Theoretical analysis of a model for globin messenger RNA accumulation during erythropoiesis

191

218

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 ␣-globin mRNA 3 untranslated region (␣-3UTR) 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 ␣-3UTR with a variety of cytosolic extracts. This so-called ␣-complex is sequence specific and is not formed on the 3UTR of either ␤-globin or growth hormone mRNAs. Furthermore, base substitutions within the C-rich stretches which destabilize ␣-globin mRNA in vivo result in a parallel disruption of the ␣-complex in vitro. Competition studies with a series of homoribopolymers reveals a striking sensitivity of ␣-complex formation to poly(C), suggesting the presence of a poly(C)-binding activity within the ␣-complex. Three predominant proteins are isolated by ␣-3UTR 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 ␣-complex formation. These data suggest that a messenger RNP complex formed by interaction of defined segments within the ␣-3UTR 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 ␣-globin mRNA.Degradation of mature mRNA in eukaryotic cells is a regulated process that can be a significant determinant of gene expression. This regulatory process is seen in fertilized oocytes (5, 56) as well as in highly differentiated tissues (13,55,60). Multiple mechanisms underlie this regulation (12,25,50). cisregulating elements can be found in the 5Ј untranslated region (5ЈUTR) (43), the coding region (41, 52), and the 3ЈUTR (28, 60) and often affect the size of the poly(A) tract (5, 39, 56). trans-acting proteins which specifically bind to certain of these cis elements have been identified (29,42,50). An emerging theme is the paramount importance of the 3ЈUTR in control of mRNA stability and subsequent determination of gene expression (28).Recent studies have emphasized a wide spectrum of functions which are mediated by the mRNA 3ЈUTR. This region can act as a trans-acting regulator of growth and differentiation in mouse myoblasts (45, 46), a localization determinant (21) and translational regulator in Drosophila embryos (22), a control element in sex determination of the germ cell precursors in Caenorhabditis elegans (3), and a translational regulator of cell cycle-related mRNAs in Xenopus laevis (56). However, the majority of studies on the 3ЈUTR focus on its role in determination of mRNA stability (15,28,56,60). The most clearly defined example of this control is the iron-responsive element loc...

mentioning

confidence: 99%

Detection and Characterization of a 3′ Untranslated Region Ribonucleoprotein Complex Associated with Human α-Globin mRNA Stability

Wang

Kiledjian

Weiss

et al. 1995

191

218

“…These half-lives have been estimated to be between 16 and 72 h in a broad range of cell culture and in vivo experiments (2,5,18,21,24,31,32). The high stability of ␣-and ␤-globin mRNAs facilitates their accumulation in transcriptionally active erythroid precursors and ensures that they remain at high levels for continued translation in subsequent, transcriptionally silent stages of terminal erythroid differentiation.…”

mentioning

confidence: 99%

Sequence Divergence in the 3′ Untranslated Regions of Human ζ- and α-Globin mRNAs Mediates a Difference in Their Stabilities and Contributes to Efficient α-to-ζ Gene Developmental Switching

Russell

Morales

Makeyev

et al. 1998

The developmental stage-specific expression of human globin proteins is characterized by a switch from the coexpression of -and ␣-globin in the embryonic yolk sac to exclusive expression of ␣-globin during fetal and adult life. Recent studies with transgenic mice demonstrate that in addition to transcriptional control elements, full developmental silencing of the human -globin gene requires elements encoded within the transcribed region. In the current work, we establish that these latter elements operate posttranscriptionally by reducing the relative stability of -globin mRNA. Using a transgenic mouse model system, we demonstrate that human -globin mRNA is unstable in adult erythroid cells relative to the highly stable human ␣-globin mRNA. A critical determinant of the difference between ␣-and -globin mRNA stability is mapped by in vivo expression studies to their respective 3 untranslated regions (3UTRs). In vitro messenger ribonucleoprotein (mRNP) assembly assays demonstrate that the ␣-and -globin 3UTRs assemble a previously described mRNP stabilitydetermining complex, the ␣-complex, with distinctly different affinities. The diminished efficiency of ␣-complex assembly on the 3UTR results from a single C3G nucleotide substitution in a crucial polypyrimidine tract contained by both the human ␣-and -globin mRNA 3UTRs. A potential pathway for accelerated -globin mRNA decay is suggested by the observation that its 3UTR encodes a shortened poly(A) tail. Based upon these data, we propose a model for -globin gene silencing in fetal and adult erythroid cells in which posttranscriptional controls play a central role by providing for accelerated clearance of -globin transcripts.

“…mRNAs encoded by the ␣-and ␤-globin gene clusters increase from less than 1% to represent over 95% of the total message and protein in the more differentiated RBCs (6). This highlevel accumulation results not only from an increase in transcription but in part from the long half-lives of globin mRNAs (24 to 60 h) and from the degradation of nonglobin mRNAs (2,7,29,33,39,44,45,49).…”

mentioning

confidence: 99%

Erythroid Cell-Specific mRNA Stability Elements in the α2-Globin 39 Nontranslated Region

Weiss

Liebhaber

1995

160

163

Very little is known about the mechanisms mediating longevities of mRNAs. As a means of identifying potential cis-and trans-acting elements which stabilize an individual mRNA, naturally occurring mutations that decreased stability of the normally long-lived globin mRNA were analyzed. Our previous studies demonstrated that a subset of mutations which allowed the translating ribosome to read through into the ␣2-globin 3 nontranslated region (NTR) targeted the mutant mRNAs for accelerated turnover in erythroid cells but not in several nonerythroid cell lines (I. M. Weiss and S. A. Liebhaber, Mol. Cell. Biol. 14:8123-8132, 1994). These results suggested that translational readthrough interfered with some feature of the ␣2-globin 3 NTR required for message stability in erythroid cells. To define the cis-acting sequences which comprise this erythroid cell-specific stability determinant, scanning mutagenesis was performed on the ␣2-globin 3 NTR, and the stability of each mutant mRNA was examined during transient expression. Three cytidine-rich regions which are required for longevity of the ␣2-globin mRNA were identified. However, in contrast to the readthrough mutations, base substitutions in these elements destabilize the message through a translation-independent mechanism. To account for these results, we propose that the cis-acting elements form a complex or determinant in the normal ␣2-globin mRNA which protects the message from degradation in erythroid cells. Disruption of this determinant, by translational readthrough or because mutations in an element prevent or inhibit its formation, targets the message for accelerated turnover in these cells.