Antisense globin RNA in mouse erythroid tissues: structure, origin, and possible function.

The aim of the experiments described in this paper was to test for the presence of antisense globin RNA in mouse erythroid tissues and, if found, to characterize these molecules. The present study made use of a multistep procedure in which a molecular tag is attached to cellular RNA by ligation with a defined ribooligonucleotide. The act of ligation preserves the termini of RNA molecules, which become the junctions between cellular RNAs and the ligated ribooligonucleotide. It also unambiguously preserves the identity of cellular RNA as a sense or antisense molecule through all subsequent manipulations. Using this approach, we identified and characterized antisense beta-globin RNA in erythroid spleen cells and reticulocytes from anemic mice. We show in this paper that the antisense globin RNA is fully complementary to spliced globin mRNA, indicative of the template/transcript relationship. It terminates at the 5' end with a uridylate stretch, reflecting the presence of poly(A) at the 3' end of the sense globin mRNA. With respect to the structure of their 3' termini, antisense globin RNA can be divided into three categories: full-size molecules corresponding precisely to globin mRNA, truncated molecules lacking predominantly 14 3'-terminal nucleotides, and extended antisense RNA containing 17 additional 3'-terminal nucleotides. The full-size antisense globin RNA contains two 14-nt-long complementary sequences within its 3'-terminal segment corresponding to the 5'-untranslated region of globin mRNA. This, together with the nature of the predominant truncation, suggests a mechanism by which antisense RNA might give rise to new sense-strand globin mRNA.

Arachidonic and docosahexaenoic acids are biosynthesized from their 18-carbon precursors in human infants.

It is becoming clear that an adequate level of long-chain highly unsaturated fatty acids in the nervous system is required for optimal function and development; however, the ability of infants to biosynthesize long-chain fatty acids is unknown. This study explores the capacity of human infants to convert 18-carbon essential fatty acids to their elongated and desaturated forms, in vivo. A newly developed gas chromatography/negative chemical ionization/mass spectrometry method employing 2H-labeled essential fatty acids allowed assessment of this in vivo conversion with very high sensitivity and selectivity. Our results demonstrate that human infants have the capacity to convert dietary essential fatty acids administered enterally as 2H-labeled ethyl esters to their longer-chain derivatives, transport them to plasma, and incorporate them into membrane lipids. The in vivo conversion of linoleic acid (18:2n6) to arachidonic acid (20:4n6) is demonstrated in human beings. All elongases/desaturases necessary for the conversion of linolenic acid (18:3n3) to docosahexaenoic acid (22:6n3) are also active in the first week after birth. Although the absolute amounts of n-3 fatty acid metabolites accumulated in plasma are greater than those of the n-6 family, estimates of the endogenous pools of 18:2n6 and 18:3n3 indicate that n-6 fatty acid conversion rates are greater than those of the n-3 family. While these data clearly demonstrate the capability of infants to biosynthesize 22:6n3, a lipid that is required for optimal neural development, the amounts produced in vivo from 18:3n3 may be inadequate to support the 22:6n3 level observed in breast-fed infants.

Parvovirus B19 promoter at map unit 6 confers autonomous replication competence and erythroid specificity to adeno-associated virus 2 in primary human hematopoietic progenitor cells.

The pathogenic human parvovirus B19 is an autonomously replicating virus with a remarkable tropism for human erythroid progenitor cells. Although the target cell specificity for B19 infection has been suggested to be mediated by the erythrocyte P-antigen receptor (globoside), a number of nonerythroid cells that express this receptor are nonpermissive for B19 replication. To directly test the role of expression from the B19 promoter at map unit 6 (B19p6) in the erythroid cell specificity of B19, we constructed a recombinant adeno-associated virus 2 (AAV), in which the authentic AAV promoter at map unit 5 (AAVp5) was replaced by the B19p6 promoter. Although the wild-type (wt) AAV requires a helper virus for its optimal replication, we hypothesized that inserting the B19p6 promoter in a recombinant AAV would permit autonomous viral replication, but only in erythroid progenitor cells. In this report, we provide evidence that the B19p6 promoter is necessary and sufficient to impart autonomous replication competence and erythroid specificity to AAV in primary human hematopoietic progenitor cells. Thus, expression from the B19p6 promoter plays an important role in post-P-antigen receptor erythroid-cell specificity of parvovirus B19. The AAV-B19 hybrid vector system may also prove to be useful in potential gene therapy of human hemoglobinopathies.

Neurotrophin 3 rescues neuronal precursors from apoptosis and promotes neuronal differentiation in the embryonic metanephric kidney.

We analyzed the developmental regulation and role of the neurotrophins during metanephric kidney morphogenesis. RNase protection assay revealed the presence of nerve growth factor, neurotrophin 3 (NT-3), and brain-derived neurotrophic factor mRNAs and the regulation of their expression during embryonic development of rat metanephros. NT-3 induced differentiation (neurite outgrowth) and survival (inhibition of apoptosis) of the neuronal precursors in cultured nephrogenic mesenchymes and neuronal differentiation in cultured whole kidneys, whereas NT-4/5, brain-derived neurotrophic factor, and nerve growth factor were without effect. The neurotrophins did not trigger tubular differentiation of isolated nephrogenic cells, which underwent apoptosis when cultured with or without the neurotrophins. NT-3 is thus an inducer of differentiation and a survival factor for renal neuronal cells, but none of the neurotrophins is a morphogen in kidney tubule induction.

Lineage specification of neuronal precursors in the mouse spinal cord.

We have investigated the differentiation potential of precursor cells within the developing spinal cord of mice and have shown that spinal cord cells from embryonic day 10 specifically give rise to neurons when plated onto an astrocytic monolayer, Ast-1. These neurons had the morphology of motor neurons and > 83% expressed the motor neuron markers choline acetyltransferase, peripherin, calcitonin gene-related peptide, and L-14. By comparison, < 10% of the neurons arising on monolayers of other neural cell lines or 3T3 fibroblasts had motor neuron characteristics. Cells derived from dorsal, intermediate, and ventral regions of the spinal cord all behaved similarly and gave rise to motor neuron-like cells when plated onto Ast-1. By using cells that expressed the lacZ reporter gene, it was shown that > 93% of cells present on the Ast-1 monolayers were motor neuron-like. Time-lapse analysis revealed that the precursors on the Ast-1 monolayers gave rise to neurons either directly or following a single cell division. Together, these results indicate that precursors in the murine spinal cord can be induced to differentiate into the motor neuron phenotype by factors produced by Ast-1 cells, suggesting that a similar factor(s) produced by cells akin to Ast-1 may regulate motor neuron differentiation in vivo.

Association of erythroid transcription factors: complexes involving the LIM protein RBTN2 and the zinc-finger protein GATA1.

The RBTN2 LIM-domain protein, originally identified as an oncogenic protein in human T-cell leukemia, is essential for erythropoiesis. A possible role for RBTN2 in transcription during erythropoiesis has been investigated. Direct interaction of the RBTN2 protein was observed in vivo and in vitro with the GATA1 or -2 zinc-finger transcription factors, as well as with the basic helix-loop-helix protein TAL1. By using mammalian two-hybrid analysis, complexes involving RBTN2, TAL1, and GATA1, together with E47, the basic helix-loop-helix heterodimerization partner of TAL1, could be demonstrated. Thus, a molecular link exists between three proteins crucial for erythropoiesis, and the data suggest that variations in amounts of complexes involving RBTN2, TAL1, and GATA1 could be important for erythroid differentiation.

Transcriptional activation of human adult alpha-globin genes by hypersensitive site-40 enhancer: function of nuclear factor-binding motifs occupied in erythroid cells.

The developmental stage- and erythroid lineage-specific activation of the human embryonic zeta- and fetal/adult alpha-globin genes is controlled by an upstream regulatory element [hypersensitive site (HS)-40] with locus control region properties, a process mediated by multiple nuclear factor-DNA complexes. In vitro DNase I protection experiments of the two G+C-rich, adult alpha-globin promoters have revealed a number of binding sites for nuclear factors that are common to HeLa and K-562 extracts. However, genomic footprinting analysis has demonstrated that only a subset of these sites, clustered between -130 and +1, is occupied in an erythroid tissue-specific manner. The function of these in vivo-occupied motifs of the alpha-globin promoters, as well as those previously mapped in the HS-40 region, is assayed by site-directed mutagenesis and transient expression in embryonic/fetal erythroid K-562 cells. These studies, together with our expression data on the human embryonic zeta-globin promoter, provide a comprehensive view of the functional roles of individual nuclear factor-DNA complexes in the final stages of transcriptional activation of the human alpha-like globin promoters by the HS-40 element.

Modulation of retinoblastoma gene in normal adult hematopoiesis: peak expression and functional role in advanced erythroid differentiation.

The retinoblastoma (RB) gene specifies a nuclear phosphoprotein (pRb 105), which is a prototype tumor suppressor inactivated in a variety of human tumors. Recent studies suggest that RB is also involved in embryonic development of murine central nervous and hematopoietic systems. We have investigated RB expression and function in human adult hematopoiesis--i.e., in liquid suspension culture of purified quiescent hematopoietic progenitor cells (HPCs) induced by growth factor stimulus to proliferation and unilinage differentiation/maturation through the erythroid or granulocytic lineage. In the initial HPC differentiation stages, the RB gene is gradually induced at the mRNA and protein level in both erythroid and granulopoietic cultures. In late HPC differentiation and then precursor maturation, RB gene expression is sustained in the erythroid lineage, whereas it is sharply downmodulated in the granulocytic series. Functional studies were performed by treatment of HPC differentiation culture with phosphorothioate antisense oligomer targeting Rb mRNA; coherent with the expression pattern, oligomer treatment of late HPCs causes a dose-dependent and selective inhibition of erythroid colony formation. These observations suggest that the RB gene plays an erythroid- and stage-specific functional role in normal adult hematopoiesis, particularly at the level of late erythroid HPCs.

Regulated production of a pleiotropic cytokine-platelet-derived growth factor--by differentiating erythroid cells in vitro and in vivo.

Erythroid progenitor growth in vitro is stimulated by exogenous platelet-derived growth factor (PDGF). We now report that both normal and transformed erythroid progenitor cells produce authentic PDGF in vitro and in vivo. Importantly, this production is highly regulated during erythropoiesis. Addition of soluble lysates from Rauscher murine erythroleukemia cells--an erythropoietin-responsive model progenitor cell line--to quiescent BALB/c 3T3 fibroblasts resulted in a mitogenic response identical to that observed with the addition of authentic recombinant PDGF. Polyclonal and monoclonal anti-PDGF antibodies immunoabsorbed 50-100% of this activity. Induction of Rauscher cell differentiation in vitro with dimethyl sulfoxide or erythropoietin for 48-72 hr markedly upregulated PDGF production by 17- to 18-fold and 14- to 38-fold, respectively. Importantly, stimulation of normal erythropoiesis in vivo in mice treated either with phenylhydrazine or with erythropoietin increased PDGF levels in the spleen by 11- to 48-fold and 20- to 34-fold, respectively. These results strongly suggest a role for erythroid cell-derived PDGF in normal erythropoiesis and provide documentation of the regulated production of a pleiotropic cytokine by erythroid cells.

Induction of ubiquitin-conjugating enzymes during terminal erythroid differentiation.

A global cellular reorganization occurs during the reticulocyte stage of erythroid differentiation. This reorganization is accomplished partly through programmed protein degradation. The selection of proteins for degradation can be mediated by covalent attachment of ubiquitin. We have cloned cDNAs encoding two ubiquitin-conjugating (E2) enzymes, E2-20K and E2-230K, and found their genes to be strongly induced during the differentiation of erythroblasts into reticulocytes. Induction of the E2-20K and E2-230K genes is specific, as transcript levels for at least two other ubiquitinating enzymes fall during erythroblast differentiation. In contrast to most proteins induced in reticulocytes, E2-20K and E2-230K enzymes are present at strongly reduced levels in erythrocytes and thus decline in abundance as reticulocyte maturation is completed. This result suggests that both enzymes function during the reticulocyte stage, when enhanced protein degradation has been observed. These data implicate regulated components of the ubiquitin conjugation machinery in erythroid differentiation.