scholarly journals Messenger RNA processing and nuclear structure: isolation of nuclear ribonucleoprotein particles containing beta-globin messenger RNA precursors.

1980 ◽  
Vol 87 (1) ◽  
pp. 47-54 ◽  
Author(s):  
T Pederson ◽  
N G Davis
Keyword(s):  
Nuclear Structure ◽  
Messenger Rna ◽  
Globin Gene ◽  
Beta Globin ◽  
Globin Mrna ◽  
Beta Globin Gene ◽  
Ribonucleoprotein Particles ◽  
Particle Form ◽  
Nuclear Rna ◽  
Friend Cells

To explore the relationships between transcription, messenger RNA (mRNA) processing, and nuclear structure, ribonucleoprotein particles containing heterogeneous nuclear RNA (hnRNP) have been purified from globin-producing mouse Friend erythroleukemia cells. These nuclear hnRNP particles sediment at 50S-200S and contain, in addition to high molecular weight hnRNA, a specific set of nuclear proteins predominated by a major component of approximately 38,000 mol wt. The hnRNP particles are free of histones and ribosomal structural proteins, indicating their purification from the two other major nucleoprotein components of the nucleus: chromatin and nucleolar ribosomal precursor RNP particles. Th authenticity of the Friend cell hnRNP particles is demonstrated by the results of reconstruction experiments with deproteinized hnRNA, and by the resistance of the articles to dissociation during isopycnic banding in Cs2SO4 gradients without prior aldehyde fixation. Hybridization analysis with cloned mouse beta-globin DNA demonstrates that hnRNP particles from induced Friend cells contain newly synthesized transcripts of the beta-globin gene. Agarose gel electrophoresis of hnRNP particle-derived RNA denatured in glyoxal followed by "Northern" transfer to diazobenzyloxymethyl paper and hybridization with 32P-labeled cloned mouse beta-globin DNA reveals the presence in hnRNP of two size classes of beta-globin gene transcripts, the larger of which corresponds to the pre-spliced 15S beta-globin mRNA precursor previously identified in whole nuclear RNA, and the smaller of which corresponds to completely processed 9S beta-globin mRNA. These results establish, for the first time, that the nuclear transcripts of a specific, well-defined eukaryotic structural gene can be isolated in an RNP particle form, and that their RNP structure persists throughout mRNA splicing.

scholarly journals IGF2BP1 Reverses Hemoglobin Switching in Adult Erythroblasts

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 639-639
Author(s):  
Laxminath Tumburu ◽  
Colleen Byrnes ◽  
Y. Terry Lee ◽  
Jaira F. de Vasconcellos ◽  
Antoinette Rabel ◽  
...  
Keyword(s):  
Gene Expression ◽  
Adult Stage ◽  
Globin Gene ◽  
Expression Patterns ◽  
Beta Globin ◽  
Globin Mrna ◽  
Beta Globin Gene ◽  
Empty Vector ◽  
Cd34 Cells ◽  
Gene And Protein Expression

Abstract During human ontogeny, high-level transcription within the beta-globin gene cluster switches sequentially from embryonic-to-fetal-to-adult genes. Beta-thalassemias and sickle-cell disease are manifested by reduced or mutated expression of the adult-stage, beta-globin gene. Research is aimed toward the eventual therapeutic goal of safely preventing or reversing the fetal-to-adult hemoglobin switch among these patient populations. To identify genes that may be involved in regulation of the fetal-to-adult erythroid switch, purified CD34(+) cells from six umbilical cord (fetal) and six adult peripheral blood samples were cultured in serum-free medium, and gene expression libraries were prepared and sequenced from CD71(+), CD235a(+) erythroblast mRNA. In total, 546 million paired-end reads with a length of 101bp were generated for a comparison of cord and adult erythroblast transcriptomes. Reads were aligned to the human reference genome (hg19), and differential gene expression was identified [false discovery rate ≤ 0.05, fold change ≥ 1.5, and reads per kilobase per million mapped reads (RPKM) ≥ 0.5]. A total of 145 genes were differentially expressed according to these criteria, with four of the top five encoding targets of the let-7 family of microRNAs. The topmost gene was insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1), which is normally involved in transcriptome regulation and developmental timing. IGF2BP1 expression was 770-fold increased in the fetal erythroblasts (RPKM > 3.0) compared with low background levels in adult erythroblasts (RPKM < 0.01). IGF2BP1 protein is present in fetal tissues including fetal liver; however, it is not detected in adult human bone marrow. A potential role for adult-stage IGF2BP1 over-expression (IGF2BP1-OE) in the regulation of globin genes and proteins was explored using lentiviral vectors designed for let-7 resistant, erythroid-specific expression of IGF2BP1 protein. IGF2BP1-OE transduced CD34(+) cells expressed the transgenic protein and maintained their ability to differentiate, accumulate hemoglobin, and enucleate ex vivo in the presence of erythropoietin. Globin mRNA and protein levels were investigated. While alpha-globin mRNA remained unchanged, gamma-globin mRNA became predominant [90% of (gamma + beta) mRNA] in IGF2BP1-OE samples [Control (empty vector) = 3.2E+06 ± 8.2E+05 copies/ng; IGF2BP1-OE = 2.0E+07 ± 5.9E+06 copies/ng; p < 0.05], and beta-globin mRNA decreased to minor levels [Control (empty vector) = 2.2E+07 ± 4.0E+06 copies/ng; IGF2BP1-OE = 2.2E+06 ± 6.2E+05 copies/ng; p < 0.05]. IGF2BP1-OE caused a pan-cellular HbF distribution by flow cytometry. Cellular fetal hemoglobin percentages [HbF/(HbF + HbA)] were measured as 5.3 ± 0.4% in donor matched control cells versus 80.3 ± 3.7% in IGF2BP1-OE cells (p < 0.05). HPLC tracings revealed that the minor HbA2 peak, composed of alpha and delta globin chains, was reduced or absent in IGF2BP1-OE. Also, IGF2BP1-OE suppressed the expression of related genes including the transcription factor BCL11A. These data demonstrate that erythroblast IGF2BP1 is silenced in humans during fetal-to-adult ontogeny, and that IGF2BP1 in adult erythroblasts reverses the developmentally related switch in beta-like globin gene and protein expression patterns. Disclosures No relevant conflicts of interest to declare.

scholarly journals A new mutation in IVS-1 of the human beta globin gene causing beta thalassemia due to abnormal splicing

Blood ◽  
1987 ◽  
Vol 70 (1) ◽  
pp. 147-151
Author(s):  
GF Atweh ◽  
C Wong ◽  
R Reed ◽  
SE Antonarakis ◽  
D Zhu ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Globin Gene ◽  
Beta Thalassemia ◽  
Normal Donor ◽  
Beta Globin ◽  
Anglo Saxon ◽  
Beta Globin Gene ◽  
A Cell ◽  
Partial Inactivation ◽  
Cryptic Splice Sites

A G to T transversion at the fifth nucleotide of the first intervening sequence (IVS-1) of the beta-globin gene has been identified in cloned beta-thalassemia genes of two unrelated individuals, one of Mediterranean and the other of Anglo Saxon ancestry. In each patient the mutation was present in a different beta globin gene framework, defined by intragenic restriction site polymorphisms, thereby suggesting the occurrence of independent mutations. The study of the RNA products of one of these cloned genes, after transfer and transient expression in HeLa cells, showed partial inactivation of the normal donor splice site of IVS-1 and activation of two major and one minor cryptic splice sites. Only one of the two major cryptic sites was utilized in a cell-free splicing extract. The effects of this mutation on messenger RNA (mRNA) splicing are similar to that of another beta thalassemia gene with a G to C transition at the same position.

Expression of the human beta-globin gene after retroviral transfer into murine erythroleukemia cells and human BFU-E cells

1988 ◽  
Vol 8 (4) ◽  
pp. 1725-1735
Author(s):  
M A Bender ◽  
A D Miller ◽  
R E Gelinas
Keyword(s):  
Globin Gene ◽  
Globin Genes ◽  
Beta Globin ◽  
Globin Mrna ◽  
Beta Globin Gene ◽  
Erythroleukemia Cells ◽  
New Gene ◽  
Normal Human ◽  
Murine Erythroleukemia ◽  
Murine Erythroleukemia Cells

Replication-defective amphotropic retrovirus vectors containing either the human beta-globin gene with introns or an intronless beta-globin minigene were constructed and used to study beta-globin expression following gene transfer into hematopoietic cells. The beta-globin genes were marked by introducing a 6-base-pair insertion into the region corresponding to the 5' untranslated region of the beta-globin mRNA to allow detection of RNA encoded by the new gene in human cells expressing normal human beta-globin RNA. Introduction of a virus containing the beta-globin gene with introns into murine erythroleukemia cells resulted in inducible expression of human beta-globin RNA and protein, while the viruses containing the minigene were inactive. The introduced human beta-globin gene was 6 to 110% as active as the endogenous mouse beta maj-globin genes in six randomly chosen cell clones. Introduction of the viruses into human BFU-E cells, followed by analysis of marked and unmarked globin RNAs in differentiated erythroid colonies, revealed that the introduced beta-globin gene was about 5% as active as the endogenous genes in these normal human erythroid cells and that again the minigene was inactive. These data are discussed in terms of the potential treatment of genetic disease by gene therapy.

scholarly journals Fetal hemoglobin silencing in humans

Blood ◽  
2006 ◽  
Vol 108 (6) ◽  
pp. 2081-2086 ◽  
Author(s):  
Patricia A. Oneal ◽  
Nicole M. Gantt ◽  
Joseph D. Schwartz ◽  
Natarajan V. Bhanu ◽  
Y. Terry Lee ◽  
...  
Keyword(s):  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Beta Thalassemia ◽  
Postnatal Life ◽  
Globin Genes ◽  
Beta Globin ◽  
Globin Mrna ◽  
Beta Globin Gene ◽  
Gamma Globin ◽  
Age Related

Abstract Interruption of the normal fetal-to-adult transition of hemoglobin expression should largely ameliorate sickle cell and beta-thalassemia syndromes. Achievement of this clinical goal requires a robust understanding of gamma-globin gene and protein silencing during human development. For this purpose, age-related changes in globin phenotypes of circulating human erythroid cells were examined from 5 umbilical cords, 99 infants, and 5 adult donors. Unexpectedly, an average of 95% of the cord blood erythrocytes and reticulocytes expressed HbA and the adult beta-globin gene, as well as HbF and the gamma-globin genes. The distribution of hemoglobin and globin gene expression then changed abruptly due to the expansion of cells lacking HbF or gamma-globin mRNA (silenced cells). In adult reticulocytes, less than 5% expressed gamma-globin mRNA. These data are consistent with a “switching” model in humans that initially results largely from gamma- and beta-globin gene coexpression and competition during fetal development. In contrast, early postnatal life is marked by the rapid accumulation of cells that possess undetectable gamma-globin mRNA and HbF. The silencing phenomenon is mediated by a mechanism of cellular replacement. This novel silencing pattern may be important for the development of HbF-enhancing therapies.

scholarly journals Two novel beta-thalassemia mutations in the 5' and 3' noncoding regions of the beta-globin gene [see comments]

Blood ◽  
1992 ◽  
Vol 79 (5) ◽  
pp. 1342-1346 ◽  
Author(s):  
SP Cai ◽  
B Eng ◽  
WH Francombe ◽  
NF Olivieri ◽  
AG Kendall ◽  
...  
Keyword(s):  
Premature Termination ◽  
Globin Gene ◽  
Polyadenylation Signal ◽  
Beta Thalassemia ◽  
Beta Globin ◽  
Globin Mrna ◽  
Noncoding Regions ◽  
Beta Globin Gene ◽  
Italian Family ◽  
Irish Family

Abstract Two novel beta-thalassemia mutations are described. The first mutation, found in an Italian family, is a G----A substitution in nucleotide (nt) +22 relative to the beta-globin gene Cap site. This mutation creates a cryptic ATG initiation codon, the utilization of which for translation would result in premature termination 36 bp 3′ downstream. The second mutation, found in an Irish family, is a T----C substitution in nt +1570, or 12 bp 5′ upstream of the AATAAA polyadenylation signal in the 3′ noncoding region. It is postulated that this mutation leads to destabilization of the encoded beta-globin mRNA.

scholarly journals Expression of the human beta-globin gene after retroviral transfer into murine erythroleukemia cells and human BFU-E cells.

1988 ◽  
Vol 8 (4) ◽  
pp. 1725-1735 ◽  
Author(s):  
M A Bender ◽  
A D Miller ◽  
R E Gelinas
Keyword(s):  
Globin Gene ◽  
Globin Genes ◽  
Beta Globin ◽  
Globin Mrna ◽  
Beta Globin Gene ◽  
Erythroleukemia Cells ◽  
New Gene ◽  
Normal Human ◽  
Murine Erythroleukemia ◽  
Murine Erythroleukemia Cells

Replication-defective amphotropic retrovirus vectors containing either the human beta-globin gene with introns or an intronless beta-globin minigene were constructed and used to study beta-globin expression following gene transfer into hematopoietic cells. The beta-globin genes were marked by introducing a 6-base-pair insertion into the region corresponding to the 5' untranslated region of the beta-globin mRNA to allow detection of RNA encoded by the new gene in human cells expressing normal human beta-globin RNA. Introduction of a virus containing the beta-globin gene with introns into murine erythroleukemia cells resulted in inducible expression of human beta-globin RNA and protein, while the viruses containing the minigene were inactive. The introduced human beta-globin gene was 6 to 110% as active as the endogenous mouse beta maj-globin genes in six randomly chosen cell clones. Introduction of the viruses into human BFU-E cells, followed by analysis of marked and unmarked globin RNAs in differentiated erythroid colonies, revealed that the introduced beta-globin gene was about 5% as active as the endogenous genes in these normal human erythroid cells and that again the minigene was inactive. These data are discussed in terms of the potential treatment of genetic disease by gene therapy.

Differential expression of alpha- and beta-globin genes in erythroleukemic cell lines

1990 ◽  
Vol 10 (7) ◽  
pp. 3591-3595
Author(s):  
N Beru ◽  
P B Maples ◽  
O Hermine ◽  
E Goldwasser
Keyword(s):  
Cell Lines ◽  
Globin Gene ◽  
Restriction Enzymes ◽  
Erythroid Differentiation ◽  
Globin Genes ◽  
Beta Globin ◽  
Globin Mrna ◽  
Beta Globin Gene ◽  
Alpha Globin ◽  
Erythroleukemic Cell

The IW32, NN10, and IW201 cell lines are erythroleukemic cell lines isolated from the spleens of mice infected with the Friend virus. IW32 and NN10 cells can be induced toward erythroid differentiation and hemoglobin synthesis by hemin or butyrate. Both cell lines contain some mature alpha- and beta-globin mRNA before induction, and addition of the inducers greatly increases the amount of globin message. Unlike IW32 and NN10 cells, IW201 cells are only partially inducible. Uninduced 201 cells contain a small amount of alpha-globin mRNA but no detectable beta-globin message. After induction, the cells contain markedly increased amounts of alpha-globin mRNA but still do not express the beta-globin gene. Southern blot analysis with 10 restriction enzymes shows that the restriction map of the beta-globin gene in IW201 cells is indistinguishable from that in IW32 and NN10 cells.

scholarly journals Two novel beta-thalassemia mutations in the 5' and 3' noncoding regions of the beta-globin gene [see comments]

Blood ◽  
1992 ◽  
Vol 79 (5) ◽  
pp. 1342-1346 ◽  
Author(s):  
SP Cai ◽  
B Eng ◽  
WH Francombe ◽  
NF Olivieri ◽  
AG Kendall ◽  
...  
Keyword(s):  
Premature Termination ◽  
Globin Gene ◽  
Polyadenylation Signal ◽  
Beta Thalassemia ◽  
Beta Globin ◽  
Globin Mrna ◽  
Noncoding Regions ◽  
Beta Globin Gene ◽  
Italian Family ◽  
Irish Family

Two novel beta-thalassemia mutations are described. The first mutation, found in an Italian family, is a G----A substitution in nucleotide (nt) +22 relative to the beta-globin gene Cap site. This mutation creates a cryptic ATG initiation codon, the utilization of which for translation would result in premature termination 36 bp 3′ downstream. The second mutation, found in an Irish family, is a T----C substitution in nt +1570, or 12 bp 5′ upstream of the AATAAA polyadenylation signal in the 3′ noncoding region. It is postulated that this mutation leads to destabilization of the encoded beta-globin mRNA.

Sign in / Sign up

Export Citation Format

Share Document