Hemoglobin Messenger Research Articles

Nonerythroid hemoglobin promotes human cumulus cell viability and the developmental capacity of the human oocyte

To determine the relationship between the levels of cumulus cell (CC) hemoglobin messenger ribonucleic acid (mRNA) and the developmental potential of the associated oocyte and whether hemoglobin protects the CCs from oxidative stress-induced apoptosis. Laboratory-based study. University laboratory and university-affiliated invitro fertilization center. Cumulus cells from the oocytes of patients who underwent invitro fertilization with intracytoplasmic sperm injection with and without preimplantation genetic testing between 2018 and2020. Studies on individual and pooled CCs collected at the time of oocyte retrieval or cultured under 20% or 5% O2. Quantitative polymerase chain reaction analysis of individual and pooled patient CC samples were used to monitor the hemoglobin mRNA levels. Reverse transcription-polymerase chain reaction arrays were used to assess genes that regulate oxidative stress in CCs associated with aneuploid and euploid blastocysts. Studies were conducted to assess the effect of oxidative stress on the rate of apoptosis, level of reactive oxygen species, and gene expression in CCs invitro. Compared with CCs associated with arrested and aneuploid blastocysts, the mRNA levels encoding the alpha and beta chains of hemoglobin increased by 2.9- and 2.3-fold in CCs associated with euploid blastocysts, respectively. The mRNA levels encoding the alpha and beta chains of hemoglobin also increased by 3.8- and 4.5-fold in CCs cultured under 5% O2 vs. 20% O2, respectively, and multiple regulators of oxidative stress were overexpressed in cells cultured under 20% O2 compared with those under 5% O2. However, the rate of apoptosis and amount of mitochondrial reactive oxidative species increased by 1.25-fold in CCs cultured under 20% O2 compared with those under 5% O2. Variable amounts of the alpha and beta chains of hemoglobin were also detected within the zona pellucida and oocytes. Higher levels of nonerythroid hemoglobin in CCs are associated with oocytes that result in euploid blastocysts. Hemoglobin may protect CCs from oxidative stress-induced apoptosis, which may enhance cumulus-oocyte interactions. Moreover, CC-derived hemoglobin may be transferred to the oocytes and protect it from the adverse effects of oxidative stress that occurs invivo and invitro.

Hypoxia Up-Regulates Expression of Hemoglobin in Alveolar Epithelial Cells

Alveolar epithelial cells are directly exposed to acute and chronic fluctuations in alveolar oxygen tension. Previously, we found that the oxygen-binding protein hemoglobin is expressed in alveolar Type II cells (ATII). Here, we report that ATII cells also express a number of highly specific transcription factors and other genes normally associated with hemoglobin biosynthesis in erythroid precursors. Because hypoxia-inducible factors (HIFs) were shown to play a role in hypoxia-induced changes in ATII homeostasis, we hypothesized that the hypoxia-induced increase in intracellular HIF exerts a concomitant effect on ATII hemoglobin expression. Treatment of cells from the ATII-like immortalized mouse lung epithelial cell line-15 (MLE-15) with hypoxia for 20 hours resulted in dramatic increases in cellular levels of HIF-2α protein and parallel significant increases in hemoglobin messenger RNA (mRNA) and protein expression, as compared with that of control cells cultured in normoxia. Significant increases in the mRNA of globin-associated transcription factors were also observed, and RNA interference (RNAi) experiments demonstrated that the expression of hemoglobin is at least partially dependent on the cellular levels of globin-associated transcription factor isoform 1 (GATA-1). Conversely, levels of prosurfactant proteins B and C significantly decreased in the same cells after exposure to hypoxia. The treatment of MLE-15 cells cultured in normoxia with prolyl 4-hydroxylase inhibitors, which mimic the effects of hypoxia, resulted in increases of hemoglobin and decreases of surfactant proteins. Taken together, these results suggest a relationship between hypoxia, HIFs, and the expression of hemoglobin, and imply that hemoglobin may be involved in the oxygen-sensing pathway in alveolar epithelial cells.

The identification of newborns using messenger RNA profiling analysis

The ability to determine the physical characteristics of an individual depositing a bloodstain at a crime scene would be an invaluable tool to investigators, akin to eyewitness information. One useful biometric that may be amenable to molecular genetic analysis is the biological age of an individual. In theory, it may be possible to determine patterns of gene expression that are age specific, thereby permitting the distinction among tissue samples originating from individuals of different ages (e.g., newborn, adolescent, middle-age, elderly). We have discovered two novel isoforms of gamma hemoglobin messenger RNA, designated HBG1n and HBG2n, which exhibit an extremely restricted pattern of gene expression, being confined to newborn individuals. Multiplex quantitative reverse transcription PCR (qRT-PCR) assays incorporating these novel mRNAs have been designed, tested, and evaluated for their potential forensic use. The results indicate that the assays provide the ability to determine whether a bloodstain originated from a newborn.

Estimating the total number of nucleotide substitutions since the common anchestor of a pair of homologous genes: Comparison of several methods and three beta hemoglobin messenger RNA's

Estimating the total number of nucleotide substitutions since the common anchestor of a pair of homologous genes: Comparison of several methods and three beta hemoglobin messenger RNA's

Translation of globin messenger RNA by the mouse ovum.

It has been demonstrated that the Xenopus oocyte can translate rabbit haemoglobin messenger RNA (mRNA) following microinjection of the message into the cell. The Xenopus oocyte has since been shown to be capable of translating a variety of messenger RNAs from different species. This system has proved useful in un-erstanding the mechanism of message translation and has also provided information about the translation capability of the Xenopus oocyte. Several other cell types, including HeLa cells and fibroblasts, can also translate exogenous message injected into the cell. However, there have been no reports of injection of mRNA into oocytes or fertilised one-cell ova of mammalian species. Nevertheless, the latter system could be of considerable use in studying the processing of exogenous messages in a mammalian system undergoing development, as well as providing insight into the way the early embryo processes injected messages and the protein products of such messages. We report here the results of injecting message into the fertilised one-cell mouse ovum and show that both mouse and rabbit globin mRNA are translated in this system.

Optical studies on the structure of hemoglobin messenger ribonucleic acid

The secondary structure of hemoglobin mRNA (HbmRNA has been investigated by optical rotatory dispersion (ORD), circular dichroism (CD) and ultraviolet absorbance spectrophotometry. The dependence on temperature or reaction with formaldehyde of the CD and absorbance are characteristic of a structure with substantial base pairings at 20°C. The presence of Cotton effects in one of the major ultraviolet absorption bands indicates a highly ordered secondary structure. The UV hyperchromism on thermal denaturation is consistent with a value of 58% double helical content.

Nucleotide Sequence Analysis of Coding and Noncoding Regions of Human β-Globin mRNA

Publisher Summary This chapter explores that the analysis of human hemoglobin messenger RNA was undertaken to determine the exact nucleotide sequence of translated and untranslated regions. Knowledge of the translated sequences of globin mRNA has important implications for determination of specific codon usage by eitkaryotic cells and for measurements of the rate of mutational events when human and subhuman species are compared. The chapter reviews the sequence analysis studies that were carried out on human β-globin mRNA. Thalassemic, sickle-cell and Hb A mRNA served as substrates for the preparation of complementary DNA by RNA-dependent DNA polymcmse. cDNA was transcribed by E. coli RNA polymerase, and the resulting cRNA was analyzed. Additional sequence information was obtained by analysis of iodinated native a-thalassemic mRNA and by digestion of cDNA with restriction endonucleases. The chapter discusses that the data derived allow assignment of over 350 nucleotides to the coding region and 43 nucleotides to the noncoding region adjacent to the termination codon. There are extensive regions of homology between the translated regions of rabbit and human β-mRNA; a short homologous region can be demonstrated between the untranslated 3'-terminal sequence of human mRNA and other animal cell mRNAs.

Potentiation of hemoglobin messenger ribonucleic acid. A step in protein synthesis initiation involving interaction of messenger with 18 S ribosomal ribonucleic acid.

The polyadenylic acid-containing messenger ribonucleic acid from rabbit reticulocyte polyribosomes, isolated by a rapid and very gentle procedure (Krystosek, A., Cawthon, M. L., and Kabat, D. (1975) J. Biol. Chem. 250, 6077-6084), sediments in a sucrose gradient in three sharp peaks, at 9 S, 17 to 18 S, and 28 S. The alpha and beta globin messenger activity follows the absorbance profile in the sucrose gradients and has its major peak at 17 to 18 S. The larger messengers are more active than 9 S messenger by approximately 2-fold per mass unit of ribonucleic acid or by at least 8-fold per molecule. The major 17 to 18 S form of globin messenger was examined further and was shown to be a 1:1 complex of 9 S messenger and 18 S ribosomal ribonucleic acid. The effect of 18 S ribosomal ribonucleic acid on translation of purified 9 S globin messenger was analyzed in a messenger-dependent protein-synthesizing system (Krystosek, A., Cawthon, M. L., and Kabat, D. (1975) J. Biol. Chem. 250, 6077-6084). In the absence of exogenous ribosomal ribonucleic acid, 9 S messenger is inefficiently translated; a large excess of messenger is required to saturate the system; and globin is synthesized mainly on di- and monoribosomes. Exogenous liver or reticulocyte 18 S ribosomal ribonucleic acid potentiates 9 S messenger translation and renders it at least 10 times more efficient. The potentiation reaction can also be accomplished by increasing the concentration of ribosomes in the assay system. However, transfer or messenger ribonucleic acids cannot carry out this reaction. It is proposed that 9 S globin messenger ribonucleic acid is an inactive molecule which is normally potentiated by specific reversible base pairing with an accessible region of ribosomal ribonucleic acid contained in a 40 S ribosomal subunit. The potentiated messenger interacts with initiation factors and with other ribosomal subunits to synthesize protein. Potentiation is the first specific function in protein synthesis demonstrated for the ribosomal ribonucleic acid portion of ribosomes.

Translational response of an E. coli “permeabilized” cell system (PCS) to hemoglobin messenger RNA

Translational response of an E. coli “permeabilized” cell system (PCS) to hemoglobin messenger RNA

Gel Chromatographic Analysis of Nascent Globin Chains: EVIDENCE OF NONUNIFORM SIZE DISTRIBUTION

Abstract A gel chromatographic analytical procedure using Bio-Gel A-0.5m in 6 m guanidine HCl-0.1 m 2-mercaptoethanol has been developed and standardized. The procedure permits the molecular weight of peptides in the size range of 2 to 146 amino acids in length to be established from the distribution coefficient of the peptide on the gel column. This procedure has been applied to an analysis of the size distribution of nascent peptides from rabbit reticulocyte ribosomes. These studies indicate that the size distribution of nascent peptides is nonuniform and that translation of hemoglobin messenger RNA molecules does not proceed at a constant rate in the reticulocyte.

Polynucleotide sequences in eukaryotic DNA and RNA that form ribonuclease-resistant complexes with polyuridylic acid

When annealed with synthetic polynucleotides and treated with ribonuclease under appropriate conditions, poly(U) forms the ribonuclease-resistant complexes poly(rA) · poly(U) (1:1), poly(dA) · 2poly(U) (1:2) and poly · (dA)poly(dT) · poly(U) (1:1:1). This forms the basis of a quantitative assay of poly(rA), poly(dA) and poly(dA) · poly(dT) sequences in unlabelled nucleic acids. Using this assay, duck haemoglobin messenger RNA is shown to contain a poly(rA) sequence approximately 100 nucleotides long. Eukaryotic DNAs contain small amounts of sequences that react with poly(U). In the case of duck DNA, these sequences are considerably shorter than the mRNA-associated sequences and are interspersed widely with other sequences. It is concluded that if duck DNA does contain poly(dA) sequences corresponding to mRNA-associated poly(rA) sequences, there are fewer than 8000 of these per haploid genome.

The translation of rabbit hemoglobin messenger RNA in a cell-free extract from chick embryo brain

The translation of rabbit hemoglobin messenger RNA in an unfractionated cytoplasmic extract from chick embryo brain was studied. This translation was not dependent upon reticulocyte-specific factors. An analysis of the product synthesized in vitro with the embryo brain cell-free extract and rabbit hemoglobin messenger RNA by carboxymethyl cellulose chromatography showed that the system was capable of synthesizing both the α and β globin chains. Analysis of the tryptic peptides of the in vitro synthesized α chain by ion-exchange chromatography showed that the embryo brain extract with rabbit hemoglobin messenger RNA was capable of synthesizing the complete α chain of rabbit hemoglobin. The results suggest that no stringent tissue-specific controls exist for the translation of globin messenger RNA and were discussed in this context.

Evidence for template-specific sites in DNA polymerases

Using rabbit hemoglobin messenger RNA as template, E. coli polymerase I produces poly (dT), poly (dA)·(dT) and antimessenger DNA products. Mild heating of the enzyme causes a differential loss in activity as indicated by three rates of inactivation for the three types of synthesis. Heat inactivation studies have also been carried out with DNA polymerases from oncogenic RNA viruses and mammalian sources using various homopolymer-oligomer pairs as primertemplates. In general, for any given enzyme these synthetic primer-templates reveal different extents of inactivation of the polymerase. These findings may be interpreted to suggest a) that the binding of DNA polymerase to various primer-templates produces conformational changes in the enzyme which are dependent on the type of template bound, or b) that many, if not all, DNA polymerases have different subsites for different templates.

Reticulocyte RNA-dependent RNA polymerase.

A cytoplasmic, microsomal bound RNA-dependent RNA polymerase has been purified 2500-fold from rabbit reticulocyte lysates. The synthesis of RNA with the purified enzyme is absolutely dependent on the addition of an RNA template. The best template is hemoglobin messenger RNA, while bacteriophage RNA and poly(A,G) are less active, and DNA is completely inactive as a template. With poly(A,G) as a template, only UTP and CTP are incorporated into polynucleotide chains, indicating that the RNA polymerase is an RNA replicase and not a terminal transferase. With messenger RNA as a template, all four ribonucleoside triphosphates are required for maximal activity. The RNA-dependent RNA polymerase reaction is extremely sensitive to low concentrations of heme, rifamycin AF/013, and ribonuclease and resistant to actinomycin D and DNase. The discovery of RNA-directed RNA synthesis in reticulocytes offers an additional site for control of gene expression in mammalian cells and provides a possible mechanism for amplification of the expression of specific genes.

Protein synthesis in rabbit reticulocytes: Evidence for the synthesis of initial dipeptides in the presence of pactamycin

The effects of pactamycin on peptide chain initiation were studied using a reconstituted system comprised of reticulocyte ribosomes, poly r(A-U-G) messenger and peptide chain initiation factors, and the transfer of methionine from precharged Met-tRNA f Met into di-, oligo- and polymethionine products was measured. In the presence of low concentrations of pactamycin (10 −6M), polymethionine synthesis in the above system was markedly reduced, and a concomitant increase in the synthesis of met-met and met-val dipeptides was observed. The latter dipeptide was presumably synthesized in response to endongenous hemoglobin messenger. Aurintricarboxylic acid and sparsomycin also inhibited polymethionine synthesis in response to poly r(A-U-G) messenger. Whereas aurintricarboxylic acid inhibited pactamycin induced dipeptide synthesis, sparsomycin had no significant effect on dipeptide synthesis in the presence of pactamycin.

Hemoglobin messenger RNA from human bone marrow. Isolation and translation in homozygous and heterozygous beta-thalassemia.

A method for isolating human hemoglobin messenger RNA (mRNA) from bone marrow cells was developed to investigate the molecular basis for the defect in globin synthesis in beta thalassemia. Active mRNA was isolated from the bone marrow cells and peripheral reticulocytes of patients with homozygous beta thalassemia, heterozygous beta thalassemia, sickle cell trait, double heterozygosity for beta thalassemia and sickle cell trait, as well as from a patient with normal hemoglobin synthesis but with an elevated reticulocyte count secondary to hereditary spherocytosis. The mRNA was prepared for assay in an mRNA-dependent rabbit reticulocyte cell-free system and the amount of alpha and beta globin chains synthesized was determined by carboxymethylcellulose column chromatography. The relative synthesis of alpha to beta chains in response to normal hemoglobin mRNA was found to be a function of the amount of mRNA added to the assay system: increasing the amount of mRNA led to a decrease in the alpha-to-beta-chain synthetic ratio. Therefore, assays were carried out at limiting concentrations of mRNA. The molecular defect in homozygous beta thalassemia was shown to be carried in the mRNA of bone marrow cells as well as in the mRNA from peripheral reticulocytes, because much less beta than alpha globin was produced in the cell-free system in response to mRNA from either type of cell. In patients doubly heterozygous for beta thalassemia and sickle cell trait, little or no synthesis of beta(A) globin occurred in the bone marrow cells or the peripheral reticulocytes. The alpha to beta(S) synthetic ratio of the intact bone marrow cells was approximately 1, while the same ratio in the peripheral reticulocytes was between 1.5 and 2. The virtual absence of translatable beta globin mRNA in the mRNA prepared from the cells of these doubly heterozygous patients further demonstrates that the molecular defect produced by the beta thalassemia gene is in the beta globin mRNA.

Haemoglobin messenger RNA in spleen of anaemic rabbits

Haemoglobin meseenger RNA was isolated from spleen erythroïd cells of anaemic rabbits. The mRNA fraction looks quite homogeneous when analyzed by sucrose gradient centrifugation; it sediments in the 9S region. When added to an ascites cell free system, spleen 9S RNA stimulates the incorporation of radioactive leucine into protein. The synthesized product has been characterized and identified as α and β chains of rabbit globin.

Haemoglobin Messenger in the Post-ribosomal Supernatant of Reticulocytes

both give rise to two additional fragments six and seven bases long with the smaller fragment existing in twice the quantity of the larger one. The individual peaks appear on the chromatograph in the same position if re-run on DEAE-Sephadex or after a second digestion with pancreatic RNAase. This finding indicates that each fragment from the RNAase TI digestion is composed of two different oligonucleotides having five and six adenylate residues respectively, and further that there is a total of four terminal sequences in the globin mRNA. Although the pancreatic RNAase used in these experiments did not degrade poly(A) or oligo(A) under the conditions of the digestion (low salt concentration) it is known that the aggregate form of the enzyme does have activity towards poly(A) at low ionic strength. Therefore the pancreatic RNAase digestions of the fragments from the RNAase TI digestion were repeated at high salt concentrations, namely 0.1 M-NaCI, 20m~-Tris-HCl buffer, pH7.0, and ~ ~ M E D T A , at 37°C for 1 h. The results were identical, indicating that the maximum length of pure poly(A) at the 3’-hydroxyl terminus is five or six bases. The proposed sequences are summarized in Table 1. The terminally labelled RNA binds to a poly(U)-cellulose column and is eluted, along with the biological activity, between 45“ and 65”C, suggesting that there may be a longer poly(A) or A-rich region in the rabbit globin mRNA, as has been shown for mouse globin mRNA (Lim & Cannellakis, 1970) and duck globin mRNA (Pemberton & Baglioni, 1972). These results suggest that a long sequence of pure poly(A) does not occur at the 3’hydroxyl terminus of the globin mRNA but does not exclude the possibility that these adenylate residues are part of a longer adenylate-rich region.

Microinjection of rabbit hemoglobin messenger RNA into amphibian oocytes and embryos.

After microinjection of hemoglobin mRNA from rabbit reticulocytes (9S mRNA) into Xenopus laevis oocytes, the cytological structure remains normal. The peroxidase reaction does not show an accumulation of synthesized hemoglobin in the germinal vesicle. Fertilized Pleurodeles Waltlii eggs synthesize rabbit hemoglobin after injection of 9S mRNA; hemoglobin synthesis decreases as development progresses. Micromeres and macromeres synthesize equal amounts of hemoglobin, despite the fact that the micromeres contain many more ribosomes. Descendants of injected blastomeres divide more slowly and do not differentiate; they ultimately undergo cytolysis.

Globin messenger sequences in high molecular weight RNA from embryonic mouse liver.

Heterogeneous high molecular weight RNA from mouse erythroblasts is shown conclusively to contain haemoglobin messenger sequences by injection into Xenopus oocytes. This gives strong support to the idea that this RNA is a precursor of messenger RNA.