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. 1999 Jul;19(7):4572-81.
doi: 10.1128/MCB.19.7.4572.

Assembly of the alpha-globin mRNA stability complex reflects binary interaction between the pyrimidine-rich 3' untranslated region determinant and poly(C) binding protein alphaCP

A N Chkheidze  1 , D L Lyakhov, A V Makeyev, J Morales, J Kong, S A Liebhaber
Affiliations

Assembly of the alpha-globin mRNA stability complex reflects binary interaction between the pyrimidine-rich 3' untranslated region determinant and poly(C) binding protein alphaCP

A N Chkheidze et al. Mol Cell Biol. 1999 Jul.

Abstract

Globin mRNAs accumulate to 95% of total cellular mRNA during terminal erythroid differentiation, reflecting their extraordinary stability. The stability of human alpha-globin mRNA is paralleled by formation of a sequence-specific RNA-protein (RNP) complex at a pyrimidine-rich site within its 3' untranslated region (3'UTR), the alpha-complex. The proteins of the alpha-complex are widely expressed. The alpha-complex or a closely related complex also assembles at pyrimidine-rich 3'UTR segments of other stable mRNAs. These data suggest that the alpha-complex may constitute a general determinant of mRNA stability. One or more alphaCPs, members of a family of hnRNP K-homology domain poly(C) binding proteins, are essential constituents of the alpha-complex. The ability of alphaCPs to homodimerize and their reported association with additional RNA binding proteins such as AU-rich binding factor 1 (AUF1) and hnRNP K have suggested that the alpha-complex is a multisubunit structure. In the present study, we have addressed the composition of the alpha-complex. An RNA titration recruitment assay revealed that alphaCPs were quantitatively incorporated into the alpha-complex in the absence of associated AUF1 and hnRNP K. A high-affinity direct interaction between each of the three major alphaCP isoforms and the alpha-globin 3'UTR was detected, suggesting that each of these proteins might be sufficient for alpha-complex assembly. This sufficiency was further supported by the sequence-specific binding of recombinant alphaCPs to a spectrum of RNA targets. Finally, density sedimentation analysis demonstrated that the alpha-complex could accommodate only a single alphaCP. These data established that a single alphaCP molecule binds directly to the alpha-globin 3'UTR, resulting in a simple binary structure for the alpha-complex.

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Figures

FIG. 1
FIG. 1
Selective recruitment of αCP into the α-complex. (A) Position of the α-complex. The α-complex (as indicated) was identified by incubating MEL cytosolic S100 extract with [32P]α3′UTR followed by RNase treatment. The sample was electrophoresed on a native polyacrylamide gel and autoradiographed. (B) Recruitment of α-CP into the α-complex. Increasing concentrations of unlabeled α3′UTR (indicated by the wedge; see Materials and Methods for concentrations) were incubated with MEL S100 extract to form the α-complex. Products of the incubations were analyzed on native gels. The position of the uncomplexed αCP in the S100 and its recruitment to the position of the α-complex were each visualized by Western analysis. (C) AUF1 was not recruited into the α-complex. AUF1 was detected with a monospecific antibody (gift from G. Brewer). The study was carried out as for panel B. (D) hnRNP K was not recruited into the α-complex. hnRNP K was detected with a corresponding monospecific antibody (38). The study was carried out as described above. (E) Recruitment of αCP-1 into the α-complex (determined as detailed for panel B). The antibody used was specific to the αCP-1 isoform (see Materials and Methods and Fig. 6A). (F) Recruitment of αCP-2 into the α-complex (determined as detailed for panel B). The antibody used was specific to the αCP-2 isoform (see Materials and Methods and Fig. 6A).
FIG. 2
FIG. 2
Recruitment of αCP but not hnRNP K into the Lox 3′UTR complex. (A) Identification of the Lox complex by EMSA using a 32P-labeled Lox 3′UTR probe. The position of the Lox complex (a doublet band) is shown in the first lane, and its sensitivity to poly(C) competition is demonstrated in the following lane. (B) Recruitment of αCP into the Lox complex. Increasing concentrations of unlabeled Lox 3′UTR (wedge) were incubated with MEL S100 extract to form the Lox complex. The incubation mixtures were analyzed on native gels. The position of the uncomplexed αCP in the S100 extract and its recruitment to the position of the Lox complex were visualized by Western analysis. (C) hnRNP K was not recruited into the Lox complex. hnRNP K was detected with a monospecific antibody. The study was carried out as described above.
FIG. 3
FIG. 3
mRNA representation of mαCP isoforms. (A) Autoradiograph of RT-PCR products. MEL RNA (left) or K562 RNA (right) was reverse transcribed and PCR-amplified by using primers that were perfect matches to all known αCP mRNAs. The reverse primer was 32P end labeled. The reaction products were electrophoresed on a 2.5% MetaPhor agarose gel and quantified by PhosphoImager analysis. The identities of the cDNA fragments encoding αCP-2, αCP-1, and αCP2-KL are as indicated and were confirmed by direct sequencing (not shown). (B) RT-PCR amplification kinetics. Relative quantities of the RT-PCR products representing each of the three αCP mRNAs in MEL and K562 cells (left and right, respectively) were determined by PhosphorImager analysis. Logarithms of band intensities were plotted against the number of PCR cycles; these plots formed straight lines for the exponential phase of amplification, and the slopes reflect amplification efficiencies (67). The similarity in slopes for different αCP isoforms shows that the efficiencies of their amplification were similar.
FIG. 4
FIG. 4
Direct, sequence-specific association of each of the recombinant αCP isoforms with the α3′UTR. (A) Direct sequence-specific binding of recombinant αCP2-KL to the α-3′UTR. 32P-labeled α3′UTR (wild type [Wt]) or homologous RNAs containing specific sets of linker scanning base substitutions (H13, H19, and H23) that either disrupt (H13 and H19) or do not interfere (H23) with α-complex formation (68, 70) were incubated with S100 extracts from K562 cells or MEL cells or with recombinant αCP (αCP2-KL). The complexes were digested with RNase T1 and applied to a native acrylamide gel. Lanes 1 and 2 represent the 3′UTR probe incubated without and with RNase T1, respectively. The subsequent lanes contain labeled RNA incubated with the indicated extracts. The position of the α-complex is noted at the left. (B) Direct binding of recombinant αCP-1 and αCP-2 to the α3′UTR (determined as detailed for panel A). In each case, the complex was fully competed by added poly(C).
FIG. 5
FIG. 5
Direct association of recombinant αCP2-KL with the 3′UTR-derived sequences from four highly stable mRNAs. [32P]RNA representing each of the following mRNAs was incubated with either MEL S100 extract (left) or recombinant αCP2-KL (right): α3′UTR, αPR, a poly(C) homoribopolymer (C17), the pyrimidine-rich 3′UTR segments of Lox, Coll, or TH, or a mutant α3′UTR (αmut). An aliquot of each incubation mixture was subjected to native gel electrophoresis. The gel was then dried and autoradiographed. The wild-type α-complex is shown in the second lane of each gel and is indicated by the arrow.
FIG. 6
FIG. 6
The α-complexes formed with S100 extracts represent a heterogeneous population containing the three αCP isoforms. (A) Selective detection of the three αCP isoforms with epitope-specific antisera. Shown are results of Western analyses of recombinant αCP-1, αCP-2, and αCP2-KL and of S100 extracts from mouse (MEL) or human (K562) erythroid cells probed with antisera specific for αCP-1, αCP-2, or an epitope shared by αCP-2 and αCP2-KL. (B) Supershift analysis of α-complexes. [32P]α3′UTR (lane 1) was incubated with K562 S100 extract and run on a native gel either alone (lane 3) or in the presence of increasing amounts of anti-αCP-1 (lanes 4 and 5), anti-αCP-2 (lanes 6 and 7), or antisera specific to αCP-2 and αCP2-KL (lanes 8 and 9). The native complex is composed of two subbands (upper [U] and lower [L]); the position of the antibody supershifted complex is indicated. Lane 2 contains probe digested with RNase prior to protein addition.
FIG. 7
FIG. 7
Binding affinities of recombinant αCPs and native cell extract for the α3′UTR. (A and B) RNA-binding affinities of proteins in the MEL S100 extract (A) and of recombinant αCP2-KL (B). Increasing amounts of each were incubated with a fixed amount of [32P]αPR probe. The amounts of αCP2-KL in the recombinant preparation and in the S100 were normalized by Western blot analysis (see Materials and Methods). The free probe and complexed probe were separated by native gel electrophoresis and quantified. (C) Plot of binding. Results of both sets of experiments are shown; the concentrations of αCP2-KL at which half of the probe is incorporated in the complex are indicated by the vertical arrows. (D) Apparent Kd (molar) for α3′UTR of each of the recombinant αCP isoforms.
FIG. 8
FIG. 8
Analysis of αCP2-KL and the α-complex sedimentation on 5 to 20% sucrose gradients. (A) Top, sedimentation analysis of the α-complex and αCP2-KL. 32P-labeled α-complex was assembled by incubating [32P]α3′UTR probe with MEL S100 extract. The gradients were centrifuged and fractionated as detailed in Materials and Methods. Aliquots of each fraction were analyzed on a native gel, and the 32P-labeled complex was detected by autoradiography. The first gel lane contained an aliquot of the loaded material. The position of the [32P]α-complex is indicated by the bracket at the left, and its poly(C) sensitivity was confirmed in the second lane. Fraction numbers are noted below the lanes. Positions of the molecular weight markers run in a parallel gradient are indicated above the gradients. Bottom, MEL S100 with no added RNA probe. The gradient containing MEL S100 extract and no added RNA probe was run as described above. Gradient fractions were analyzed by SDS-PAGE, and αCP was detected by Western blotting with chicken anti-αCP antibody. Positions of molecular weight standards are indicated at the left. (B) Standard curve for sucrose gradient sedimentation. Positions of standards (see Materials and Methods) are indicated by the open circles. Positions of respective peak centers for αCP2-KL and α-complex are indicated by arrows. Sedimentation coefficients of standards, sw,20 (10−13 s−1), are also indicated. Ald, aldolase; Ova, ovalbumin; myo, myoglobin. (C) Sedimentation profiles of the α-complexes assembled with S100 extract (top) or with recombinant αCP2-KL (middle) compared with uncomplexed αCP (bottom). Note identical sedimentation profiles of the native and recombinant α-complexes and their positioning to the right of the uncomplexed αCP.
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References

    1. Aasheim H C, Loukianova T, Deggerdal A, Smeland E B. Tissue specific expression and cDNA structure of a human transcript encoding a nucleic acid binding [oligo(dC)] protein related to the pre-mRNA binding protein K. Nucleic Acids Res. 1994;22:959–964. - PMC - PubMed
    1. Atwater J A, Wisdom R, Verma I M. Regulated mRNA stability. Annu Rev Genet. 1990;24:519–641. - PubMed
    1. Aviv H, Voloch Z, Bastos R N, Levy S. Biosynthesis and stability of globin mRNA in cultured erythroleukemic Friend cells. Cell. 1976;8:495–503. - PubMed
    1. Bastos R N, Aviv H. Theoretical analysis of a model for globin messenger RNA accumulation during erythropoiesis. J Mol Biol. 1977;110:205–218. - PubMed
    1. Bastos R N, Volloch Z, Aviv H. Messenger RNA population analysis during erythroid differentiation: a kinetical approach. J Mol Biol. 1977;110:191–203. - PubMed

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