by five and six residue changes respectively. 6. A ribonuclease-resistant hairpin loop is a principle feature of secondary structure models proposed for this molecule. 7. Sequence heterogeneity may occur at one position at a very low level (approximately 0.01) in X. laevis 5.8-S rRNA, while none was detected in X. borealis or HeLa cell 5.8-S rRNA.     

Calmodulin synthesis and accumulation during oogenesis and maturation of Xenopus laevis oocytes

The calmodulin levels in stage 6 Xenopus oocytes averaged 89 +/- 24 (SD) ng/oocyte and had largely accumulated by stage 3 of oogenesis. From stage 3 to early stage 6, calmodulin levels did not increase further. However, in large stage 6 oocytes (greater than 1.25 mm diam) calmodulin levels again rose to a level as high as 121 ng/oocyte. Calmodulin levels did not change during the maturation of stage 6 oocytes and the results of measurements on animal and vegetal oocyte halves from control and mature oocytes showed no evidence of a redistribution of calmodulin during maturation. Measurements of calmodulin synthesis in stages 1 and 2 oocytes, stage 4 oocytes, and stage 6 oocytes indicated that calmodulin was being synthesized continuously during oogenesis and that the rate of synthesis increased during oogenesis. In stage 1 and 2 oocytes (combined), the synthesis rate was 3.5 pg/hr/oocyte; in stage 4 oocytes it was 48 pg/hr/oocyte, and in large stage 6 oocytes the rate had increased to 160 pg/hr/oocyte. These changes in the rates of synthesis were discussed as they relate to the pattern of calmodulin accumulation during oogenesis.