Globin Messenger Research Articles

Contribution of polyadenylic acid sequences to the maintenance of secondary structure of globin messenger RNA

Purified rabbit hemoglobin mRNA (HbmRNA) has been characterized by cell-free translation in vitro and by denaturing (glyoxal) gel electrophoresis. Enzymatic deadenylation yields shorter molecules, susceptible of translation but unable to serve as a template for reverse transcriptase. Comparison between thermal denaturation parameters of intact and deadenylated HbmRNA in phosphate buffer alone before and after reaction with formaldehyde (900 × 10 min) and in phosphate buffered 4M guanidinium chloride indicates a highly ordered secondary structure, 10% of which is given by single stranded base stacking, in intact HbmRNA. Deadenylated HbmRNA displays very little single stranded base stacking, suggesting that most of this contribution to the overall structure of intact HbmRNA is given by its poly (A) sequence.

In vivo degradation of rat globin messenger RNA during maturation of reticulocytes.

The degradation of globin mRNA in rat reticulocytes maturing in the peripheral blood was investigated. Poly(A) and non poly(A) portions of mRNA molecules were determined quantitatively by hybridization with radioactive poly(U) and complementary DNA, respectively. During the degradation of mRNA in vivo, it was shown that (1) globin mRNA and the bulk of RNA decrease in parallel, (2) the average chain length of poly(A) segments in the mRNA does not change, (3) the percentage of poly(A) (-) globin mRNA in total globin mRNA does not change, and (4) fragments of large molecular weight do not accumulate. Possible mechanisms of degradation of globin mRNA in the reticulocytes are discussed on the basis of these observations.

Quantitation and localization of globin messenger RNA in rabbit reticulocyte lysate.

A sensitive and quantitative method is described for the determination of globin mRNA distribution in rabbit reticulocyte lysate. The method uses high resolution sucrose density gradient centrifugation followed by [5'-3H]polyuridylate hybridization to poly(A)-mRNA in gradient fractions. Polyadenylate, purified globin mRNA, and ribonuclease-treated lysate are used to standardize the hybridization assay. It is demonstrated that changes of mRNA and ribosomal distribution do not affect quantitation of the total mRNA localization and Met-tRNAf which suggest that the monitoring of Met-tRNAf binding alone may not be sufficient to assess the mechanisms of control which affect the initiation of protein synthesis.

Defects in DNA and globin messenger RNA in homozygotes for hemoglobin Lepore.

Globin messenger RNA (mRNA) isolated from three patients homozygous for hemoglobin Lepore is shown to have a marked reduction of the amount of beta-like globin mRNA (Lepore-globin mRNA sequences) compared with alpha-globin mRNA by molecular hybridization. The relative amounts of alpha- and Lepore mRNA are similar to the amounts of alpha- and Lepore globin synthesized in intact cells and by isolated mRNA in a cell-free system. It is also demonstrated that Lepore-globin mRNA can completely hybridize to full-length or nearly full-length beta-globin specific complementary DNA and protect it from nuclease digestion, indicating close homology between the delta-mRNA sequences present in Lepore mRNA and the beta-complementary-DNA probe. We have also quantitated the numbers of beta-like globin gene sequences in genomic Lepore DNA by molecular hybridization and demonstrated a reduction in their number consistent with the Lepore gene being a delta beta-gene fusion product.

Recognition of Messenger RNA in Eukaryotic Protein Synthesis

Equilibrium studies of the interaction between messenger RNA and the rabbit reticulocyte initiation factor that binds methionyl‐tRNAf, eIF‐2, were performed, using filtration through nitrocellulose membranes to measure complex formation. The formation of complexes between the initiation factor and messenger RNA is shown to be consistent with a simple, bimolecular equilibrium. Binding of native, translatable 125I‐labeled globin messenger RNA to the factor is tight, with an apparent dissociation constant of approximately 5 × 10 −10 M at physiological salt concentration. This binding does not involve the poly(A) sequence at the 3′ end of globin messenger RNA, nor the 90‐nucleotide sequence adjacent to it, for poly(A)‐free derivatives of globin messenger RNA prepared by controlled, processive phosphorolysis bind the initiation factor with an affinity essentially equal to that of native globin messenger RNA. In contrast to other cases of protein‐nucleic‐acid interaction, binding of initiation factor eIF‐2 to messenger RNA is as tight at 0.15 M KCI as it is at 0.01 M KCI and is consistent with a preference for the RNA conformation existing at physiological salt concentration. 32P‐labeled bacteriophage R17 RNA, a messenger RNA that can be translated, although at relatively low efficiency, in a mammalian cell‐free system, also forms an equimolar complex with eIF‐2, but binds 10 to 15‐fold less tightly than globin messenger RNA. These results show that the eukaryotic initiation factor that binds methionyl‐tRNAf recognizes a sequence in globin messenger RNA that does not include the poly(A) moiety or most of the untranslated sequence at the 3′ end of the molecule. The data suggest a relationship between the affinity of a given messenger RNA species for initiation factor eIF‐2 and its efficiency as a template in protein synthesis.

Evidence for the protection of specific RNA sequences in globin messenger ribonucleoprotein particles.

Purified 15 S globin mRNA-protein (mRNP) complexes obtained by EDTA dissociation of duck reticulocytes polyribosomes were digested with the calcium dependant Staphylococcus aureus nuclease (EC 3. 1. 4. 7.). 25% of the globin mRNA sequences were resistant to extensive nuclease digestion as determined by TCA precipitation of the digested 15 S particles labelled in vivo with tritiated uridine. Polyacrylamide gel electrophoresis of the RNA from nuclease digested 15 S particles showed that the protected oligoribonucleotides were distributed into two distinct size classes of 25,000 and 12,000 MW. Comparison between in vitro iodine-labelled 9 S globin mRNA extracted from Staphylococcal nuclease digested 15 S mRNP particles was carried out by fingerprinting. Mapping of T1 ribonuclease digests by high-voltage electrophoresis and homochromatography showed that specific oligoribonucleotides were protected against nuclease attack by proteins of the 15 S mRNP.

The influence of Rauscher Leukemia Virus (R-MuLV) on the differentiation of red blood cells in BALB/c mice

The influence of Rauscher Leukemia Virus infection of BALB/c mice on the differentiation of red blood cells has been investigated. Twenty-one days after infection, the total number of red blood cells in the spleen is increased 20 times in contrast to the bone marrow which is largely unaffected. The cellular composition of these hemopoietic organs has been analysed using a number of markers of erythroid maturation, i.e. heme and hemoglobin synthesis and the presence of globin messenger RNAs. These studies show that most of the blasts present in leukemic spleens are immature as judged by morphological criteria and do not contain globin mRNA, whereas the more mature blasts contain globin mRNA as determined autoradiographically by in situ hybridization to globin cDNA. The number of mature blasts, comprising about one-third of the immature ones, is both in absolute terms and per mg tissue higher than in control spleens. Pulse labelling with 59Fe shows, that heme and hemoglobin synthesis takes place in leukemic spleens. The rate of heme synthesis in leukemic spleens, as judged by short-term incorporation of 59Fe and 14C-glycine is intermediate between the values for spleens from anemic and control animals. After a pulse, labelled 59Fe is first bound to non-hemoglobin proteins. After a chase, this protein-bound 59Fe is transferred to hemoglobin as erythroid differentiation occurs in spleens of normal and anemic mice. However, this transfer of 59Fe to hemoglobin does not occur in leukemic spleen cells during a pulse/chase in vivo. These results could mean that these immature blasts die or that the maturation is inhibited in the enlarged spleens, whereas only a minor portion differentiates in smaller basophilic erythroblasts, which mature into reticulocytes.

Nonuniform size distribution of nascent peptides: The role of messenger RNA

The origin of the nonuniform size distribution of nascent rabbit globin peptides has been investigated in the reticulocyte lysate and wheat germ cell-free protein synthesis systems. Increasing the concentrations of the cellular components involved in protein synthesis failed to alter the elution pattern observed upon chromatographic analysis of reticulocyte lysate nascent chains. Nascent chains isolated from a globin messenger RNA-directed wheat germ cell-free system showed a nonuniform size distribution of nascent peptides similar to that of the rabbit reticulocyte nascent chains. These observations indicate that the nonuniformity of the globin nascent chains arises from a unique property of the messenger RNA being translated and not from limiting concentrations of a component or components of the reticulocyte protein synthesis system.

Site-directed mutagenesis in DNA: Generation of point mutations in cloned β globin complementary DNA at the positions corresponding to amino acids 121 to 123

We have utilized the principle of site-directed mutagenesis, previously applied to the RNA of bacteriophage Qβ, to generate nucleotide transitions in a predetermined region of DNA. Plasmid PβG, which contains an almost complete DNA copy of rabbit β globin messenger RNA, was nicked at the EcoRI site which is located within the globin gene, in a region corresponding to amino acids 121 and 122. Substrate-limited nick translation using DNA polymerase I and N 4 -hydroxydCTP, dCTP and dATP led to the replacement of TMP residues by the nucleotide analog in the immediate vicinity of the nicks. The substituted DNA was amplified in vivo, treated with EcoRI and retransfected. 1.9% of the amplified DNA was found to be EcoRI-resistant. Nucleotide sequence analysis of the critical region of six EcoRI-resistant isolates revealed that two plasmids had one, three had two and one had three A · T → G · C transitions, all located within the substituted region. No point mutations (< 3 × 10 −3%) were found in control preparations; however, a small number of deletion mutants, lacking the EcoRI site, were isolated.

Terminal stages of erythroid differentiation: persistent production of globin messenger RNA is necessary to sustain globin synthesis

The kinetic relationship between the globin mRNA accumulation and the rate of synthesis of globin chains was studied during the terminal stages of differentiation in erythroid cells derived from the yolk sac of mouse fetuses. RNA derived from the whole cells and from different cell compartments were hybridized to DNA complementary to embryonic globin mRNA. The relative proportion of embryonic globin RNA molecules and their absolute number per cell were estimated on the 11th, 12th, and 13th days of mouse fetal development. During erythroid terminal differentiation globin mRNA became progressively predominant on polyribosomes along with the progressive specialization of cell functions. The number of embryonic globin RNA molecules per cell remained constant while yolk sac erythroid cells underwent two rounds of cell division. These data indicate that the transcription of globin genes is operative throughout the last stages of terminal differentiation and that there is no detectable storage of globin RNA sequences in these cells. The rates of accumulation of mRNA molecules and of globin synthesis both seem correlated to the length of the cell cycle.

Terminal Stages of Erythroid Differentiation: Persistent Production of Globin Messenger RNA is Necessary to Sustain Globin Synthesis

The kinetic relationship between the globin mRNA accumulation and the rate of synthesis of globin chains was studied during the terminal stages of differentiation in erythroid cells derived from the yolk sac of mouse fetuses. RNA derived from the whole cells and from different cell compartments were hybridized to DNA complementary to embryonic globin mRNA. The relative proportion of embryonic globin RNA molecules and their absolute number per cell were estimated on the 11th, 12th, and 13th days of mouse fetal development. During erythroid terminal differentiation globin mRNA became progressively predominant on polyribosomes along with the progressive specialization of cell functions. The number of embryonic globin RNA molecules per cell remained constant while yolk sac erythroid cells underwent two rounds of cell division. These data indicate that the transcription of globin genes is operative throughout the last stages of terminal differentiation and that there is no detectable storage of globin RNA sequences in these cells. The rates of accumulation of mRNA molecules and of globin synthesis both seem correlated to the length of the cell cycle.

Purification of myosin mRNP translational control RNA and its inhibition of myosin and globin messenger translation

Purification of myosin mRNP translational control RNA and its inhibition of myosin and globin messenger translation

Translational recognition of the 5'-terminal 7-methylguanosine of globin messenger RNA as a function of ionic strength

The translation of rabbit globin mRNA in cell-free systems derived from either wheat germ or rabbit reticulocyte was studied in the presence of various analogues of the methylated 5' terminus (cap) as a function of ionic strength. Inhibition by these analogues was strongly enhanced by increasing concentrations of KCl, K(OAc), Na(OAc), or NH4(OAc). At appropriate concentrations of K(OAc), both cell-free systems were equally sensitive to inhibition by m7GTP. At 50 mM K(OAc), the reticulocyte system was not sensitive to m7GMP or m7GTP, but at higher concentrations up to 200 mM K(OAc), both nucleotides caused strong inhibition. The compound in m7G5'ppp5'Am was inhibitory at all concentrations of K(OAc) ranging from 50 to 200 mM, although more strongly so at the higher concentrations. Over the same range of nucleotide concentrations, the compounds GMP, GTP, and G5'ppp5'Am were not inhibitors. The mobility on sodium dodecyl sulfate-polyacrylamide electrophoresis of the translation product was that of globin at all K(OAc) concentrations in the presence of m7GTP. Globin mRNA from which the terminal m7GTP group had been removed by chemical treatment (periodate-cyclohexylamine-alkaline phosphatase) or enzymatic treatment (tobacco acid pyrophosphatase-alkaline phosphatase) was translated less efficiently than untreated globin mRNA at higher K(OAc) concentrations, but retained appreciable activity at low K(OAc) concentrations.

Organization of transcribed regions of chromatin.

The endonuclease DNase II preferentially attacks a limited and tissue-specific portion of chromosomal DNA. This material may be separated from the bulk of chromatin DNA by virtue of its solubility in 2 mM MgCl2. The Mg2+ soluble fraction forms a specific subset of DNA sequences and is enriched four to sevenfold in sequences coding for cytoplasmic poly(A)-containing RNA and globin messenger RNA (in globin-producing cells). The bulk (70--90%) of rapidly labelled RNA is found associated with the Mg2+-soluble fraction. Transcriptionally active, Mc2+-soluble chromatin is organized into repeating subunits of DNA (200 +/- 5 base pairs) and histone. Mc2+-soluble active subunits differ from the subunits or nucleosomes of non-transcribed regions in many respects: namely, chemical composition (non-histone protein and RNA), sedimentation properties, differential sensitivity to DNase I and the single-strand-specific nuclease S1, and optical melting behaviour. These results suggest that chromatin subunits adopt a new configuration during the process of transcription.

All factors required for protein synthesis are retained on heparin bound to Sepharose.

1. Postmitochondrial supernatants of rabbit reticulocyte lysates were chromatographed on heparin bound to Sepharose 4B, and the fraction retained on affinity columns was separated by subsequent gel filtration on Sepharose 4B into three fractions, two of them active in protein synthesis. 2. The heavier fraction sedimented at 40S and contained more than 10% RNA. This consisted predominantly of a 12S component, with smaller amounts of the 9S and 4S RNA species. The lighter fraction (18-20S) was composed of proteins with less than 1% RNA. 3. Different enzymic activities were associated with these fractions. 4. In the presence of both fractions, efficient translation took place on combined ribosomal subunits of rat liver with added cofactors. Globin messenger ribonucleoprotein stimulated this translation 5-6-fold. 5. Relatively large complexes of all factors required for protein synthesis are apparently isolated from reticulocytes by affinity chromatography on heparin-Sepharose 4B. Such complexes may occur naturally in the cytoplasm of mammalian cells.

Cordycepin. An inhibitor of newly synthesized globin messenger RNA.

The effect of cordycepin (3'-deoxyadenosine) on newly synthesized globin mRNA in cultured mouse fetal liver erythroid cells is investigated. At cordycepin concentrations that do not inhibit amino acid incorporation into acid-precipitable material, the quantity of pulse-labeled (radioactive) globin mRNA nucleotide sequences is reduced by 90%, as compared to adenosine-treated controls. The reduction of radioactivity in globin-specific RNA sequences is greater than the inhibition of total RNA synthesis in experiments in which the labeling times range from 6 to 60 min. Control experiments demonstrate that cordycepin does not reduce the recovery of total cell RNA or steady state (unlabeled) globin mRNA. The hybridization assay used to detect radioactive globin mRNA sequences is independent of the cellular location or the number of 3'-terminal adenylate residues in the mRNA-containing molecules. These data thus indicate that cordycepin inhibits newly synthesized mRNA as effectively as it inhibits ribosomal and transfer RNA synthesis.

Purification and structural properties of the precursors of the globin messenger RNAs

A three-step procedure is described for the purification of globin mRNAs and their precursors from nucleated mouse erythroid cells. The steps are affinity chromatography on oligo(dT)-cellulose, electrophoresis in a 4.5% polyacrylainide gel containing 98% formamide, and hybridization to globin cDNA-cellulose. The β globin mRNA precursor ( M r, 600,000), its putative cleavage intermediate ( M r 280,000), and the α globin precursor ( M r, 280,000) have been purified from pulselabeled fetal mouse liver cells with recoveries of approximately 20%. The 280,000 M r, β-specific RNA has not been separated from the α-precursor. The purity of each RNA fraction was estimated by hybridizing the fetal liver RNAs and authentic reticulocyte 10 S globin [ 125I]mRNA to excess, unlabeled globin cDNA and comparing the maximum extent of annealing of each RNA. The purities of the 600,000 M r RNA (β-precursor) and the 280,000 M r RNAs as calculated by this method are 78% and 100%, respectively. More than 90% of each RNA fraction is recovered intact following cDNA-cellulose hybridization. There is a single globin mRNA moiety in each precursor molecule.

Precursors of alpha and beta globin messenger RNAs

Several structural properties of the pulse-labeled globin messenger RNA precursor molecules that sediment faster than steady-state (10 S) globin mRNA (Curtis & Weissmann, 1976; Ross, 1976; Kwan et al., 1977; Bastos & Aviv, 1977) are investigated. The radioactive, globin-specific RNA resolves into two peaks during electrophoresis in polyacrylamide gels prepared in 98% formamide. The molecular weights of the larger and the smaller RNAs are 600,000 and 280,000, respectively, as compared with the average molecule weight of cytoplasmic globin mRNA, 200,000. When RNA from cells labeled for 10 minutes is analyzed by formamide gel electrophoresis, a precursor larger than 600,000 M r is not detected. Most, if not all, of the molecules in the 600,000 and 280,000 M r RNA fractions are polyadenylated. The 600,000 M r RNA contains β globin mRNA sequences, but little, if any, a globin sequences; the 280,000 M r RNA contains both α and β sequences. At steady-state the 14-day-old mouse fetal liver erythroblast contains approximately 50 molecules of the 600,000 M r RNA, 1000 molecules of the 280,000 M r RNAs, and 60,000 molecules of mature globin mRNA. The half-lives of the 600,000 and 280,000 M r RNAs calculated using steady-state levels are 2 minutes and 17 minutes, respectively. These data demonstrate that β globin gene sequences are transcribed into a molecule (β-precursor) that is threefold larger than mature β globin mRNA. In contrast, the largest transcript of the α globin genes detected in these experiments is only 1.4-fold larger than mature α globin mRNA. The possibility that the 280,000 M r, β-specific RNA is a processing intermediate generated by cleavage of the 600,000 M r β-precursor is discussed, along with alternative hypotheses.

Functional stabilisation of HeLa cell histone messenger RNAs injected into Xenopus oocytes by 3'-OH polyadenylation.

WE have shown that, when injected into Xenopus oocytes, poly (A) free globin messenger RNA is translated for a short period of time and then rapidly degraded1–3, while, under the same conditions, native mRNA shows a marked stability1,2,4. The poly (A) segment itself is responsible for the stability of native globin mRNA, since its readdition to previously de-adenylated mRNA restores the stability of the message5. We have shown also that the poly (A) stretch must contain a minimum number of adenylate residues to ensure its protective function6. It was interesting to see whether the concept of the stabilisation of eukaryotic mRNAs by 3′-OH polyadenylation is general. This can be done by looking at the effect of the presence of a 3′-OH poly (A) segment on the stability of various mRNAs. Here we study the stability of HeLa cell poly (A)-free histone mRNAs injected into Xenopus oocytes, and the effect of 3′-OH polyadenylation on this stability.

Structure of recombinant plasmids containing synthetic human foetal globin gene sequences

In vitro synthesized duplex DNA complementary to human foetal globin messenger RNA was integrated into bacterial plasmids and amplified by transformation of Escherichia coli. Recombinants carrying globin DNA were identified by hybridization of foetal globin messenger RNA to bacterial DNA in situ and by liquid hybridization of purified plasmids to specific globin complementary DNA probes. Heteroduplex mapping revealed either a simple insertion loop at the position of the EcoRI site of the parental plasmid DNA. We provide evidence to suggest that these deletions are the result of a site-specific nicking activity of the EcoRI preparations used in the formation of recombinant plasmids.