Histone messenger RNA synthesis and accumulation during early development in the echiuroid worm, Urechis caupo

RNA isolated from Urechis caupo mature oocytes and embryos was analyzed for the presence of histone messenger RNAs (mRNAs) by in vitro translation and by filter blot hybridization to determine the contribution of maternal and newly transcribed histone mRNAs to the pattern of histone synthesis during early development. Histone mRNAs were not detected in mature oocyte RNA which suggests that relatively few if any maternal histone mRNAs are sequestered in the mature oocytes. Histone mRNAs were detected in cleavage-stage RNA and increased in amount from midcleavage through late gastrula stages. The in vitro translation analysis also demonstrated that the amount of H1 histone mRNA in late cleavage- and early blastula-stage embryos exceeds that of the individual core histone mRNAs. The disproportionate accumulation of individual histone mRNAs correlates with the noncoordinate synthesis of H1 and core histones which occurs during early embryogenesis.     

Differential expression of histone sequences in Drosophila following heat shock

The expression of the sequences encoding the four nucleosomal histone proteins was examined following heat shock of a variety of Drosophila cells and was found to be highly differential. In Drosophila melanogaster KC-O cells grown in suspension culture, there is a continuation of the synthesis of all four of the nucleosomal histone proteins following heat shock. Analysis of RNA from these cells confirms that histone messengers are transcribed and located on polysomes. This exact same pattern of histone protein synthesis occurs in KC-O cells grown to low density on plates. In contrast, KC-O cells grown to high density on plates exhibit a dramatic elevation of H2b protein synthesis relative to the synthesis of the other core histones. Organs from D melanogaster third instar larvae were examined to ascertain whether histone protein synthesis continues following heat shock in the organism. Different tissue types exhibited differential histone synthesis. Imaginal disks excised from heat-shocked larvae continue to synthesize nucleosomal histones in a variable fashion. In contrast, neither fat bodies, brains, nor salivary glands continues to synthesize core histone proteins at a significant level. D hydei plated cell cultures and larval tissues fail to synthesize histones at any detectable level following a heat shock. Based on these observations, we propose that there is a differential synthesis of nucleosomal proteins in Drosophila that is highly dependent on the state of the cells prior to the heat shock.     

HSP70 mRNA translation in chicken reticulocytes is regulated at the level of elongation

During heat shock of chicken reticulocytes the synthesis of a single heat shock protein, HSP70, increases greater than 10-fold, while the level of HSP70 mRNA increases less than 2-fold during the same period. Comparison of the in vivo levels of HSP70 and beta-globin synthesis with their mRNA abundance reveals that the translation of HSP70 mRNA is repressed in normal reticulocytes and is activated upon heat shock. In its translationally repressed state HSP70 mRNA is functionally associated with polysomes based on sedimentation analysis of polysomes from untreated or puromycin-treated cells and by analysis of in vitro "run-off" translation products using isolated polysomes. Treatment of control and heat shocked cells with the initiation inhibitor pactamycin reveals that elongation of the HSP70 nascent peptide is not completely arrested, but is slower in control cells. Furthermore, the inefficient translation of HSP70 mRNA in vivo is not due to the lack of an essential translation factor; HSP70 mRNA is efficiently translated in chicken reticulocyte translation extracts as well as in heterologous rabbit reticulocyte extracts. Our results reveal that a major control point for HSP70 synthesis in reticulocytes is the elongation rate of the HSP70 nascent peptide.     

Selective translation of heat shock mRNA in Drosophila melanogaster depends on sequence information in the leader.

One of the effects of a temperature increase above 35 degrees C on Drosophila melanogaster is a rapid switch in selectivity of the translational apparatus. Protein synthesis from normal, but not from heat shock, mRNA is much reduced. Efficient translation at high temperature might be a result of the primary sequence of heat shock genes. Alternatively a mRNA modification mechanism, altered as a consequence of heat shock, might allow for efficient high temperature translation of any mRNA synthesized during a heat shock. The gene for alcohol dehydrogenase (Adh) was fused to the controlling elements of a heat shock protein 70 (hsp70) gene. Authentic Adh mRNA, synthesized from this fusion gene at elevated temperatures was not translated during heat shock. A second Adh fusion gene in which the mRNA synthesized contained the first 95 nucleotides of the Hsp70 non-translated leader sequence gave rise, at high temperature, to mRNA which was translated during the heat shock. Thus, the signal(s) in the mRNAs controlling translation efficiency at heat shock temperatures is encoded within the heat shock genes.     

The heat-shock response in Drosophila KC 161 cells. mRNA competition is the main explanation for reduction of normal protein synthesis

The effect of heat shock on protein synthesis in the Drosophila melanogaster KC 161 tissue culture cell line was examined with a view to investigating the mechanism underlying the acute reduction in normal cellular protein synthesis typical of heat-shocked Drosophila cells. However, at 36-37 degrees C, the optimum temperature for induction of the 70-kDa heat-shock protein, this cell line did not show such a response. The synthesis of a very limited number of proteins was abruptly turned off following heat shock in the presence or absence of actinomycin, but the rate of synthesis of the majority of normal cellular proteins declined slowly over a three-hour period. Incubation of heat-shocked cells in hypertonic media increased the relative proportion of protein synthesis directed towards heat-shock proteins (as opposed to normal cellular proteins). Incubation with low concentrations of cycloheximide had the converse effect and resulted in a preferential increase in the size of polysomes translating normal cellular mRNAs, greater than the increase in size of polysomes synthesising heat-shock proteins. Heat shock also resulted in some mRNAs being almost completely displaced from polysomes into the postribosomal supernatant. These observations suggest that competition between normal cellular mRNAs and increasing amounts of heat-shock mRNAs with a higher affinity for the translation machinery was the main explanation for the gradual reduction in the synthesis of normal cellular proteins, although a slight reduction in overall translation initiation rates cannot be excluded as a subsidiary cause. The results demonstrate that the acute reduction in normal cellular protein synthesis seen in other Drosophila cell lines is not an integral and necessary feature of the heat-shock response in this organism, which makes it unlikely that the mechanism of this acute shut-off is intimately connected with the mechanism of induction of heat-shock mRNAs.     

Stability of histone mRNAs is related to their location in polysomes

Synthesis of histone mRNAs is closely coupled to DNA synthesis. Following inhibition of DNA synthesis in L6 myoblasts with cytosine arabinoside, a coordinate and exaggerated rate of degradation of histone mRNAs occurs while other mRNAs, encoding ribosomal protein L32 and actin, are unaffected. Inhibition of protein synthesis by puromycin, emetine, or cycloheximide stabilizes histone mRNAs and results in their accumulation. When inhibition of DNA synthesis was followed immediately by inhibition of protein synthesis, the exaggerated rate of decay of the existing subspecies of histone H4 mRNAs was prevented and histone mRNA accumulated. If inhibition of protein synthesis was delayed longer than 3 minutes following inhibition of DNA synthesis, the ability to accumulate H4 mRNAs was lost. Furthermore, new protein synthesis was required to activate the mechanism which specifically destabilized histone mRNA. Puromycin was able to prevent the exaggerated rate of degradation of the various subspecies of H4 mRNA when added up to 15 min after inhibition of DNA synthesis, whereas emetine was effective only when added up to 5 min following inhibition of DNA synthesis. These data suggest that histone H4 mRNAs in polysomes are better targets than those released from polysomes for the specific mechanism which destabilizes histone mRNAs upon inhibition of DNA synthesis.     

Synthesis of heat shock proteins in Drosophila melanogaster embryos

The pattern of polypeptides synthesized in a cell-free protein synthesizing system containing polysomes isolated from heat-shocked (37 C) Drosophila embryos showed significant differences when compared with the pattern obtained when polysomes from normal embryos were used. The synthesis of normal embryonal proteins was reduced and the heat shock proteins were the major products of elongation. After short, 10 min, heat treatment mainly quantitative changes were observed suggesting that normal mRNAs were still present on polysomes, and their products could be completed in vitro in the heterologous cell-free system. The mRNAs coding for normal embryonal proteins were present in almost unchanged amounts in heat-shocked embryos as could be judged from the pattern of proteins synthesized in heterologous cell-free system supplemented with cytoplasmic RNA from normal and heat-shocked embryos. Thus the change in protein synthesis in heat-shocked embryos is not associated with degradation of normal embryonal mRNAs but with their inaccessibility for translation.     

Heat Shock Proteins and Their mRNAs in Dry and Early Imbibing Embryos of Wheat

Two-dimensional gels of in vitro translation products of mRNAs isolated from quiescent wheat (Triticum aestivum) embryos demonstrate the presence of mRNAs encoding heat shock proteins (hsps). There were no detectable differences in the mRNAs found in mature embryos from field grown, from 25°C growth chamber cultivated, or from plants given 38°C heat stresses at different stages of seed development. The mRNAs encoding several developmentally dependent (dd) hsps were among those found in the dry embryos. Stained two-dimensional gels of proteins extracted from 25°C growth chamber cultivated wheat embryos demonstrated the presence of hsps, including dd hsps. A study of the relationship of preexisting hsp mRNAs and the heat shock response during early imbibition was undertaken. Heat shocks (42°C, 90 minutes) were administered following 1.5, 16, and 24 hours of 25°C imbibition. While the mRNAs encoding the low molecular weight hsps decayed rapidly upon imbibition, the mRNAs for dd hsps persisted longer and were still detectable following 16 hours of imbibition. After 1.5 hours of imbibition, the mRNAs for the dd hsps did not accumulate in response to heat shock, even though the synthesis of the proteins was enhanced. Thus, an applied heat shock appeared to lead to the preferential translation of preexisting dd hsp mRNAs. The mRNAs for the other hsps, except hsp 70, were newly transcribed at all of the imbibition times examined. The behavior of the hsp 70 group of proteins during early imbibition was examined by RNA gel blot analysis. The mRNAs for the hsp 70 group were detectable at moderate levels in the quiescent embryo. The relative level of hsp 70 mRNA increased after the onset of imbibition at 25°C and remained high through 25.5 hours of prior imbibition. The maximal levels of these mRNAs at 25°C was reached at 17.5 hours of imbibition. Heat shock caused modest additional accumulation of hsp70 mRNA at later imbibition times.     

Changing rates of histone mRNA synthesis and turnover in Drosophila embryos

The rates of synthesis and turnover of histone mRNA in Drosophila embryos were determined by hybridization of in vivo and in vitro labeled embryonic RNA to Drosophila histone DNA of the recombinant plasmid cDm500. There is a large store of maternal histone mRNA, equivalent to at least 7 X 10(7) copies of each of the five classes of histone mRNA per embryo. Embryonic synthesis of histone mRNA begins at 90 min after oviposition, making the histone genes among the first to be transcribed by embryonic nuclei. Embryonic histone mRNA accumulates rapidly during the blastoderm and gastrula stages. The peak in the rate of histone mRNA synthesis per embryo coincides with the peak in the rate of DNA synthesis per embryo, which occurs at 6 hr after oviposition. After 6 hr, as the rate of DNA synthesis per embryo decreases, the rate of histone mRNA synthesis and the total mass of histone mRNA per embryo both drop sharply. The rate of histone mRNA synthesis per gene falls more than 60 fold in the first 13 hr after oviposition, from 1.3 -2.5 copies per gene-min at 2 hr to 0.02-0.03 copies per gene-min at 13 hr. From measurements of the mass of histone mRNA per embryo and of the rate of accumulation of newly synthesized histone mRNA at a number of stages of early embryogenesis we determined that the cytoplasmic half-life of histone mRNA decreases approximately 7 fold during early Drosophila development, from 2.3 hr at blastoderm to 20 min by the end of gastrulation. Thus the level of expression of histone genes in Drosophila development is controlled not only by the size of the maternal mRNA pool and changes in the rate of histone mRNA synthesis, but also by changes in the rate of histone mRNA turnover.     

Translational regulation of Hsp90 mRNA. AUG-proximal 5'-untranslated region elements essential for preferential heat shock translation

Heat shock in Drosophila results in repression of most normal (non-heat shock) mRNA translation and the preferential translation of the heat shock mRNAs. The sequence elements that confer preferential translation have been localized to the 5'-untranslated region (5'-UTR) for Hsp22 and Hsp70 mRNAs (in Drosophila). Hsp90 mRNA is unique among the heat shock mRNAs in having extensive secondary structure in its 5'-UTR and being abundantly represented in the non-heat shocked cell. In this study, we show that Hsp90 mRNA translation is inefficient at normal growth temperature, and substantially activated by heat shock. Its preferential translation is not based on an IRES-mediated translation pathway, because overexpression of eIF4E-BP inhibits its translation (and the translation of Hsp70 mRNA). The ability of Hsp90 mRNA to be preferentially translated is conferred by its 5'-UTR, but, in contrast to Hsp22 and -70, is primarily influenced by nucleotides close to the AUG initiation codon. We present a model to account for Hsp90 mRNA translation, incorporating results indicating that heat shock inhibits eIF4F activity, and that Hsp90 mRNA translation is sensitive to eIF4F inactivation.     

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.     

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.