Impaired ferritin mRNA translation in primary erythroid progenitors: shift to iron-dependent regulation by the v-ErbA oncoprotein.

In immortalized cells of the erythroid lineage, the iron-regulatory protein (IRP) has been suggested to coregulate biosynthesis of the iron storage protein ferritin and the erythroid delta-aminolevulinate synthase (eALAS), a key enzyme in heme production. Under iron scarcity, IRP binds to an iron-responsive element (IRE) located in ferritin and eALAS mRNA leaders, causing a block of translation. In contrast, IRP-IRE interaction is reduced under high iron conditions, allowing efficient translation. We show here that primary chicken erythroblasts (ebls) proliferating or differentiating in culture use a drastically different regulation of iron metabolism. Independently of iron administration, ferritin H (ferH) chain mRNA translation was massively decreased, whereas eALAS transcripts remained constitutively associated with polyribosomes, indicating efficient translation. Variations in iron supply had minor but significant effects on eALAS mRNA polysome recruitment but failed to modulate IRP-affinity to the ferH-IRE in vitro. However, leukemic ebls transformed by the v-ErbA/v-ErbB-expressing avian erythroblastosis virus showed an iron-dependent reduction of IRP mRNA-binding activity, resulting in mobilization of ferH mRNA into polysomes. Hence, we analyzed a panel of ebls overexpressing v-ErbA and/or v-ErbB oncoproteins as well as the respective normal cellular homologues (c-ErbA/TRalpha, c-ErbB/EGFR). It turned out that v-ErbA, a mutated class II nuclear hormone receptor that arrests erythroid differentiation, caused the change in ferH mRNA translation. Accordingly, inhibition of v-ErbA function in these leukemic ebls led to a switch from iron-responsive to iron-independent ferH expression.

Structure and function of the iron-responsive element from human ferritin L chain mRNA.

We report the cloning and functional characterization of the iron responsive element (IRE) of human ferritin light (L) chain mRNA from a cDNA library of primary human T lymphocytes. Comparison of this palindromic cDNA element to the IRE predicted from the reported genomic sequence revealed significant differences, resulting in a stem-loop structure with lower stability than the IRE of the heavy (H) chain mRNA. Nevertheless, the L subunit IRE mediated efficient binding of the iron regulatory protein (IRP) in a manner comparable to that of human ferritin H chain mRNA in vitro. In accordance with previous observations on H form transcripts, the cis-acting regulatory IRE motif of human ferritin L chain mRNA was capable of repressing translation under iron deprivation but permitted mobilization of the transcripts into polysomes following iron repletion in vivo.

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.

Polyubiquitin RNA characteristics and conditional induction in sea urchin embryos.

A cDNA of the sea urchin Strongylocentrotus purpuratus was identified as encoding polyubiquitin and used to detect a single gene with transcripts containing multiple ubiquitin coding units. Polyubiquitin transcripts exist as a 3.2-kb RNA in polyribosomes and as three higher molecular weight RNAs in purified nuclei. The amount of polyubiquitin RNA is essentially constant at 10(4) -10(5) transcripts per embryo during the egg-to-blastula period and then declines during further development. Heat shock elicits a transient increase in the level of polyubiquitin RNA, while Zn(II) ions induce a sustained accumulation, that is influenced by developmental parameters: One round of Zn(II) induction elicits the accumulation of the nuclear 7.6- and 5.6-kb RNAs, as well as the 3.2-kb polysomal RNA; however, a second round of induction yields only the 5.6- and 3.2-kb RNAs, suggestive of a change in pre-mRNA size or processing. Polyubiquitin RNA is expressed equally in ectodermal and mesoendodermal tissues and is induced in both tissue fractions by treatment of pluteus larvae with Zn(II). However, in isolated and cultured tissue fractions, polyubiquitin RNA is not inducible by Zn(II), in contrast to the full inducibility of metallothionein mRNAs. Polyubiquitin RNA induction thus appears to be conditioned by the integrity of the embryo, as well as by previous exposure to inducer.

Human NAD(+)-dependent mitochondrial malic enzyme. cDNA cloning, primary structure, and expression in Escherichia coli.

Mitochondrial NAD(+)-dependent malic enzyme (EC 1.1.1.40) is expressed in rapidly proliferating cells and tumor cells, where it is probably linked to the conversion of amino acid carbon to pyruvate. In this paper, we report the cDNA cloning, amino acid sequence, and expression in Escherichia coli of functional human NAD(+)-dependent mitochondrial malic enzyme. The cDNA is 1,923 base pairs long and contains an open reading frame coding for a 584-amino acid protein. The molecular mass is 65.4 kDa for the unprocessed precursor protein. Comparison of the amino acid sequence of the human protein with the published NADP(+)-dependent mammalian cytosolic or plant chloroplast malic enzymes reveals highly conserved regions interrupted with long stretches of amino acids without significant homology. Expression of the processed protein in E. coli yielded an enzyme with the same kinetic and allosteric properties as malic enzyme purified from human cells.

Spermidine labels proteins during sea urchin embryogenesis.

We have previously described the presence of a protein containing intact, covalently bound spermidine during very early embryogenesis of the sea urchin (Strongylocentrotus purpuratus). Proteins containing other polyamine metabolites also appear as embryogenesis proceeds. These proteins which contain label derived from exogenous radioactive spermidine show a characteristic pattern which changes during the course of embryonic development. We document for the first time that hypusine, the polyamine metabolite which is a characteristic component of the eukaryotic protein translation initiation factor eIF-4D, is present in more than one species of macromolecule. In addition, N1-acetylspermidine has also been identified as a significant intracellular metabolite of spermidine during embryogenesis.

Association of 7 SL RNA and an SRP-like particle with polysomes and endoplasmic reticulum in the developing sea urchin embryo.

We have identified the sea urchin cognate of the mammalian signal recognition particle (SRP). This particle contains the diagnostic 7 SL small RNA, sediments at a similar velocity to that reported for the mammalian particle, and is found associated with the ER and polysomes. We have examined its subcellular localization during embryogenesis in order to determine whether it could serve in a translational regulatory capacity for a subset of the stored maternal mRNAs. In these studies the 7 SL RNA was used as a marker for the particle, since we determined that the 7 SL RNA exists exclusively within the SRP-like particle at all developmental stages. The relative distribution of the SRP among cytoplasmic structures changes dramatically during development. This represents an actual change in subcellular localization because the 7 SL RNA level remains nearly constant per embryo until the pluteus stage, when it increases slightly. In eggs, the SRP exists almost entirely free in the cytoplasm as an 11 S particle. Very soon after fertilization and throughout development there is an increase in the association of the particle with rapidly sedimenting structures, until by the pluteus stage greater than 90% of the SRP exists in a bound state. The nature of the associations is complex, and the bound structures include, at least in part, ribosomes, polysomes, and microsomes. The SRP is associated with microsomal membranes in gastrula (36 hr) but not in blastula (12 hr) or earlier embryos. Using the criteria of sensitivity to Triton X-100, we determined that 16% of the SRP in a 10,000g cytoplasmic fraction was bound to membranes in a microsomal (endoplasmic reticulum)-containing fraction in the gastrula. In contrast, less than 1% was membrane associated in the blastula. The SRP was also found in a ribosome-polysome fraction in 12-, 36-, and 48-hr embryos, but not in eggs. Finally, a small but significant portion of the SRP was found associated with monosomes in cleavage stage embryos. The possible role the SRP could play in the elongation arrest of stored maternal messages for secreted proteins is discussed.

Effects of training and visual distance on speechreading performance.

The purpose of this study was to investigate the effects of training on subjects' ability to speechread sentences at varying visual distances of 3-6 ft., 6-12 ft., and 12-18 ft. 15 college women who received training obtained higher sentence speechreading scores than the 15 untrained women. The emphasis placed on kinesthetic feedback may have assisted the trained subjects' performance; however, distance up to 18 ft. did not appear to affect subjects' ability to speechread sentences, regardless of training.

A new type of prosome-like particle, composed of small cytoplasmic RNA and multimers of a 21-kDa protein, inhibits protein synthesis in vitro.

A large fraction of the translationally repressed non-globin messenger RNA in duck erythroblasts is present in non-polyribosomal free mRNP structures which sediment in the 30-40-S range ('35 S'). In 0.5 M KCl, they form core complexes which show a pronounced peak at about 32 S containing mRNA and a discrete spherical RNP particle with a diameter of about 12 nm and the typical morphology of a prosome [H.-P. Schmid et al. (1984) EMBO J. 3, 29-34]. Buoyant density measurements and chromatography on oligo(dT)-cellulose indicate that this particle is bound to mRNA; it can be released from the mRNA by treatment of the free mRNP fraction with SDS. This prosome-like particle inhibits the translation of mRNA in vitro. It is composed primarily of multimers of a single 21-kDa protein and at least one species of RNA of about 80-100 nucleotides. It is resistant to dissociation by 2 M CS2SO4 and 1% SDS; the 21-kDa protein is not attacked by proteinase K unless the particle is extracted with phenol prior to treatment with the protease. The small RNA moiety of the particle hybridizes to the poly(A)-rich mRNA derived from the free mRNPs, as well as to polyribosomal mRNA. These data indicate that prosomes may serve to regulate mRNA translation; they show furthermore that prosome-like particles (about 600 kDa mass) may be built of up to 25 molecules of a single specific protein, rather than of the entire set of about 20 prosomal proteins previously identified.

Sea urchin prosome: characterization and changes during development.

A cytoplasmic particle displaying properties in common with a structure present in duck erythroblasts, termed the prosome, has been isolated from eggs and embryos of two species of sea urchin. This particle was partially purified by sedimentation in sucrose gradients containing 0.5 M KCl, and some of its physical properties and its behavior during early development were determined. The prosome sediments between 16 and 19 S and has a buoyant density of 1.30 g/cm3 in Cs2SO4 gradients. Biochemically, the particle is characterized as 20-25 polypeptides of molecular size 24-35 kDa with about 10 small RNAs. A monoclonal antibody directed against the 27-kDa protein of duck erythroblast prosome recognizes a 27-kDa protein of the sea urchin prosome. We have used this protein, as representative of the prosome, to immunologically determine the level and the subcellular localization of the particle during development. Immunoblotting and cellular fractionation studies show that the 27-kDa prosome polypeptide is present almost entirely in the postribosomal supernatant of unfertilized egg lysates. After fertilization and during early development, the total amount of 27-kDa protein per embryo remains constant, but the amount in the postribosomal supernatant decreases; there is a concomitant increase in the level of the 27-kDa protein in a rapidly sedimenting, particulate fraction containing nuclei. Immunofluorescence studies further show that the 27-kDa protein is located mainly in the cytoplasm of eggs and two-cell embryos. The subcellular location of the prosome, therefore, appears to change during development. In vivo labeling experiments have failed to detect the synthesis of either the prosome proteins or RNAs in eggs and embryos up to 48 hr of development, suggesting that this cytoplasmic particle is not synthesized de novo in early embryogenesis and thus is metabolically stable. The prosome is thus a normal cellular constituent of the sea urchin and is most likely synthesized during oogenesis and stored in the unfertilized egg.

Translational initiation factors from sea urchin eggs and embryos: functional properties are highly conserved.

We have used three mammalian in vitro assays for translational initiation (globin synthesis, methionyl-puromycin synthesis, and ternary complex formation), consisting of defined components, to ask whether sea urchin (Strongylocentrotus purpuratus) egg and embryo translational components are active in heterologous assays for mammalian components, and to determine to what extent these activities are evolutionarily conserved. A "pH 5 enzyme" fraction prepared from unfertilized eggs and embryos efficiently replaced the rat liver pH 5 fraction in a globin synthesis assay, indicating that the elongation and termination factors and the aminoacyl-tRNAs are compatible with the mammalian translational machinery. The classical schemes for mammalian initiation factor purification yielded low or no detectable activities in the ribosomal salt washes, so a novel procedure was developed to partially purify initiation factors from sea urchin eggs and embryos before testing for activity. A 12,000 g homogenate from unfertilized eggs was fractionated by step elution from phosphocellulose at 100, 300, 600, and 1,200 mM salt. Initiation factor activities were found in each salt step as predicted for the mammalian counterparts. The following activities have been detected: eIF2, eIF3/4F, eIF4A, eIF4B, eIF4C, eIF4D, and eIF5. Further fractionation of each elution step yielded preparations enriched in specific initiation factor activities. However, denaturing polyacrylamide gel electrophoresis of the fractions gave complex polypeptide patterns and no clearly identifiable bands corresponding to the mammalian initiation factor polypeptides. In spite of the conservation of factor activity, crude and affinity purified polyclonal antibodies to the mammalian factors did not cross-react with the sea urchin preparations. The demonstration that initiation factor activities are sufficiently conserved to allow their being assayed is the first step in our dissection of the translational machinery of eggs and embryos, and in the complete analysis of the regulation of translation during early development.

Spermidine is bound to a unique protein in early sea urchin embryos.

Spermidine is rapidly taken up and becomes bound to protein during the very early hours of sea urchin embryogenesis. During the first 6 hr after fertilization of freshly obtained sea urchin eggs (Strongylocentrotus purpuratus), which are incubated in the presence of exogenous [3H]-spermidine, up to 7% of the total cell-associated spermidine appears uniquely as spermidine bound in macromolecular form. This unique protein containing spermidine migrates as a single radioactive band in gel electrophoresis. It has a Mr of approximately equal to 30,000 and is readily distinguishable from the protein initiation factor eIF-4D, which has a Mr of 18,000, the only other identifiable protein known to date to be posttranslationally modified by polyamines.

Individual regulation of the accumulation of H1 mRNA and core histone mRNAs in sea urchin embryos.

The relative cytoplasmic accumulation of the individual histone mRNAs in sea urchins was determined by gel analysis of 3H-labeled cytoplasmic RNA isolated from embryos of the early cleavage through the mesenchyme blastula stages. A number of separate determinations showed that H1 mRNA accumulates at a molar ratio of 0.5 or less compared with each of the H2 or H3 core histone mRNAs through approximately the first 12 h of embryonic development. After this time, the accumulation of H1 mRNA increases relative to the core histone mRNAs, and approximately equimolar amounts of the histone mRNAs are produced by about the 14-h stage. The equimolar synthesis of H1 mRNA appears to be transient, returning to 0.5-molar levels several hours later. The increase in H1 mRNA accumulation, relative to the core histone RNAs, is coincident with the transition from expression of the early (alpha) sea urchin histone gene set to the late histone genes. Since all five of the early histone genes occur in a 1:1 ratio within repeating units, the data suggest that the genes within a single repeat, or their immediate products, are individually regulated. Gel analysis of the proteins synthesized in vivo by embryos demonstrates that the pattern of synthesis of the histone proteins reflects the changing ratios of the histone mRNAs.

Utilization of maternal and embryonic histone RNA in early sea urchin development.

Histone RNA in early sea urchin embryos is derived from maternal stores and from new transcription. We show that the sedimentation of maternal free RNPs, containing histone RNA, is somewhat more rapid than the sedimentation of the newly made histone RNPs. Yet, prior to the 2- to 4-cell stage, both the maternally derived and the newly synthesized histone RNA are localized to the same extent in the non-polysomal-free RNPs, and the timing of their recruitment into embryonic polysomes appears to be the same. The levels of hybridization of histone probe to RNAs in cleaving embryos increases severalfold in intensity, and the increase occurs primarily in the polysomes. These data suggest that new transcription may provide an important contribution to the total histone RNA mass by as early as the 32- to 64-cell stage of development.

Nonrandom distribution of histone mRNAs into polysomes and nonpolysomal ribonucleoprotein particles in sea urchin embryos.

In the early sea urchin embryo, newly synthesized cytoplasmic histone mRNA is found both on polysomes and free of ribosomes as nonpolysomal messenger ribonucleoprotein particles (free RNPs). The distribution of newly synthesized histone mRNAs between translating and nontranslating compartments is nonrandom and dependent on the developmental stage. Gel electrophoresis and autoradiography of polysomal and free RNP RNA from embryos at various stages show that (i) the fraction of total newly synthesized histone mRNA that is in polysomes is greater than the fraction of total newly synthesized poly(A)+mRNA that is in polysomes, at all stages examined, and (ii) among the five histone mRNAs. H1 mRNA and H4 mRNA are relatively more enriched in the free RNPs than are the mRNAs for H2A, H2B, and H3. These data suggest that histone mRNA, as a class, is more efficiently utilized as a template than the average mRNA and, of the cytoplasmic histone mRNAs, the mRNAs for histones H2A, H2B, and possibly H3 are selected more frequently for translation than those for H1 and H4. Cell-free translations of polysomal and free RNP RNAs yield different ratios of in vitro histone products, consistent with the RNA distribution data. To test the possibility that the in vivo distribution of the histone mRNAs is the consequence of different intrinsic initiation capabilities of the individual mRNAs, ribosome-binding assays were carried out and unequal binding abilities of the histone mRNAs in the reticulocyte lysate were shown. A translational level component in the regulation of histone synthesis in the sea urchin embryo is indicated.

Analysis of the Escherichia coli ribosome-ribosomal subunit equilibrium using pressure-induced dissociation.

Hydrostatic pressure can be used to perturb the ribosome-ribosomal subunit equilibrium. We have used glutaraldehyde fixation and subsequent sucrose gradient analysis to determine the equilibrium concentrations of Escherichia coli 70 S, 50 S, and 30 S particles at pressures from 1 to 1400 atm. This method is shown to be sufficiently rapid and free of interfering ribosomal aggregation artifacts when performed at Mg2+ concentrations below 8 mM. We show directly that the E. coli ribosome is in equilibrium with its subunits and that the pressure-sensitive reaction is appropriately described by the expression: In Kp = ln K0 + (P delta V/RT), where Kp and K0 are the equilibrium constants at pressure P and 1 atm, respectively, and delta V is the change in molecular volume that occurs during the reaction. The method provides values for K0 under different conditions, and the effects of Mg2+ ion can be readily ascertained. K0 and delta V were also estimated by a method of fitting computer-generated sucrose gradient profiles to experimental profiles. Determination of delta H0, delta S0, and delta V0 at 5 mM Mg2+ are presented. The results are discussed in the context of previous thermodynamic studies of the E. coli ribosome.

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.

A potential DNA replication complex isolated from sea urchin embryos by renografin gradient centrifugation.

A complex containing deoxyribonucleic acid, protein and lipid has been isolated from the nuclei of the sea urchin Stronglocentrotus pupuratus by gentle lysis of nuclei and centrifugation of the lysate in a renografin gradient. The complex is similar in several respects to DNA-membrane complexes isolated by the sarkosyl M-band technique [1]. These include the following: 1, most of the cellular DNA is present in the complex but the bulk of the DNA may be removed by sonication or nuclease treatment; 2, nascent DNA is preferentially found in the complex; and 3, destruction of membrane/protein components removes DNA found in the complex.

Persistent cytoplasmic location of a DNA polymerase beta in sea urchins during development.

A subcellular localization study of a low molecular weight DNA polymerase beta indicates that this enzyme, as well as a high molecular weight DNA polymerase alpha, is found in large quantities in the cytoplasm of Strongylocentrotus purpuratus eggs. The two enzyme activities are distinguished by DEAE-sievorptive chromatography and by their differential activities with activated DNA and oligo(dT)10 . poly(dA)200 primer-templates. Using an enucleation procedure, it is concluded that an extremely low proportion if any, of both polymerases is present in the egg nucleus. At blastula stage, a period of rapid cell proliferation, similar studies of DNA polymerase subcellular localization using two different methods of nuclear isolation indicate that the DNA polymerase beta remains largely cytoplasmic while the alpha enzyme is found to be predominantly nuclear. Since the results for the alpha enzyme agree with previous reports (Loeb, L.A. (1969) J. Biol. Chem. 244, 1672) and since one method of nuclear isolation, using hypotonic solutions, enables us to recover both DNA polymerase alpha and beta activities in isolated mouse L-cell nuclei, the enzyme quantitation of isolated sea urchin nuclei is considered accurate. Thus, although there is a translocation of the polymerase alpha from a cytoplasmic to nuclear site during early embryonic development, such a massive relocalization of the polymerase beta does not occur.