Expression of maternal and paternal histone genes during early cleavage stages of the echinoderm hybrid Strongylocentrotus purpuratus × Lytechinus pictus

Interspecific hybrids of the sea urchins Strongylocentrotus purpuratus (♀) and Lytechinus pictus (♂) were used to estimate the contributions of the maternal and paternal genomes to histone mRNA synthesis during early development. Radiolabeled histone mRNAs from the two sea urchin species were identified by hybridization to cloned histone genes from both S. purpuratus and L. pictus and shown to be electrophoretically distinguishable. The synthesis of maternal and paternal histone mRNA in these hybrid embryos is evident as early as the two-cell stage. By at least the 16-cell stage, both maternal and paternal histone mRNAs are associated with polysomes. The relative amounts of the maternal and paternal histone mRNAs synthesized by the zygote appear to be similar.     

Sea urchin small RNA ribonucleoprotein particles: identification, synthesis, and subcellular localization during early embryonic development.

Small RNAs in sea urchins were examined in order to characterize developmental changes in their level, subcellular localization, synthesis, and association with proteins and other RNAs. Small RNAs such as the U snRNAs, 5S and 5.8S rRNAs, and 7S RNAs were identified by their mobility on highly cross-linked acrylamide gels. In addition, 7SL and U1 RNAs were identified by northern blot hybridization to cloned human and sea urchin probes, respectively. The level, subcellular localization, and association with proteins or RNA do not change for most small RNAs from fertilization to blastula, even though this is the time when the stored maternal pool of many small RNAs is being supplemented and replaced by embryonically synthesized RNAs. New embryonic synthesis of small RNAs was first detected at the 8-12 hr blastula stage. Although the predicted subsets of the total small RNA pool can be found in the appropriate subcellular compartments, newly synthesized small RNAs have a predominantly cytoplasmic localization: All of the newly synthesized small RNAs were found to be constituents of small RNPs. The RNPs containing newly synthesized small RNAs had sedimentation rates indistinguishable from their maternal counterparts. Thus, on the basis of sedimentation rate, no gross differences could be detected between maternal and embryonic small RNP pools. These small RNPs include a cytoplasmic RNP containing newly synthesized U1 snRNA and the sea urchin signal recognition particle (SRP) containing the 7SL, RNA. We have also identified a small RNP bearing the 5S rRNA which is present in both eggs and embryos. The presence of multiple, abundant, small RNAs and RNPs that are maintained at constant levels in particular subcellular fractions throughout development suggests that small RNAs may be involved in many more cellular activities than have so far been described.     

Synthesis of histone mRNA sequences in isolated nuclei of cleavage stage sea urchin embryos

A qualitative assay for detection of histone mRNA sequences in nuclear RNA was developed using actinomycin D-CsCl gradients to separate histone DNA from bulk DNA by differences in buoyant density. A significant amount of RNA synthesized in vitro in isolated nuclei from early blastula stage sea urchin embryos hybridized coincident with the histone DNA satellite, and this hybridization was competed out by unlabeled “9S” polysomal RNA purified from embryos at the same stage of development. The biogenesis of these histone mRNA sequences appeared similar as observed during in vivo and in vitro synthesis. Nuclear RNA from embryos pulse labeled in vivo was found to lack histone sequences, suggesting a rapid exit time for these sequences from the nucleus. Attempts to study the exit of histone sequences from isolated nuclei labeled in vitro also suggested a rapid exit time for histone sequences. The histone sequences were synthesized to a much lesser extent in isolated nuclei from late blastula stage embryos, as anticipated from the much reduced amount of histone mRNA labeled on polysomes at this stage.     

Cytogenetic analysis of oocytes and early preimplantation embryos from XO mice.

The cytoplasm of early sea urchin embryos contains nonribosomal, high molecular weight RNA both associated with ribosomes in polysomes and free of ribosomes in particles termed free RNP. In a 1-hr labeling period, 50% of the newly synthesized RNA enters the pool of ribosome-free RNP particles during the cleavage stages, and this percentage decreases until less than 20% of the new RNA in the mesenchyme blastula stage is found in the free RNP. mRNA from both polysomes and free RNP contain poly(A)(+) and poly(A)(?) species. During the cleavage stages only 8–10% of the RNA from each fraction is polyadenylated; however, in the blastula, 40–50% of the nonhistone polysomal RNA is polyadenylated while only 22–30% of the free RNP RNA is polyadenylated. At any developmental stage, the poly(A)(+)RNA from the free RNA and polysomes have identical sedimentation profiles; this is also the case for the poly(A)(?)RNA except for the absence of the 9 S histone mRNA from the free RNP. Changes in poly(A)(+)RNA content and sedimentation profiles during development occur simultaneously in the free RNP and the polysomes. Kinetic studies of these two RNP populations as well as nuclear RNP show that the bulk of the free RNP are not unusually stable cytoplasmic components. The free RNP decay with a half-life of about 40 min while nuclear RNA and polysomal RNA display half-lives of about 12 and 65 min, respectively. Further, the rate of synthesis of the free RNP is not consistent with their being the only precursors for polysomes. Our estimates of the rates of synthesis for nuclear RNA, polysomes, and free RNP are, respectively, 1.1 × 10^?15, 2.2 × 10^?16, and 5.0 × 15^?17 g/min/nucleus. The data on free RNP is discussed in terms of translational regulation of protein synthesis in the developing sea urchin.     

Messenger ribonucleoprotein particles in developing sea urchin embryos

Messenger RNA entering polysomes during early development of the sea urchin embryo consists of both oogenetic and newly transcribed sequences. Newly transcribed mRNA enters polysomes rapidly while oogenetic mRNA enters polysomes from a pool of stable, nontranslatable messenger ribonucleoprotein particles (mRNPs) derived from the unfertilized egg. Protein content may relate to differences in the regulation of newly transcribed and oogenetic mRNAs. Oogenetic poly(A)⁺ mRNA was found to be present in both polysomal and subpolysomal fractions of cleavage stage and early blastula stage embryos. This mRNA was found to be present in subpolysomal mRNPs with a density of 1.45 g/cm³ in Cs₂SO₄. Poly(A)⁺ mRNPs released from polysomes of embryos cultured in the presence of actinomycin D sedimented in a broad peak centered at 55 S and contained RNA of 21 S. The density of these particles was sensitive to the method of release; puromycin-released mRNPs had a density of 1.45 g/cm³, while EDTA caused a shift in density to 1.55 g/cm³, indicating a partial loss of protein. The results with newly synthesized mRNAs contrast sharply. Newly transcribed mRNA in subpolysomal mRNPs had a density of 1.55–1.66 g/cm³, a density approaching that of deproteinized RNA. Messenger RNA released from polysomes either by EDTA or puromycin was examined to determine the possible existence of polysomal mRNPs. When [³H]uridine-labeled mRNA was released from late cleavage stage embryo polysomes by either technique, and centrifuged on sucrose gradients, two broad peaks were found. One peak centered at 30 S contained 21 S mRNA while the other at 15 S contained 9 S histone mRNA. When these fractions were fixed with formaldehyde, they banded on Cs₂SO₄ gradients at a density of 1.60–1.66 g/cm³, very similar to that of pure RNA. We conclude that the newly transcribed mRNA may be present in stable mRNPs containing up to 10% protein in either subpolysomal or polysomal fractions. These mRNPs are clearly distinguishable from the protein-rich mRNPs containing oogenetic mRNAs.     

Loss of Xist imprinting in diploid parthenogenetic preimplantation embryos.

We have analysed Xist expression patterns in parthenogenetic and control fertilised preimplantation embryos by using RNA FISH. In normal XX embryos, maternally derived Xist alleles are repressed throughout preimplantation development. Paternal alleles are expressed as early as the 2-cell stage. In parthenogenetic embryos, we observed Xist RNA expression and accumulation from the morula stage onwards, indicating loss of maternal imprinting. In the majority of cells, expression was from a single allele, indicating that X chromosome counting occurs to establish appropriate monoallelic Xist expression. We discuss these data in the context of models for regulation of imprinted and random X inactivation.