Translation of IGBP1 mRNA Contributes to the Regulation of Expansion and Differentiation of Erythroid Progenitors.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 72-72
Author(s):  
Godfrey Grech ◽  
Montserrat Blazquez-Domingo ◽  
Andrea Kolbus ◽  
Hartmut Beug ◽  
Bob Lowenberg ◽  
...  
Keyword(s):  
Gene Expression ◽  
Signaling Pathways ◽  
Binding Protein ◽  
Expression Profiles ◽  
Constitutive Expression ◽  
Mrna Translation ◽  
Erythroid Differentiation ◽  
Scaffolding Protein ◽  
Initiation Factor ◽  
Erythroid Progenitors

Abstract Erythroid progenitors can be expanded in vitro in the presence of erythropoietin (Epo), stem cell factor (SCF) and dexamethasone, while they differentiate to enucleated erythrocytes in presence of Epo only. Our study aims to identify (i) signaling pathways that control expansion of erythroid progenitors and (i) genes regulated by these signaling pathways. Since (i) SCF strongly activates phosphotidylinositol 3 kinase (PI3K) and (ii) inhibition of PI3K with LY294002 induces terminal differentiation of erythroid progenitors under Epo and SCF stimulation, SCF seems to enhance renewal divisions of erythroid progenitors via a PI3K-dependent mechanism. An important PI3K-dependent process in cell proliferation is regulation of mRNA translation. PI3K controls the activity of mTOR (mammalian target of rapamycin), whose activation results in phosphorylation of eIF4E (eukaryote Initiation Factor 4E)-binding protein (4E-BP). Fully phosphorylated 4E-BP releases eIF4E, which can subsequently bind eIF4G, the scaffolding protein of the eIF4F cap-binding and scanning complex. In particular mRNAs with a structured UTR (untranslated region) require optimal availability of eIF4E to be translated. SCF, but not Epo can induce full phosphorylation of 4E-BP and efficient formation of the eIF4F cap-binding complex. Overexpression of eIF4E inhibited erythroid differentiation as if SCF were present, indicating that SCF-induced release of eIF4E from 4E-BP is an important mechanism regulating the balance between progenitor expansion and differentiation. A major step in mRNA translation controlled by eIF4F is polysome recruitment. To identify genes whose expression is regulated by signaling-induced polysome recruitment, we compared total and polysome-bound mRNA from factor deprived and Epo-, SCF- or Epo plus SCF restimulated progenitors on gene-expression micro-arrays (Affymetrix). The profiling was performed with 4 biological replicates and candidate genes were selected using ANOVA. In subsequent cluster analysis we combined these data with (polysomal) expression profiles of differentiating erythroid cells. Thus we identified a cluster containing genes, upregulated in part or completely at the level of mRNA polysome recruitment and downregulated during erythroid differentiation. Targets involved in signal transduction and gene expression were analyzed in more detail. Polysome recruitment of 15/17 targets tested so far appeared to be dependent on PI3K activation and eIF4E expression. Constitutive expression of these targets in erythroid progenitors revealed that one target in particular was able to inhibit erythroid differentiation comparable to overexpression of eIF4E. This target was IGBP1 (Immunoglobulin binding protein 1). IGBP1 binds to and modulates the activity of the catalytic subunit of PP2A, a major cellular serine/threonine phosphatase, which also dephosphorylates 4E-BP. Overexpression of IGBP1 does not inhibit 4E-BP dephosphorylation in absence of factor, but enhances phosphorylation of 4E-BP in presence of Epo. Nevertheless, constitutive expression of IGBP1 does not increase polysome association of structured mRNAs. The multiple functions of PP2A suggest that the potent inhibition of erythroid differentiation by IGBP1 may be due to deregulation of several cellular mechanisms.

The Regulation of mRNA Translation Controls the Balance between Expansion and Differentiation of Erythroid Progenitors.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 462-462
Author(s):  
Godfrey Grech ◽  
Montserrat Balzquez-Domingo ◽  
Andreas Kolbus ◽  
Walbert Bakker ◽  
Helmut Dolznig ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Translation Initiation ◽  
Expression Profiles ◽  
Mrna Translation ◽  
Pi3k Inhibitor ◽  
Initiation Factor ◽  
Erythroid Progenitors ◽  
Pi3k Inhibitor Ly294002 ◽  
Gene Transcripts ◽  
Dependent Mechanism

Abstract Erythroid progenitors can be expanded in vitro in the presence of erythropoietin (Epo), stem cell factor (SCF) and dexamethasone, while they differentiate to enucleated erythrocytes in presence of Epo only. Our study aims to identify (i) signaling pathways that control expansion of erythroid progenitors and (ii) genes regulated by these signaling pathways. Since SCF strongly activates phosphotidylinositol 3 kinase (PI3K) and inhibition of PI3K with LY294002 induces terminal differentiation of erythroid progenitors under Epo and SCF stimulation, SCF seems to enhance renewal divisions of erythroid progenitors via a PI3K-dependent mechanism. An important PI3K-dependent mechanism is the regulation of cap-binding factor eIF4E (eukaryotic Initiation Factor 4E), a rate limiting step in translation initiation. eIF4E is sequestered by 4E-BP (4E-Binding Protein), but released when 4E-BP is phosphorylated by mTOR (mammalian target of rapamycin), a target of the PI3K/PKB pathway. We show that moderate overexpression of eIF4E (up to 2-fold) markedly delays Epo-induced differentiation and allows for an increased number of renewal divisions instead. Moreover, eIF4E overexpressing cells are unresponsive to the PI3K inhibitor LY294002 under progenitor expansion conditions. These data suggest that mRNAs with a structured untranslated region (UTR), that are only efficiently translated at optimal eIF4E levels, contribute to maintain proliferation of hematopoietic progenitors. To identify the genes whose expression is regulated by signaling-induced polysome recruitment, we analysed gene-expression profiles on micro-arrays (Affymetrix, MGU74), in which we compared total and polysome-bound mRNA from factor deprived and Epo-, SCF- or Epo plus SCF restimulated erythroid progenitors. Profiling was done with 4 biological replicates and candidate genes were selected using ANOVA. In subsequent cluster analysis we combined these data with (polysomal) expression profiles of differentiating erythroid cells and identified a cluster containing genes, upregulated in response to Epo/SCF specifically at the level of mRNA polysome recruitment and downregulated during erythroid differentiation. A selection of gene transcripts was validated by RT-PCR and Northern blot analysis using sub-polysomal and polysomal mRNA fractions isolated from cells stimulated with Epo and SCF in presence and absence of the PI3K inhibitor LY294002. Polysome recruitment of 10 transcripts tested so far, appeared to be PI3K dependent. In cells overexpressing eIF4E, polysome-association of these mRNAs was increased under conditions of suboptimal PI3K/PKB activation. In conclusion, we have demonstrated that regulation of translation initiation by eIF4E is an important pathway stimulated by SCF to delay differentiation and to maintain expansion of erythroid progenitors. We identified gene-transcripts of which polysome recruitment is regulated by SCF-induced PI3K/PKB activity. Currently we are evaluating the role of selected genes involved in signal transduction mechanisms (acp1, igbp1) and DNA binding (rbms1, YB1) in the control of expansion and differentiation of erythroid progenitors.

scholarly journals Igbp1 is part of a positive feedback loop in stem cell factor–dependent, selective mRNA translation initiation inhibiting erythroid differentiation

Blood ◽  
2008 ◽  
Vol 112 (7) ◽  
pp. 2750-2760 ◽  
Author(s):  
Godfrey Grech ◽  
Montserrat Blázquez-Domingo ◽  
Andrea Kolbus ◽  
Walbert J. Bakker ◽  
Ernst W. Müllner ◽  
...  
Keyword(s):  
Stem Cell ◽  
Translation Initiation ◽  
Positive Feedback ◽  
Stem Cell Factor ◽  
Binding Protein ◽  
Constitutive Expression ◽  
Mrna Translation ◽  
Erythroid Differentiation ◽  
Translation Initiation Factor ◽  
Initiation Factor

Abstract Stem cell factor (SCF)–induced activation of phosphoinositide-3-kinase (PI3K) is required for transient amplification of the erythroblast compartment. PI3K stimulates the activation of mTOR (target of rapamycin) and subsequent release of the cap-binding translation initiation factor 4E (eIF4E) from the 4E-binding protein 4EBP, which controls the recruitment of structured mRNAs to polysomes. Enhanced expression of eIF4E renders proliferation of erythroblasts independent of PI3K. To investigate which mRNAs are selectively recruited to polysomes, we compared SCF-dependent gene expression between total and polysome-bound mRNA. This identified 111 genes primarily subject to translational regulation. For 8 of 9 genes studied in more detail, the SCF-induced polysome recruitment of transcripts exceeded 5-fold regulation and was PI3K-dependent and eIF4E-sensitive, whereas total mRNA was not affected by signal transduction. One of the targets, Immunoglobulin binding protein 1 (Igbp1), is a regulatory subunit of protein phosphatase 2A (Pp2a) sustaining mTOR signaling. Constitutive expression of Igbp1 impaired erythroid differentiation, maintained 4EBP and p70S6k phosphorylation, and enhanced polysome recruitment of multiple eIF4E-sensitive mRNAs. Thus, PI3K-dependent polysome recruitment of Igbp1 acts as a positive feedback mechanism on translation initiation underscoring the important regulatory role of selective mRNA recruitment to polysomes in the balance between proliferation and maturation of erythroblasts.

Gene Expression Profiles, Differentiation and Lineage Commitment of Human Myeloid Progenitors.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4120-4120
Author(s):  
Louise Edvardsson ◽  
Josefina Dykes ◽  
Tor Olofsson
Keyword(s):  
Gene Expression ◽  
Culture System ◽  
Bone Marrow Cells ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Erythroid Differentiation ◽  
Surface Expression ◽  
Erythroid Progenitors ◽  
Colony Forming Capacity ◽  
Myeloid Progenitors

Abstract With the objective to further elucidate the mechanism behind commitment to erythroid and neutrophil lineages, we isolated by cell sorting common myeloid progenitors (CMPs), granulocyte/monocyte progenitors (GMPs) and megakaryocyte/erythrocyte progenitors (MEPs), based on the surface expression of CD123 (IL3R-α) and CD45RA on human CD34+ bone marrow cells (first described by Manz et al. PNAS, 2002;99:11872). Methylcellulose cultures supporting the growth of myeloid and erythroid progenitors, and real-time RT-PCR mapping the gene expression of Flt3, c-kit, TpoR, GATA-2, GATA-1, SCL, NF-E2, EpoR, ABO, β-globin, GPA, PU.1, C/EBPα, C/EBPε, G-CSFR, proteinase 3 (PR3) and lactoferrin, were used to validate and characterize the progenitors and their progeny. Cell sorted progenitors were labeled with CFDA, SE to track cell division and cultured in suspension to induce neutrophil or erythroid differentiation (SCF+G-CSF for neutrophil and Epo+IL–3+GM–CSF for erythroid culture). After 3–5 days, cells that had gone through 1–8 divisions were sorted and changes in clonogenicity and gene expression were studied. The CMP-population retained some clonogenicity after as many divisions as were tested (at the most six divisions in erythroid and eight in neutrophil culture) and the CMPs differentiated along the lineage defined by the culture system, as evidenced both by the methylcellulose cultures and an increasing expression of GATA-1 and EpoR in erythroid and PU.1, G-CSFR and PR3 in neutrophil cultures, respectively. On the other hand, the GMP-population displayed granulocyte/monocyte (G/M)-differentiation irrespective of the culture system used, although it divided fewer times and lost its clonogenic capacity faster in erythroid culture. Little or no clonogenicity remained after 4–5 divisions in erythroid culture, while some colony-forming capacity remained even after seven divisions in neutrophil culture (maximum number tested). The increased expression of the granulopoiesis-associated genes was also less pronounced in the erythroid culture. The MEP-population dominated by erythroid differentiation capacity retained colony-forming capacity for at least 6–7 divisions in both erythroid and neutrophil cultures, although with a higher overall clonogenicity in erythroid culture. Unexpectedly, however, MEPs were restricted to G/M-differentiation when cultured in neutrophil culture. In cells from erythroid culture the expression of GATA-1, EpoR and β-globin increased, while a corresponding pattern was seen for PU.1, G-CSFR and PR3 in neutrophil culture. Overall, our data support the progenitor classification, based on the surface expression of CD123 and CD45RA, with regard to CMP and GMP populations but question it with regard to the MEP-population. The change in differentiation course for the MEPs in neutrophil culture could be a result of an initially present G/M-potential or a less strict commitment susceptible to cytokine-induced redifferentiation.

scholarly journals CELF1 is an EIF4E binding protein that promotes translation of epithelial-mesenchymal transition effector mRNAs

2019 ◽  
Author(s):  
Arindam Chaudhury ◽  
Rituraj Pal ◽  
Natee Kongchan ◽  
Na Zhao ◽  
Yingmin Zhu ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Mammalian Cells ◽  
Rna Binding ◽  
Epithelial Mesenchymal Transition ◽  
Binding Protein ◽  
Mrna Translation ◽  
Scaffolding Protein ◽  
Initiation Factor ◽  
Eukaryotic Initiation Factor ◽  
Mesenchymal Transition

AbstractMounting evidence is revealing a granularity within gene regulation that occurs at the level of mRNA translation. Within mammalian cells, canonical cap-dependent mRNA translation is dependent upon the interaction between the m7G cap-binding protein eukaryotic initiation factor 4E (eIF4E) and the scaffolding protein eukaryotic initiation factor 4G (eIF4G), the latter of which facilitates pre-translation initiation complex assembly, mRNA circularization, and ultimately ribosomal scanning. In breast epithelial cells, we previously demonstrated that the CELF1 RNA-binding protein promotes the translation of epithelial to mesenchymal transition (EMT) effector mRNAs containing GU-rich elements (GREs) within their 3’ untranslated regions (UTRs). Here we show that within this context, CELF1 directly binds to both the eIF4E cap-binding protein and Poly(A) binding protein (PABP), promoting translation of GRE-containing mRNAs in mesenchymal cells. Disruption of this CELF1/eIF4E interaction inhibits both EMT induction and experimental metastasis. Our findings illustrate a novel way in which non-canonical mechanisms of translation initiation underlie transitional cellular states within the context of development or human disease.

scholarly journals Molecular Network Profiling in Intestinal- and Diffuse-Type Gastric Cancer

Cancers ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 3833
Author(s):  
Shihori Tanabe ◽  
Sabina Quader ◽  
Ryuichi Ono ◽  
Horacio Cabral ◽  
Kazuhiko Aoyagi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gastric Cancer ◽  
Drug Resistance ◽  
Signaling Pathways ◽  
Pathway Analysis ◽  
Rna Binding ◽  
Binding Protein ◽  
Diffuse Type ◽  
Mesenchymal Transition ◽  
Diffuse Type Gastric Cancer

Epithelial-mesenchymal transition (EMT) plays an important role in the acquisition of cancer stem cell (CSC) feature and drug resistance, which are the main hallmarks of cancer malignancy. Although previous findings have shown that several signaling pathways are activated in cancer progression, the precise mechanism of signaling pathways in EMT and CSCs are not fully understood. In this study, we focused on the intestinal and diffuse-type gastric cancer (GC) and analyzed the gene expression of public RNAseq data to understand the molecular pathway regulation in different subtypes of gastric cancer. Network pathway analysis was performed by Ingenuity Pathway Analysis (IPA). A total of 2815 probe set IDs were significantly different between intestinal- and diffuse-type GC data in cBioPortal Cancer Genomics. Our analysis uncovered 10 genes including male-specific lethal 3 homolog (Drosophila) pseudogene 1 (MSL3P1), CDC28 protein kinase regulatory subunit 1B (CKS1B), DEAD-box helicase 27 (DDX27), golgi to ER traffic protein 4 (GET4), chromosome segregation 1 like (CSE1L), translocase of outer mitochondrial membrane 34 (TOMM34), YTH N6-methyladenosine RNA binding protein 1 (YTHDF1), ribonucleic acid export 1 (RAE1), par-6 family cell polarity regulator beta (PARD6B), and MRG domain binding protein (MRGBP), which have differences in gene expression between intestinal- and diffuse-type GC. A total of 463 direct relationships with three molecules (MYC, NTRK1, UBE2M) were found in the biomarker-filtered network generated by network pathway analysis. The networks and features in intestinal- and diffuse-type GC have been investigated and profiled in bioinformatics. Our results revealed the signaling pathway networks in intestinal- and diffuse-type GC, bringing new light for the elucidation of drug resistance mechanisms in CSCs.

scholarly journals A potential role for a novel ZC3H5 complex in regulating mRNA translation in Trypanosoma brucei

2020 ◽  
Vol 295 (42) ◽  
pp. 14291-14304
Author(s):  
Kathrin Bajak ◽  
Kevin Leiss ◽  
Christine Clayton ◽  
Esteban Erben
Keyword(s):  
Gene Expression ◽  
Trypanosoma Brucei ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Protein ◽  
Affinity Purification ◽  
Critical Role ◽  
Rna Binding Protein ◽  
Mrna Translation

In Trypanosoma brucei and related kinetoplastids, gene expression regulation occurs mostly posttranscriptionally. Consequently, RNA-binding proteins play a critical role in the regulation of mRNA and protein abundance. Yet, the roles of many RNA-binding proteins are not understood. Our previous research identified the RNA-binding protein ZC3H5 as possibly involved in gene repression, but its role in controlling gene expression was unknown. We here show that ZC3H5 is an essential cytoplasmic RNA-binding protein. RNAi targeting ZC3H5 causes accumulation of precytokinetic cells followed by rapid cell death. Affinity purification and pairwise yeast two-hybrid analysis suggest that ZC3H5 forms a complex with three other proteins, encoded by genes Tb927.11.4900, Tb927.8.1500, and Tb927.7.3040. RNA immunoprecipitation revealed that ZC3H5 is preferentially associated with poorly translated, low-stability mRNAs, the 5′-untranslated regions and coding regions of which are enriched in the motif (U/A)UAG(U/A). As previously found in high-throughput analyses, artificial tethering of ZC3H5 to a reporter mRNA or other complex components repressed reporter expression. However, depletion of ZC3H5 in vivo caused only very minor decreases in a few targets, marked increases in the abundances of very stable mRNAs, an increase in monosomes at the expense of large polysomes, and appearance of “halfmer” disomes containing two 80S subunits and one 40S subunit. We speculate that the ZC3H5 complex might be implicated in quality control during the translation of suboptimal open reading frames.

scholarly journals Activation of mRNA translation in rat cardiac myocytes by insulin involves multiple rapamycin-sensitive steps

2000 ◽  
Vol 278 (4) ◽  
pp. H1056-H1068 ◽  
Author(s):  
Lijun Wang ◽  
Xuemin Wang ◽  
Christopher G. Proud
Keyword(s):  
Protein Synthesis ◽  
Protein Kinase ◽  
Signaling Pathways ◽  
Glycogen Synthase ◽  
Mrna Translation ◽  
Mitogen Activated Protein Kinase ◽  
Initiation Factor ◽  
Translation Factors ◽  
P70 S6k ◽  
Eef2 Kinase

Insulin acutely activates protein synthesis in ventricular cardiomyocytes from adult rats. In this study, we have established the methodology for studying the regulation of the signaling pathways and translation factors that may be involved in this response and have examined the effects of acute insulin treatment on them. Insulin rapidly activated the 70-kDa ribosomal S6 kinase (p70 S6k), and this effect was inhibited both by rapamycin and by inhibitors of phosphatidylinositol 3-kinase. The activation of p70 S6k is mediated by a signaling pathway involving the mammalian target of rapamycin (mTOR), which also modulates other translation factors. These include the eukaryotic initiation factor (eIF) 4E binding proteins (4E-BPs) and eukaryotic elongation factor 2 (eEF2). Insulin caused phosphorylation of 4E-BP1 and induced its dissociation from eIF4E, and these effects were also blocked by rapamycin. Concomitant with this, insulin increased the binding of eIF4E to eIF4G. Insulin also activated protein kinase B (PKB), which may lie upstream of p70 S6k and 4E-BP1, with the activation of the different isoforms being in the order α>β>γ. Insulin also caused inhibition of glycogen synthase kinase 3, which lies downstream of PKB, and of eEF2 kinase. The phosphorylation of eEF2 itself was also decreased by insulin, and this effect and the inactivation of eEF2 kinase were attenuated by rapamycin. The activation of overall protein synthesis by insulin in cardiomyocytes was substantially inhibited by rapamycin (but not by inhibitors of other specific signaling pathways, e.g., mitogen-activated protein kinase), showing that signaling events linked to mTOR play a major role in the control of translation by insulin in this cell type.

scholarly journals Translation Initiation Factor 4E Inhibits Differentiation of Erythroid Progenitors

2005 ◽  
Vol 25 (19) ◽  
pp. 8496-8506 ◽  
Author(s):  
Montserrat Blázquez-Domingo ◽  
Godfrey Grech ◽  
Marieke von Lindern
Keyword(s):  
Target Genes ◽  
Mrna Translation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Erythroid Progenitors ◽  
Major Pathway ◽  
Pi3k Inhibitor Ly294002 ◽  
Specific Mrnas ◽  
Phosphoinositide 3 Kinase

ABSTRACT Stem cell factor (SCF) delays differentiation and enhances the expansion of erythroid progenitors. Previously, we performed expression-profiling experiments to link signaling pathways to target genes using polysome-bound mRNA. SCF-induced phosphoinositide-3-kinase (PI3K) appeared to control polysome recruitment of specific mRNAs associated with neoplastic transformation. To evaluate the role of mRNA translation in the regulation of expansion versus differentiation of erythroid progenitors, we examined the function of the eukaryote initiation factor 4E (eIF4E) in these cells. SCF induced a rapid and complete phosphorylation of eIF4E-binding protein (4E-BP). Overexpression of eIF4E did not induce factor-independent growth but specifically impaired differentiation into mature erythrocytes. Overexpression of eIF4E rendered polysome recruitment of mRNAs with structured 5′ untranslated regions largely independent of growth factor and resistant to the PI3K inhibitor LY294002. In addition, overexpression of eIF4E rendered progenitors insensitive to the differentiation-inducing effect of LY294002, indicating that control of mRNA translation is a major pathway downstream of PI3K in the regulation of progenitor expansion.

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