Changes in the polysome content of developing Xenopus laevis embryos

A method for preparing polysomes from all embryonic stages of Xenopus laevis is described. In the oocyte only about 1–2% of the total ribosomes are present in polysomes, the remainder being a developmental reserve. Upon conversion to an egg the polysome content rises by up to 3-fold, and by about a further 2-fold after fertilization. There is only a small further increase during cleavage, but by the tailbud stage, when organogenesis begins, there is a more rapid rise. Most of the ribosomes are incorporated into polysomes by stage 42, shortly before feeding begins.At very early stages, the changes in polysome content seem to mirror the changes in protein synthesis. At later stages the polysome contents reported here provide the only available guide to changes in the rate of protein synthesis. Judged by polysome content, the stage 42 tadpole seems to make protein about 20 times faster than the unfertilized egg, though it contains very few more ribosomes. The relationship between polysome content and the synthesis of various types of RNA is discussed.     

Translational control in early sea urchin embryogenesis: initiation factor eIF4F stimulates protein synthesis in lysates from unfertilized eggs of Strongylocentrotus purpuratus

We have used cell-free translation systems from unfertilized eggs and embryos of the sea urchin Strongylocentrotus purpuratus to analyze the mechanisms limiting protein synthesis in early embryogenesis. Unfertilized egg lysates supplemented with nuclease-treated reticulocyte lysate were stimulated 2-4-fold in incorporation of radioactive amino acid into protein. Thirty-minute zygote lysates supplemented in this way were not stimulated. These results suggested that a component limiting translation in the unfertilized egg lysate was provided by the nuclease-treated lysate and that this component was no longer limiting protein synthesis following fertilization. In view of these results, partially fractionated lysates and individual purified translational components from mammalian cells were tested for stimulation of the unfertilized egg lysate. A 1000000g supernatant devoid of ribosomal subunits also stimulated the unfertilized egg lysate. Thus, the stimulation was not due to the addition of active ribosomal subunits but to soluble elements in the reticulocyte lysate. Of the soluble components tested, only the cap-binding protein complex eIF4F caused a dramatic stimulation of the unfertilized egg lysate (2-3.5-fold). The 30-min zygote lysate was not stimulated by eIF4F or by any of the other components tested, supporting the hypothesis that a block in the translational machinery is removed at fertilization. A rabbit reticulocyte shift assay was used to analyze whether mRNA is limiting in early development. When unfertilized egg lysate was added to the shift assay, there was no shift in radioactivity from 43S to 80S complexes, indicating the unfertilized egg mRNA is not available for translation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of alpha- and beta-tubulin genes during development of sea urchin embryos

Mature unfertilized eggs of the sea urchin Lytechinus pictus contain multiple alpha-tubulin mRNAs, which range in size from 1.75 to 4.8 kb, and two beta-tubulin mRNAs, 1.8 and 2.25 kb. These mRNAs were found at similar levels throughout the early cleavage stages. RNA gel blot hybridizations showed that prominent quantitative and qualitative changes in tubulin mRNAs occurred between the early blastula and hatched blastula stages. The overall amounts of alpha- and beta-tubulin mRNAs increased two- to fivefold between blastula and pluteus. These increases were due mainly to a rise in a 1.75-kb alpha RNA and a new 2.0-kb beta RNA. Other, minor changes also occurred during subsequent development. All size classes of alpha- and beta-tubulin RNAs in early and late embryos contained poly(A)+ translatable sequences. As reported earlier, some of each of the alpha RNAs, but neither of the beta RNAs, are translated in the egg and a small portion of each of the stored alpha and beta RNAs is recruited onto polysomes within 30 min of fertilization. In the work described here, subsequent development up to the morula stage was accompanied by a gradual recruitment of tubulin mRNAs into polysomes. By the early blastula stage, most of the maternal tubulin sequences were associated with polysomes. In contrast to the gradual recruitment of maternal sequences throughout cleavage, the tubulin mRNAs which appeared at the blastula stage showed no delay in entering polysomes. The exact fraction of each mRNA that was translationally active at later stages varied somewhat among the individual mRNAs. From the differential hybridization patterns of egg, embryo, and testis RNAs to various tubulin cDNA and genomic DNA probes, it is concluded that at least one gene producing maternal alpha mRNA is different from a second one which is expressed only in testis. Each of the three embryonic beta RNAs is encoded by a different beta gene; at least two of these different beta genes are also expressed in testis.     

Distribution of messenger ribonucleic acid in polysomes and nonpolysomal particles of sea urchin embryos: translational control of actin synthesis

We have used cell-free translation and two-dimensional gel electrophoresis to examine the complexities of the polysomal and cytoplasmic nonpolysomal [ribonucleo-protein (free RNP)] messenger ribonucleic acid (mRNA) populations of sea urchin eggs and embryos. We show that all species of mRNA detected by this method are represented in both the polysomes and free RNPs; essentially all messages present in polysomes are also in the free RNP fraction. However, the cytoplasmic distribution is clearly nonrandom since some templates are relatively concentrated in the free RNPs and others are predominantly in the polysomes. The polypeptides synthesized under the direction of unfertilized egg mRNA are qualitatively indistinguishable from those made by using embryonic mRNA, indicating that the complexity of the abundant class mRNA remains unchanged from egg through early development. However large changes in the abundancies of specific mRNAs occur, and changes are detected in the polysomal/free RNP distribution of some mRNAs through development. The differences in the realtive abundancies of specific mRNAs between polysomes and free RNPs and the developmental changes that take place indicate significant cytoplasmic selection of mRNA for translation. Three different forms of actin (termed alpha, beta, and gamma) were identified among the translation products. Messages for all three are present in the unfertilized egg and early cleavage embryo, yet the gamma form is preferentially located in the polysomes and the alpha and beta in the free RNPs. The relative concentrations of the three change greatly during development as do their relative distributions into polysomes and free RNPs. Examinations of in vivo labeled proteins largely support the in vitro findings. The results indicate that the synthesis of actin mRNAs increases greatly during development and that the expression of the actin mRNAs is partly controlled at the translation level during early development.     

Sequence-specific adenylations and deadenylations accompany changes in the translation of maternal messenger RNA after fertilization of Spisula oocytes

A dramatic change in the pattern of protein synthesis occurs within ten minutes after fertilization of Spisula oocytes. This change is regulated entirely at the translational level. We have used DNA clones complementary to five translationally regulated messenger RNAs to follow shifts in mRNA utilization at fertilization and to characterize alterations in mRNA structure that accompany switches in translational activity in vivo. Four of the mRNAs studied are translationally inactive in the oocyte. After fertilization two of these mRNAs are completely recruited onto polysomes, and two are partially recruited. All four of these mRNAs have very short poly(A) tracts in the oocyte; after fertilization the poly(A) tails lengthen considerably. In contrast, a fifth mRNA, that encoding alpha-tubulin mRNA, is translated very efficiently in the oocyte and is rapidly lost from polysomes after fertilization. Essentially all alpha-tubulin mRNA in the oocyte is poly(A)+ and a large portion of this mRNA undergoes complete deadenylation after fertilization. These results reveal a striking relationship between changes in adenylation and translational activity in vivo. This correlation is not perfect, however. Evidence for and against a direct role for polyadenylation in regulating these translational changes is discussed. Changes in poly(A) tails are the only alterations in mRNA sizes that we have been able to detect. This indicates that, at least for the mRNAs studied here, translational activation is not due to extensive processing of larger translationally incompetent precursors. We have also isolated several complementary DNA clones to RNAs encoded by the mitochondrial genome. Surprisingly, the poly(A) tracts of at least two of the mitochondrial RNAs also lengthen in response to fertilization.     

Translational regulation of histone synthesis in the sea urchin strongylocentrotus purpuratus

The pattern and schedule of histone synthesis in unfertilized eggs and early embryos of the sea urchin Strongylocentrotus purpuratus were studied using two-dimensional gel electrophoresis. After fertilization there is an abrupt change in the pattern of histone variant synthesis. Although both cleavage-stage (CS) variants. However, after fertilization, both CS and alpha messages are translated. Since alpha histone mRNA isolated from unfertilized eggs can be translated in vitro, the synthesis of alpha histone subtypes appears to be under translational control. Although the synthesis of alpha subtypes is shown here to occur before the second S phase after fertilization, little or no alpha histone is incorporated into chromatin at this time. Thus, early chromatin is composed predominantly of CS variants probably recruited for the most part from the large pool of CS histones stored in the unfertilized egg.     

The mobilization of maternal histone messenger RNA after fertilization of the sea urchin egg

The extent of protein, RNA and DNA synthesis in early cleavage stages of the sea urchin embryo (Parechinus angulosus) was determined. A histone mRNA specific cDNA was used in hybridization experiments to investigate the cytoplasmic localization of maternal histone mRNA in the unfertilized sea urchin egg and first cleavage stage embryo. In the unfertilized egg histone mRNA was localized exclusively in ribonucleoprotein particles with none in ribosomes or polyribosomes. This distribution changed after fertilization, in particular, coupled with the first cleavage telophase there was a significant transfer of histone mRNA from the ribonucleoprotein fraction to the polyribosomes. The results indicate mRNA specific translational control mechanisms.     

Simultaneous synthesis,translation, and storage of mRNA including histone mRNA in sea urchin eggs

The kinetics of accumulation of RNA labeled with uridine and the time course of change in the specific activity of the UTP pool were used to estimate the rate constants for synthesis and decay of RNA synthesized in unfertilized eggs of the sea urchin Lytechinus pictus. The rate of synthesis per haploid genome is similar to that in embryos. Most of the RNA is turning over with a half-life of about 5 hr, and an average of 11 pg of newly synthesized RNA accumulates at steady state. About 3.7% of the RNA in the polysomes of the egg is newly synthesized and this RNA has the heterogeneous size distribution expected for mRNA. Thus most, probably all, of the mRNA translated in the egg is also synthesized in the egg. Little, if any, of the RNA synthesized in the egg enters polysomes following fertilization. Thus the egg synthesizes a population of mRNA which is unstable and translated, but it also contains a more stable, untranslated population of previously synthesized, stored mRNA, which is translated only after fertilization. Since the two populations of mRNA code for the same abundant proteins (Brandhorst, B. P. (1976). Develop. Biol., 52, 310–317), there is a temporal separation in the metabolism and function of coexisting mRNA molecules of identical coding sequence. Among the mRNAs synthesized and translated in the egg are histone mRNAs having the same electrophoretic mobilities and rates of synthesis per genome as those synthesized in rapidly cleaving embryos. Thus the synthesis, entry into the cytoplasm, and translation of histone mRNA are not restricted to the S phase of the cell cycle or the period of cell division.     

Multiple levels of regulation of protein synthesis at fertilization in sea urchin eggs

Fertilization of sea urchin eggs results in a large stimulation of protein synthesis. This increase in protein synthesis is mediated by the mobilization of stored maternal mRNA (mRNPs) into polysomes, but the details of the molecular mechanisms which regulate this process are not well understood. Using a sea urchin egg cell-free translation system, evidence has been obtained which indicates that the capacity to initiate protein synthesis on new mRNAs is limited. Addition of exogenous mRNAs failed to stimulate overall protein synthesis, whereas supplementing the system with a nuclease-treated reticulocyte lysate, an S-100 supernatant fraction, or purified eIF-2 stimulated nearly twofold. In addition, the levels of 43 S preinitiation complexes containing a 40 S ribosomal subunit and methionyl-tRNA were increased at pH 7.4 compared to pH 6.9, or when reticulocyte S-100 was added. However, other experiments showed clearly that mRNA availability may also regulate translation in the sea urchin egg. Sea urchin lysates only stimulated poorly the nuclease-treated reticulocyte lysate system, and the mRNPs in the sea urchin lysate did not bind to reticulocyte 43 S preinitiation complexes. Since purified sea urchin egg mRNA was active in both assays, the bulk of sea urchin mRNA must be masked in the egg, and remain masked in the in vitro assays. Thus, protein synthesis appears to be regulated at both the level of mRNA availability and the activity of components of the translational machinery.     

Regulation of mRNA entry into polysomes. Parameters affecting polysome size and the fraction of mRNA in polysomes

The kinetics of labeled histone mRNA entry into polysomes was studied in nuclease-treated reticulocyte lysates. Added mRNA rapidly bound 1 or 2 ribosomes. However, the formation of full size polysomes required at least 16 min. The amount of mRNA bound to ribosomes reached a maximum (73%) within 2 min after mRNA addition and then declined slowly for the remainder of the experiment. Two initiation inhibitors, aurintricarboxylic acid and 7-methylguanosine 5'-triphosphate, were found to affect polysome size and the fraction of mRNA in polysomes in an opposite manner. These results suggest that initiation and reinitiation events may be intrinsically different. The relatively long time period required for the formation of large polysomes can be explained by large polysomes having higher initiation and/or reinitiation rates or slower elongation rates. These possibilities are not mutually exclusive. The results suggest that there exist several levels of control which can regulate polysome size and the fraction of mRNA in polysomes.