Erythroid cell-specific determinants of alpha-globin mRNA stability.

Abstract

Although globin mRNAs are considered prototypes of highly stable messages, the mechanisms responsible for their longevity remain largely undefined. As an initial step in identifying potential cis-acting elements or structures which contribute to their stability, we analyzed the defect in expression of a naturally occurring a2-globin mutant, aConstant Spring (CS). The CS mutation is a single-base change in the translation termination codon (I!AA-WCAA) that allows the ribosome to read through into the 3' nontranslated region (NTR). The presence of CS mRNA in transcriptionally active erythroid precursors and its absence (relative to normal a-globin mRNA) in the more differentiated transcriptionally silent erythrocytes suggest that this mutation disrupts some feature of the a-globin mRNA required for its stability. Using a transient transfection system, we demonstrate that in murine erythroleukemia cells the CS mRNA is unstable compared with the normal a2-globin mRNA. The analyses of several other naturally occurring and site-directed mutant a-globin genes in murine erythroleukemia cells indicate that entry of a translating ribosome into the 3' NTR targets the message for accelerated degradation in erythroid cells. In contrast, both the CS and a2-globin mRNAs are stable in several nonerythroid cell lines. These results suggest that translational readthrough disrupts a determinant associated with the a2-globin 3' NTR which is required for mRNA stability in erythroid cells.While the major roles of transcription and processing in the control of gene expression have long been acknowledged (13), a number of systems which demonstrate a primary role for regulation at the level of mRNA stability have recently been described (reviewed in references 2, 7, 42, and 44). In eucaryotes, mRNAs have a wide range of half-lives, from as short as a few minutes to as long as several days. These differences in mRNA stability can have significant impacts on the levels of gene expression (20,48). A variety of elements and mechanisms appear to be responsible for modulating mRNA halflives. Features governing the turnover of individual mRNAs have been attributed to specific cis-acting RNA sequences and structures and to trans-acting proteins (10,27,36,41,45,46,55,57,63). An additional level of complexity is possible as an mRNA is translated, since the interaction of cis-and transacting elements with the ribosome and its associated components or with higher-order RNA structures could modulate mRNA turnover (47,52).The at-and P-globin gene clusters are useful model systems to study mechanisms responsible for mRNA stability. Globin genes are expressed in highly specialized, terminally differentiated erythrocytes (RBC) and can account for as much as 95% of the soluble proteins (17,43). During RBC differentiation the nucleus is extruded (17) and globin mRNAs increase from less than 1% to more than 98% of the total mRNA (6). This high-level accumulation of globin mRNA is dependent not only on increased transcription but also on the long half-lives...

Post-Transcriptional Regulation of the GAP-43 Gene by Specific Sequences in the 3′ Untranslated Region of the mRNA

“…In addition, several studies have revealed that a number of variables need to be considered in the interpretation of mRNA stability data, including the effect of translation on mRNA turnover (Savant-Bhonsale and Cleveland, 1992;Nishizawa and Okamoto, 1994;Chen et al, 1995). For example, minor changes in mRNA sequence, because of the introduction of point mutations or specific deletions, may affect mRNA stability indirectly via a perturbation of the RNA secondary structure that leads to changes in translation efficiency (Weiss and Liebhaber, 1994). In view of this potential problem, we examined the ability of different 3Ј UTR deletion mutants used in this study to generate GAP-43 protein in transfected cells and found that all of them were translated effectively.…”

The poly(C)-binding proteins: A multiplicity of functions and a search for mechanisms

“…It has long been noted that certain RNA-binding proteins can be characterized, grouped, and purified on the basis of their binding to nucleic acid homopolymers (Swanson & Dreyfuss, 1988)+ A defined set of RNAbinding proteins is characterized by high affinity and sequence-specific interaction with poly(C)+ These poly(C)-binding proteins (PCBPs) comprise two subsets in mammalian cells; hnRNPs K/J (Matunis et al+, 1992) and the aCP proteins (a-complex proteins)+ The exact structural and genetic relationship of hnRNP K and J remains to be fully defined+ The aCPs are encoded at four dispersed loci, with additional isoforms generated via alternative splicing (Makeyev & Liebhaber, 2000)+ The PCBPs studied in greatest detail are hnRNP K, aCP-1, and aCP-2+ The latter two proteins are alternatively referred to as PCBP1 and PCBP2 or hnRNP-E1 and hnRNP-E2 (Kiledjian et al+, 1995;Leffers et al+, 1995)+ Recent studies, summarized in this review, reveal that the PCBPs are involved in a remarkable array of biological processes+ Members of this protein family are linked to mRNA stabilization (Weiss & Liebhaber, 1994Holcik & Liebhaber, 1997), translational silencing (Ostareck et al+, 1997;Collier et al+, 1998), and translational enhancement (Blyn et al+, 1997;Gamarnik & Andino, 1997)+ These proteins also appear to be involved as determinants of transcriptional controls (Michelotti et al+, 1996;Tomonaga & Levens, 1996) and apoptotic pathways (Charroux et al+, 1999;Zhu & Chen, 2000) and constitute candidate structural components of recombination complexes within retrotransposon long terminal repeats (Goller et al+, 1994) and in telomere complexes (Lacroix et al+, 2000)+ The common denominator of PCBP activities appears to reflect binding to C-rich single-strand motifs+ Determining how such interactions might factor into this broad array of diverse biologic functions constitutes a major challenge to current research efforts+ Initial studies of PCBPs have been previously reviewed Ostareck-Lederer et al+, 1998)+ This article will focus with particular attention on three areas of recent interest: (1) identification and characterization of novel PCBP isoforms and posttranslational modifications that may underlie their functional diversity; (2) new insights into the evolutionary history of the PCBPs that may shed light on their conserved structure-function relationships, and (3) the expanding spectrum of PCBP functions in transcriptional and posttranscriptional controls+…”

“…PCBPs have been implicated in a wide spectrum of posttranscriptional controls+ Initial insights into the roles emerged from studies of human a-globin mRNA stabilization (Weiss & Liebhaber, 1994+ Highlevel stability of a-globin mRNA is essential to full expression of globin proteins during the 2-3 days of transcriptionally silent terminal erythroid differentiation+ Stabilization of a-globin mRNA is tightly linked to formation of a binary complex between a single molecule of aCP and a pyrimidine-rich motif within the a-globin 39 UTR ("a-complex"; Fig+ 4A; Kiledjian et al+, 1995;Wang et al+, 1995;Chkheidze et al+, 1999)+ Interruption of any of the three C-rich patches within this region disrupts the a-complex assembly in vitro and decreases a-globin mRNA stability in vivo (Wang et al+, 1995;Weiss & Liebhaber 1995)+ aCP-1 and aCP-2 can independently bind to the human a-globin mRNA 39 UTR to form the a-complex as demonstrated in vitro+ This binding is specific to aCPs as hnRNP K lacks this binding activity (Chkheidze et al+, 1999)+ The respective K d values of recombinant aCP-1 (51 nM) and aCP-2 (0+8 nM) FIGURE 4. Examples of functions mediated by the PCBPs+ Six distinct examples of PCBP function are shown+ In each case, the PCBP isoform of interest is indicated as a filled circle+ A specific example is given for each of these indicated functions and each is expanded upon in the text+ The mRNAs are indicated in diagrammatic format with tick marks indicating the initiation and termination codons+ The cellular mRNAs are also shown with cap (filled circles) and poly(A) tails+ The polioviral mRNA lacks a cap structure; the two IRES structures of specific interest, secondary structures known as Domain I and Domain IV, are shown+ Question marks indicate unidentified protein factors and/or assumed protein-protein interactions (as discussed in the text)+ 272…”

Control of mRNA translation and stability in haematopoietic cells: The function of hnRNPs K and E1/E2

“…A single base substitution in the human a-globin mRNA termination codon, the ConstantSpring mutation, allows the read-through of the translating ribosome into the 3'UTR and leads to destabilization of the mRNA, presumably by displacement of the a-complex (Weiss and Liebhaber, 1994). Interestingly, analysis of the a−complex composition identified hnRNP E1 and hnRNP E2, also known as aCP1 and aCP2, as the main components (Kiledjian et al, 1995) (Fig.…”