Auf-1 and YB-1 Independently Regulate β-Globin mRNA Stability Through Interaction with Poly(A) Binding Protein

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1020-1020
Author(s):  
Sebastiaan van Zalen ◽  
Alyssa A Lombardi ◽  
Grace R Jeschke ◽  
Elizabeth O Hexner ◽  
J. Eric Russell
Keyword(s):  
Mrna Stability ◽  
Half Life ◽  
High Stability ◽  
Binding Protein ◽  
K562 Cells ◽  
Erythroid Progenitors ◽  
Globin Mrna ◽  
Hematopoietic Stem ◽  
Knock Down

Abstract Abstract 1020 The normal expression of Hb A in humans requires the high-level stability of α - and β-globin mRNAs in both transcriptionally active and transcriptionally silenced erythroid progenitors. In contrast to α -globin–whose stability is known to be enhanced by an mRNA-protein (mRNP) complex that assembles on a specific pyrimidine-rich track within its 3'UTR–the structure(s) and mechanism(s) that underlie the high stability of human β-globin mRNA remain poorly defined. We recently identified two RNA-binding proteins, AUF-1 and YB-1, that regulate levels of β-globin mRNA in erythroid progenitors by assembling a cytoplasm-restricted mRNP 'β-complex' on its 3'UTR. The function of the β-complex was predicted by in vitro analyses that mapped its binding to a cis-acting determinant of β-globin mRNA stability, and by in vivo siRNA studies demonstrating that simultaneous knockdown of AUF-1 and YB-1 coordinately ablated the β-complex and coordinately reduced the accumulation of β-globin mRNA in K562 cells. The biological importance of the β-complex was subsequently confirmed in human hematopoietic stem cells, where shRNA-mediated knock-down of AUF-1 or YB-1 effected lower levels of β-globin mRNA in cells induced to the erythroid lineage, again implicating their participation in post-transcriptional mechanism(s) regulating the stability of β-globin mRNA. To unambiguously link β-complex activity to β-globin mRNA half-life, we conducted formal in vivo mRNA stability analyses in K562 cells using a β-globin mRNA-specific tetracycline-conditional transcriptional chase strategy. A derivative β-globin mRNA carrying a 5-nt substitution that totally disrupts β-complex assembly (βMut mRNA) displayed a lower half-life than wild-type β-globin mRNA (βWT mRNA), confirming the participation of the β-complex in post-transcriptional regulatory processes. Parallel poly(A) tail length analyses indicated a possible mechanism for this activity, revealing that the βMut mRNA had a shortened steady-state poly(A) tail that truncated faster than the poly(A) tail on βWT mRNA, suggesting a functional interaction between the β-complex and poly(A) tail-associated factors. This observation is fully consistent with the known importance of deadenylation to processes regulating the decay of heterologous mRNAs in several other experimental systems. Subsequent studies supported our model for β-complex/poly(A) tail interaction: electrophoretic gel mobility-shift analyses demonstrated that the β-complex readily assembles on polyadenylated β-globin 3'UTRs but not on corresponding deadenylated 3'UTRs, while RNA affinity capture experiments using K562 cytoplasmic extracts demonstrated that a polyadenylated βWT 3'UTR retains poly(A) binding protein (PABP), while a similar β-complex-deficient βMut 3'UTR fails to bind PABP. Ongoing co-immunoprecipation studies are expected to determine whether the β-complex and PABP are tethered by an interval of mRNA or, alternately, interact directly via a protein-protein interaction. Based upon our previous structural and functional analyses indicating that AUF-1 and YB-1 act redundantly to regulate the cytoplasmic level of β-globin mRNA, we are currently investigating the hypothesis that these two factors also display redundant interactions with the poly(A) tail and its trans-acting binding factors. Our initial RNA affinity analyses confirm this expectation, demonstrating that K562 extracts depleted of either AUF-1 or YB-1 (using an shRNA-knock-down strategy) both maintained the ability to assemble a β-complex as well as facilitate PABP binding to a the polyadenylated βWT 3'UTR. We are presently testing AUF-1 and YB-1 for corresponding functional redundancy (i.e., their abilities to independently induce βWT mRNA stability) using in vivo mRNA tethering experiments in which AUF-1 or YB-1 can be structurally modified to promote their independent interaction with the β-complex binding site. Altogether, these experiments demonstrate that the β-complex, through its component mRNA-binding factors AUF-1 and YB-1, effects the high stability of β-globin mRNA by interacting with PABP. A detailed structural and mechanistic description of this process will be invaluable to the design of novel therapeutics for patients with congenital disorders of β-globin gene expression. Disclosures: No relevant conflicts of interest to declare.

scholarly journals AUF-1 and YB-1 are critical determinants of β-globin mRNA expression in erythroid cells

Blood ◽  
2012 ◽  
Vol 119 (4) ◽  
pp. 1045-1053 ◽  
Author(s):  
Sebastiaan van Zalen ◽  
Grace R. Jeschke ◽  
Elizabeth O. Hexner ◽  
J. Eric Russell
Keyword(s):  
Posttranscriptional Regulation ◽  
Rna Binding ◽  
Globin Gene ◽  
K562 Cells ◽  
Erythroid Cells ◽  
Erythroid Progenitors ◽  
Globin Mrna ◽  
Affinity Enrichment

Abstract The normal accumulation of β-globin protein in terminally differentiating erythroid cells is critically dependent on the high stability of its encoding mRNA. The molecular basis for this property, though, is incompletely understood. Factors that regulate β-globin mRNA within the nucleus of early erythroid progenitors are unlikely to account for the constitutively high half-life of β-globin mRNA in the cytoplasm of their anucleate erythroid progeny. We conducted in vitro protein-RNA binding analyses that identified a cytoplasm-restricted β-globin messenger ribonucleoprotein (mRNP) complex in both cultured K562 cells and erythroid-differentiated human CD34+ cells. This novel mRNP targets a specific guanine-rich pentanucleotide in a region of the β-globin 3′untranslated region that has recently been implicated as a determinant of β-globin mRNA stability. Subsequent affinity-enrichment analyses identified AUF-1 and YB-1, 2 cytoplasmic proteins with well-established roles in RNA biology, as trans-acting components of the mRNP. Factor-depletion studies conducted in vivo demonstrated the importance of the mRNP to normal steady-state levels of β-globin mRNA in erythroid precursors. These data define a previously unrecognized mechanism for the posttranscriptional regulation of β-globin mRNA during normal erythropoiesis, providing new therapeutic targets for disorders of β-globin gene expression.

scholarly journals Detection and characterization of a 3' untranslated region ribonucleoprotein complex associated with human alpha-globin mRNA stability.

1995 ◽  
Vol 15 (3) ◽  
pp. 1769-1777 ◽  
Author(s):  
X Wang ◽  
M Kiledjian ◽  
I M Weiss ◽  
S A Liebhaber
Keyword(s):  
Complex Formation ◽  
Mrna Stability ◽  
Untranslated Region ◽  
Binding Protein ◽  
Erythroid Cell ◽  
Globin Mrna ◽  
Rnp Complex ◽  
Alpha Globin

The highly stable nature of globin mRNA is of central importance to erythroid cell differentiation. We have previously identified cytidine-rich (C-rich) segments in the human alpha-globin mRNA 3' untranslated region (alpha-3'UTR) which are critical in the maintenance of mRNA stability in transfected erythroid cells. In the present studies, we have detected trans-acting factors which interact with these cis elements to mediate this stabilizing function. A sequence-specific ribonucleoprotein (RNP) complex is assembled after incubation of the alpha-3'UTR with a variety of cytosolic extracts. This so-called alpha-complex is sequence specific and is not formed on the 3'UTR of either beta-globin or growth hormone mRNAs. Furthermore, base substitutions within the C-rich stretches which destabilize alpha-globin mRNA in vivo result in a parallel disruption of the alpha-complex in vitro. Competition studies with a series of homoribopolymers reveals a striking sensitivity of alpha-complex formation to poly(C), suggesting the presence of a poly(C)-binding activity within the alpha-complex. Three predominant proteins are isolated by alpha-3'UTR affinity chromatography. One of these binds directly to poly(C). This cytosolic poly(C)-binding protein is distinct from previously described nuclear poly(C)-binding heterogeneous nuclear RNPs and is necessary but not sufficient for alpha-complex formation. These data suggest that a messenger RNP complex formed by interaction of defined segments within the alpha-3'UTR with a limited number of cytosolic proteins, including a potentially novel poly(C)-binding protein, is of functional importance in establishing high-level stability of alpha-globin mRNA.

Cytoplasmic Regulators of β-Globin mRNA Are Structurally Modified During Erythroid Terminal Differentiation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1093-1093
Author(s):  
Sebastiaan van Zalen ◽  
Grace R Jeschke ◽  
Elizabeth Hexner ◽  
J. Eric Russell
Keyword(s):  
Terminal Differentiation ◽  
K562 Cells ◽  
Regulatory Control ◽  
Erythroid Progenitors ◽  
Globin Mrna ◽  
Cd34 Cells ◽  
High Level ◽  
Mrna Binding

Abstract Abstract 1093 The high-level accumulation of β globin in mature erythrocytes requires a correspondingly high level of its encoding mRNA in terminally differentiating erythroid progenitors. We recently identified two RNA-binding proteins–AUF1 and YB1–that appear to regulate levels of β-globin mRNA in these cells by assembling a cytoplasm-restricted RNA-protein 'β-complex' on its 3'UTR. The function of the β-complex was predicted by in vitro analyses mapping it to a cis-acting determinant of β-globin mRNA stability, and subsequently validated by in vivo siRNA studies demonstrating that simultaneous knockdown of AUF1 and YB1 ablated the β-complex and coordinately reduced the accumulation of β-globin mRNA in K562 cells. Although both AUF1 and YB1 are ubiquitously expressed, studies in cultured cells and in Epo-induced CD34+ primary cells indicated that their β-globin mRNA-specific regulatory properties are restricted to erythroid cells during later stages of terminal differentiation. Based upon these observations, we reasoned that AUF1 and YB1 undergo erythroid and differentiation stage-restricted alterations that permit their assembly into the mRNA-regulatory β-complex. Our analyses of AUF1 focused on three structural isoforms, observed in K562 cells, resulting from alternative pre-mRNA processing events that retain or exclude exon 7. GST-AUF1 fusion isoforms that retain exon 7 fail to bind the β-globin 3'UTR in vitro, while related isoforms that exclude exon 7 bind the 3'UTR with high efficiency. These results, which implicate the importance of exon 7 exclusion to AUF1 function, were subsequently validated in intact K562 cells using an AUF1 isotype-specific siRNA knockdown strategy. In these in vivo experiments, a reduction in exon 7-excluded AUF1 effected a two-fold decrease in steady-state β-globin mRNA, while similar reductions in exon 7-retained AUF1 isoforms had no measurable effect. The isotype-specific mRNA-binding characteristics of AUF1 may be particularly important during terminal differentiation: Epo-induced CD34+ cells display an increase in exon 7-excluded AUF1, paralleling their capacity to assemble a regulatory β-complex in vitro. Among several possible mechanisms, we asked whether the isoform-specific function of AUF1 might relate to the unusually high number (20) of phosphorylation-capable residues encoded by exon 7. In vitro analyses were fully consistent with this possibility, demonstrating that the β-globin mRNA-binding activity of exon 7-retained AUF1 could be restored by prior dephosphorylation. These experiments suggest that post-transcriptional regulation of β-globin mRNA during erythroid differentiation is likely to be effected by alternative splicing of AUF1 pre-mRNA that eliminates phosphorylation-active exon 7 amino-acids in the translated protein. Based upon these results, we reasoned that related post-translational processes might similarly regulate the β-globin mRNA-binding specificity of YB1. Our analyses focused on a specific residue (Ser102) that is a known target for regulatory phosphorylation and can be experimentally identified using a Ser102 phospho-specific YB1 antibody. In in vitro studies with K562 cytoplasmic extract, which contains both phospho- and dephospho- forms of YB1, we observed that only dephospho-YB1 adheres to the β-globin 3'UTR; likewise, in in vivo studies of CD34+ cells we noted a substantial increase in the ratio of dephospho:phospho-YB1 following Epo induction. Both experiments indicate the likely importance of this post-translational process to the function of YB1 during terminal differentiation. Confirmatory studies are currently being conducted in vivo using an epitope-tagged YB1 containing a position 102 Ser->Ala substitution. Collectively, our analyses indicate that the β-globin mRNA-binding specificities of AUF1 and YB1–and, hence, their corresponding regulatory activities–are determined by post-transcriptional and -translational events. This work suggests mechanisms through which erythroid progenitors can maintain dynamic regulatory control during an interval when transcriptional processes are beginning to silence, and identifies new pathways that can be therapeutically targeted in patients with congenital disorders of β-globin gene expression. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Monoclonal antibodies detecting antigenic determinants with restricted expression on erythroid cells: from the erythroid committed progenitor level to the mature erythroblast

Blood ◽  
1984 ◽  
Vol 63 (6) ◽  
pp. 1376-1384 ◽  
Author(s):  
T Yokochi ◽  
M Brice ◽  
PS Rabinovitch ◽  
T Papayannopoulou ◽  
G Stamatoyannopoulos
Keyword(s):  
Monoclonal Antibodies ◽  
K562 Cells ◽  
Erythroid Differentiation ◽  
Antigenic Determinants ◽  
Erythroid Progenitors ◽  
Cell Surface Antigens ◽  
Cytotoxicity Assays ◽  
Complement Dependent Cytotoxicity

Two new cell surface antigens specific for the erythroid lineage were defined with cytotoxic IgM monoclonal antibodies (McAb) (EP-1; EP-2) that were produced using BFU-E-derived colonies as immunogens. These two antigens are expressed on in vivo and in vitro derived adult and fetal erythroblasts, but not on erythrocytes. They are not detectable on resting lymphocytes, concanavalin-A (Con-A) activated lymphoblasts, granulocytes, and monocytes or granulocytic cells or macrophages present in peripheral blood or harvested from CFU-GM cultures. Cell line and tissue distributions distinguish McAb EP-1 and EP-2 from all previously described monoclonal antibodies. McAb EP-1 (for erythropoietic antigen-1) inhibits the formation of BFU-E and CFU-E, but not CFU-GM, colonies in complement-dependent cytotoxicity assays. By cell sorting analysis, about 90% of erythroid progenitors (CFU-E, BFU-E) were recovered in the antigen-positive fraction. Seven percent of the cells in this fraction were progenitors (versus 0.1% in the negative fraction). The expression of EP-1 antigen is greatly enhanced in K562 cells, using inducers of hemoglobin synthesis. McAb EP-2 fails to inhibit BFU-E and CFU-E colony formation in complement-dependent cytotoxicity assays. EP-2 antigen is predominantly expressed on in vitro derived immature erythroblasts, and it is weakly expressed on mature erythroblasts. The findings with McAb EP-1 provide evidence that erythroid progenitors (BFU-E and CFU-E) express determinants that fail to be expressed on other progenitor cells and hence appear to be unique to the erythroid lineage. McAb EP-1 and EP-2 are potentially useful for studies of erythroid differentiation and progenitor cell isolation.

scholarly journals Monoclonal antibodies detecting antigenic determinants with restricted expression on erythroid cells: from the erythroid committed progenitor level to the mature erythroblast

Blood ◽  
1984 ◽  
Vol 63 (6) ◽  
pp. 1376-1384 ◽  
Author(s):  
T Yokochi ◽  
M Brice ◽  
PS Rabinovitch ◽  
T Papayannopoulou ◽  
G Stamatoyannopoulos
Keyword(s):  
Monoclonal Antibodies ◽  
K562 Cells ◽  
Erythroid Differentiation ◽  
Antigenic Determinants ◽  
Erythroid Progenitors ◽  
Cell Surface Antigens ◽  
Cytotoxicity Assays ◽  
Complement Dependent Cytotoxicity

Abstract Two new cell surface antigens specific for the erythroid lineage were defined with cytotoxic IgM monoclonal antibodies (McAb) (EP-1; EP-2) that were produced using BFU-E-derived colonies as immunogens. These two antigens are expressed on in vivo and in vitro derived adult and fetal erythroblasts, but not on erythrocytes. They are not detectable on resting lymphocytes, concanavalin-A (Con-A) activated lymphoblasts, granulocytes, and monocytes or granulocytic cells or macrophages present in peripheral blood or harvested from CFU-GM cultures. Cell line and tissue distributions distinguish McAb EP-1 and EP-2 from all previously described monoclonal antibodies. McAb EP-1 (for erythropoietic antigen-1) inhibits the formation of BFU-E and CFU-E, but not CFU-GM, colonies in complement-dependent cytotoxicity assays. By cell sorting analysis, about 90% of erythroid progenitors (CFU-E, BFU-E) were recovered in the antigen-positive fraction. Seven percent of the cells in this fraction were progenitors (versus 0.1% in the negative fraction). The expression of EP-1 antigen is greatly enhanced in K562 cells, using inducers of hemoglobin synthesis. McAb EP-2 fails to inhibit BFU-E and CFU-E colony formation in complement-dependent cytotoxicity assays. EP-2 antigen is predominantly expressed on in vitro derived immature erythroblasts, and it is weakly expressed on mature erythroblasts. The findings with McAb EP-1 provide evidence that erythroid progenitors (BFU-E and CFU-E) express determinants that fail to be expressed on other progenitor cells and hence appear to be unique to the erythroid lineage. McAb EP-1 and EP-2 are potentially useful for studies of erythroid differentiation and progenitor cell isolation.

The Role of Nitric Oxide in γ-Globin Induction by Hydroxyurea.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3794-3794
Author(s):  
Tzu-Fang Lou ◽  
Ashley Williams ◽  
Wei Li ◽  
Betty S. Pace
Keyword(s):  
Nitric Oxide ◽  
Sickle Cell ◽  
Fetal Hemoglobin ◽  
K562 Cells ◽  
Fold Increase ◽  
Heme Iron ◽  
Guanosine Monophosphate ◽  
Erythroid Progenitors ◽  
Globin Mrna ◽  
No Donor

Abstract Hydroxyurea (HU) has been shown to induce fetal hemoglobin (HbF) synthesis through activation of the soluble guanosine cyclase/cyclic guanosine monophosphate signaling pathway. The release of NO from HU by heme iron is thought to be involved in this mechanism of HbF induction. Studies completed in sickle cell patients confirmed increased serum NO levels after oral HU therapy but NO generation in red blood cells and the effect on γ-gene transcription have not been extensively investigated. Therefore, we performed studies to quantify NO generated by HU in K562 cells and normal erythroid progenitors as a mechanism for γ-globin activation. NO levels were measured after drug treatments using the Nitric Oxide Assay Kit (Calbiochem) and γ-globin mRNA was measured using quantitative PCR. HU (100μM) increased NO 1.4 to 1.8-fold at 24–72 hrs in K562 cells compared to a 2.0 to 2.5-fold increase in NO produced by the known NO donor, deta-nonoate (DE; 400μM). NO levels were also measured in erythroid progenitors grown in liquid cultures; a 1.6-fold increase in NO was produced by 30μM HU after 48 hrs with comparable increases produced by 200μM DE. To understand the effects of HU on normal NO synthesis from L-arginine through the action of NO synthase (NOS), we performed studies with the NOS inhibitor, NG-Monomethyl-L-arginine (L-NMMA). Interestingly, HU increased NO levels 2.5-fold at 24 hrs when combined L-NMMA compared a 1.4-fold increase produced by HU alone; this pattern persisted up to 72 hrs. Parallel with these findings γ-globin activation by HU was augmented approximately 25% by L-NMMA; DE combined with L-NMMA did not produce the same effect. These data suggest a novel mechanism for NOS regulation by HU compared to DE. Subsequent studies were completed to determine if HbF synthesis could be augmented by combining NO donors since they have different mechanisms of action. HbF levels in K562 cells were measured by ELISA (Bethyl Laboratories) and normalized by total hemoglobin and protein. Treatment with HU or DE increased HbF 3.6-fold and 4.6-fold respectively; when HU was combined with DE an additive 7.6-fold increase in HbF was produced. These data confirm that HU treatment lead to NO generation in K562 cells and normal erythroid progenitors which plays a role in its mechanism of γ-globin activation. HU combined with DE had an additive effect on HbF synthesis. These findings are relevant to current research efforts to develop novel HbF inducers for therapy in sickle cell patients.

Design and Validation of Genes Encoding Hyperstable β-Globin mRNAs.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4059-4059
Author(s):  
Osheiza Abdulmalik ◽  
J. Eric Russell
Keyword(s):  
Sickle Cell Disease ◽  
Mrna Stability ◽  
Half Life ◽  
Fold Increase ◽  
Globin Genes ◽  
Wild Type ◽  
Cell Disease ◽  
Globin Mrna ◽  
Stem Loop ◽  
The Stability

Abstract 4059 Poster Board III-994 Transgenic approaches to β thalassemia and sickle cell disease require viral vectors that express high levels of therapeutic β-like globin proteins. We recently proposed that the overall expression of these transgenes would likely be improved by structural modifications that prolong the cytoplasmic half-lives of their encoded mRNAs. Relevant experiments from our laboratory have previously linked the constitutively high stability of β-globin mRNA to a region of its 3'UTR that appears to interact with at least two distinct cytoplasmic mRNA-stabilizing factors, and is predicted to form an imperfect stem-loop (SL) structure. Based upon these findings, we conducted enzymatic secondary-structure mapping studies of the β-globin 3'UTR, unequivocally validating the existence of the predicted functional stem-loop element. We subsequently reasoned that the constitutive half-life of β-globin mRNA might be prolonged by the insertion of multiple SL motifs into its 3'UTR, resulting in increased levels of the mRNA–and its encoded β-globin product–in terminally differentiating erythroid cells. To test this hypothesis, we constructed full-length β-globin genes containing either wild-type 3'UTRs, or variant 3'UTRs that had been modified to contain either two or three tandem SL motifs. Each gene was identically linked to a tetracycline-suppressible promoter, permitting pulse-chase mRNA stability analyses to be conducted in vivo in intact cultured cells. Erythroid-phenotype K562 cells were transiently transfected with SL-variant and control wild-type β-globin genes, exposed to tetracycline, and levels of β-globin mRNA determined by qRT-PCR at defined intervals using tet-indifferent β-actin mRNA as internal control. Relative to wild-type β-globin mRNA, SL-duplicate β-globin mRNAs displayed a position-dependent two-fold increase in cytoplasmic half-life; SL-triplicate β-globin mRNAs did not exhibit any additional stability. These experiments confirm the existence of a defined SL structure within the β-globin 3'UTR, and demonstrate that duplication of this motif can substantially increase the stability of β-globin mRNA. We subsequently designed a series of experiments to elucidate post-transcriptional processes involved in mRNA hyperstability. These studies required the construction of HeLa cells that stably express either wild-type β-globin mRNA (11 subclones) or SL-duplicate β-globin mRNAs (10 subclones). Preliminary analyses indicate an approximate 1.5-fold increase in the median steady-state expression of SL-duplicate genes, consistent with a prolongation in the half-life of its encoded mRNA. While formal mRNA stability studies are not yet complete, early data appear to replicate results from experiments conducted in transiently transfected cells. We have also initiated structural studies to link differences in the stability of SL-variant β-globin mRNA to alterations in its poly(A) tail. Using an RNase H-based strategy, we identified a previously unknown poly(A)-site heterogeneity–of undetermined significance–affecting both wild-type and SL-duplicate β-globin mRNAs. Finally, we recently isolated fifty-four K562 subclones expressing SL-duplicate or control β-globin mRNAs; parallel analyses of these cells will permit the cell-specificity of β-globin SL-directed mRNA stabilization to be investigated in detail. Results from each of these studies will be immediately applicable to the design of high-efficiency therapeutic transgenes for β thalassemia and sickle-cell disease. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Regulation of thrombospondin-1 expression through AU-rich elements in the 3′UTR of the mRNA

2011 ◽  
Vol 16 (1) ◽  
Author(s):  
Asa Mcgray ◽  
Timothy Gingerich ◽  
James Petrik ◽  
Jonathan Lamarre
Keyword(s):  
Gene Expression ◽  
Mrna Stability ◽  
Half Life ◽  
Rna Binding ◽  
Binding Protein ◽  
Site Directed Mutagenesis ◽  
Hek293 Cells ◽  
Luciferase Reporter ◽  
Thrombospondin 1 ◽  
Mrna Half Life

AbstractThrombospondin-1 (TSP-1) is a matricellular protein that participates in numerous normal and pathological tissue processes and is rapidly modulated by different stimuli. The presence of 8 highly-conserved AU rich elements (AREs) within the 3′-untranslated region (3′UTR) of the TSP-1 mRNA suggests that post-transcriptional regulation is likely to represent one mechanism by which TSP-1 gene expression is regulated. We investigated the roles of these AREs, and proteins which bind to them, in the control of TSP-1 mRNA stability. The endogenous TSP-1 mRNA half-life is approximately 2.0 hours in HEK293 cells. Luciferase reporter mRNAs containing the TSP-1 3′UTR show a similar rate of decay, suggesting that the 3′UTR influences the decay rate. Site-directed mutagenesis of individual and adjacent AREs prolonged reporter mRNA halflife to between 2.2 and 4.4 hours. Mutation of all AREs increased mRNA half life to 8.8 hours, suggesting that all AREs have some effect, but that specific AREs may have key roles in stability regulation. A labeled RNA oligonucleotide derived from the most influential ARE was utilized to purify TSP-1 AREbinding proteins. The AU-binding protein AUF1 was shown to associate with this motif. These studies reveal that AREs in the 3′UTR control TSP-1 mRNA stability and that the RNA binding protein AUF1 participates in this control. These studies suggest that ARE-dependent control of TSP-1 mRNA stability may represent an important component in the control of TSP-1 gene expression.

scholarly journals An mRNA Stability Complex Functions with Poly(A)-Binding Protein To Stabilize mRNA In Vitro

1999 ◽  
Vol 19 (7) ◽  
pp. 4552-4560 ◽  
Author(s):  
Zuoren Wang ◽  
Nancy Day ◽  
Panayiota Trifillis ◽  
Megerditch Kiledjian
Keyword(s):  
Mrna Stability ◽  
Binding Protein ◽  
Major Protein ◽  
Globin Mrna ◽  
Ideal System ◽  
Complex Functions ◽  
Cytosolic Extract ◽  
Protein Components ◽  
The Stability

ABSTRACT The stable globin mRNAs provide an ideal system for studying the mechanism governing mammalian mRNA turnover. α-Globin mRNA stability is dictated by sequences in the 3′ untranslated region (3′UTR) which form a specific ribonucleoprotein complex (α-complex) whose presence correlates with mRNA stability. One of the major protein components within this complex is a family of two polycytidylate-binding proteins, αCP1 and αCP2. Using an in vitro-transcribed and polyadenylated α-globin 3′UTR, we have devised an in vitro mRNA decay assay which reproduces the α-complex-dependent mRNA stability observed in cells. Incubation of the RNA with erythroleukemia K562 cytosolic extract results in deadenylation with distinct intermediates containing a periodicity of approximately 30 nucleotides, which is consistent with the binding of poly(A)-binding protein (PABP) monomers. Disruption of the α-complex by sequestration of αCP1 and αCP2 enhances deadenylation and decay of the mRNA, while reconstitution of the α-complex stabilizes the mRNA. Similarly, PABP is also essential for the stability of mRNA in vitro, since rapid deadenylation resulted upon its depletion. An RNA-dependent interaction between αCP1 and αCP2 with PABP suggests that the α-complex can directly interact with PABP. Therefore, the α-complex is an mRNA stability complex in vitro which could function at least in part by interacting with PABP.

scholarly journals Hematopoietic stem cells differentiate into restricted myeloid progenitors before cell division

2018 ◽  
Author(s):  
Tatyana Grinenko ◽  
Anne Eugster ◽  
Lars Thielecke ◽  
Beata Ramazs ◽  
Anja Krueger ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Cell Division ◽  
Hematopoietic Stem Cells ◽  
Embryonic Stem ◽  
Erythroid Progenitors ◽  
Multipotent Stem Cells ◽  
Hematopoietic Stem ◽  
Myeloid Progenitors

SummaryHematopoietic stem cells (HSCs) continuously replenish all blood cell types through a series of differentiation steps that involve the generation of lineage-committed progenitors as well as necessary expansion due to repeated cell divisions. However, whether cell division in HSCs precedes differentiation is unclear. To this end, we used an HSC cell tracing approach and Ki67RFP knock-in mice to assess simultaneously divisional history, cell cycle progression, and differentiation of adult HSCs in vivo. Our results reveal that HSCs are able to differentiate into restricted progenitors, especially common myeloid progenitors, restricted megakaryocyte-erythroid progenitors (PreMEs) and pre-megakaryocyte progenitors (PreMegs), without undergoing cell division and even before entering the S phase of the cell cycle. Additionally, the phenotype of the undivided but differentiated progenitors correlated with expression of lineage-specific genes that manifested as functional differences between HSCs and restricted progenitors. Thus, HSC fate decisions appear to be uncoupled from physical cell division. Our results facilitate a better understanding of the mechanisms that control fate decisions in hematopoietic cells. Our data, together with separate findings from embryonic stem cells, suggest that cell division and fate choice are independent processes in pluripotent and multipotent stem cells.

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