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, 21 (17), 5879-88

Structural and Functional Analysis of an mRNP Complex That Mediates the High Stability of Human Beta-Globin mRNA

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Structural and Functional Analysis of an mRNP Complex That Mediates the High Stability of Human Beta-Globin mRNA

J Yu et al. Mol Cell Biol.

Abstract

Human globins are encoded by mRNAs exhibiting high stabilities in transcriptionally silenced erythrocyte progenitors. Unlike alpha-globin mRNA, whose stability is enhanced by assembly of a specific messenger RNP (mRNP) alpha complex on its 3' untranslated region (UTR), neither the structure(s) nor the mechanism(s) that effects the high-level stability of human beta-globin mRNA has been identified. The present work describes an mRNP complex assembling on the 3' UTR of the beta-globin mRNA that exhibits many of the properties of the stability-enhancing alpha complex. The beta-globin mRNP complex is shown to contain one or more factors homologous to alphaCP, a 39-kDa RNA-binding protein that is integral to alpha-complex assembly. Sequence analysis implicates a specific 14-nucleotide pyrimidine-rich track within its 3' UTR as the site of beta-globin mRNP assembly. The importance of this track to mRNA stability is subsequently verified in vivo using mice expressing human beta-globin transgenes that contain informative mutations in this region. In combination, the in vitro and in vivo analyses indicate that the high stabilities of the alpha- and beta-globin mRNAs are maintained through related mRNP complexes that may share a common regulatory pathway.

Figures

FIG. 1
FIG. 1
hα- and hβ-globin mRNA 3′ UTRs assemble comigrating mRNP complexes in vitro. An EMSA was performed by incubating 32P-labeled α and β 3′ UTR probes in MEL cell cytoplasmic (S-100) extract and resolving the RNase-resistant mRNP products on a nondenaturing 6.5% polyacrylamide gel. Extract-free reactions assembled in the absence (lanes 1 and 4) or presence (lanes 2 and 5) of added RNase were run in parallel as controls. The composition of each reaction is indicated at the top of the autoradiograph, and the migration of the mRNP complexes is shown on the left.
FIG. 2
FIG. 2
Assembly of α- and β-globin mRNP complexes is inhibited by homodeoxyribopoly(C) but not by other homodeoxyribopolymers. EMSA reactions were performed on 32P-labeled α and β 3′ UTRs supplemented with 100 μg of unlabeled competitor homodeoxyribopolymer (dC, dA, dG, and dT). Control lanes demonstrate migration of RNase-undigested 3′ UTRs (lanes 1 and 8) and RNase-digested 3′ UTRs (lanes 2 and 9) in extract-free reactions. The components of each reaction are indicated at the top of the autoradiograph, and the positions of the α and β mRNP complexes are shown on the left.
FIG. 3
FIG. 3
Binding specificity of trans-acting factors for α and β 3′ UTRs. EMSA analyses were performed using in vitro-transcribed 32P-labeled α-globin 3′ UTRs (A) and β-globin 3′ UTRs (B) incubated in MEL cell S-100 extract. Reactions were supplemented with defined quantities of unlabeled α- or β-mRNA 3′ UTR competitor. The composition of each reaction mixture is indicated above the autoradiograph, and the positions of the α and β complexes are shown on the left.
FIG. 4
FIG. 4
Fully assembled α and β complexes exchange α- and β-globin 3′ UTRs. In vitro-transcribed α- and β-globin 3′ UTRs were incubated in MEL cell S-100 extract and then resolved on a single polyacrylamide gel. (Left) A reaction utilizing 32P-labeled α 3′ UTR indicates the migration of the α complex (lane 3). Undigested and fully digested probes, as well as a homodeoxyribopoly(C)-competed reaction, were included as controls (lanes 1, 2, and 4). The composition of each reaction is indicated above the autoradiograph, and the position of the α complex is shown on the left. (Right) On the same gel, reactions utilizing α 3′ UTRs (lanes 6, 9, and 12) and β 3′ UTRs (lanes 7, 10, and 13) were resolved in triplicate. Control lanes 5, 8, and 11 contained S-100 extract alone. The complexes were transferred to a nylon membrane and were subsequently probed with either 32P-labeled α 3′ UTR, 32P-labeled β 3′ UTR, or an unrelated [32P]mRNA control (indicated at bottom), and autoradiographs were exposed. The composition of each reaction is indicated above the autoradiograph. A double-headed arrow emphasizes comigration of the α complex and the membrane-bound mRNP complexes that exchange 32P-labeled α and β 3′ UTRs.
FIG. 5
FIG. 5
Anti-αCP antibodies bind the mRNP β complex. 32P-labeled β 3′ UTR was incubated in MEL cell S-100 extract in the absence (lanes 1 and 2) or presence (lanes 3 to 5) of anti-αCP antibodies, and the reactions were resolved on a nondenaturing polyacrylamide gel. FF1 and FF3 denote rabbit antisera raised against αCP-1 and αCP-2 isoforms, respectively. The reaction shown in lane 2 was supplemented with homodeoxyribopoly(C) to verify the position of the β complex. The composition of each reaction is indicated at the top of the gel, and the positions of the native and supershifted β complexes are given on the left.
FIG. 6
FIG. 6
The β-globin 3′ UTR contains three extended PREs. The β-globin 3′ UTR is displayed with the native UAA termination codon and AAUAAA polyadenylation signal doubly underlined. β-Globin mRNAs carrying naturally occurring +2 frameshift antitermination mutations terminate translation at an in-frame UAA 10 codons into the 3′ UTR (underlined; see text). Pyrimidine-rich tracks designated β PRE-1, -2, and -3 are boldfaced and labeled.
FIG. 7
FIG. 7
Construction of transgenes encoding βWT and βRT β-globin mRNAs. (A) Transgene structures. The βWT and βRT transgenes comprise the full-length hβ-globin gene, including native promoter (P) and 3′ enhancer (E) elements, linked in their native orientation to a β μLCR (LCR) (63). The two transgenes are identical except for two tandem mutations (asterisks) in exon 3 of the βRT transgene (54). Tick marks indicate the positions of the native translation initiation and termination codons. (B) Structures of βWT and βRT mRNAs. The 5′-cap (●), poly(A) tail (An), and native translation initiation and termination codons (tick marks) are indicated. Asterisks denote the positions of tandem mutations in the βRT mRNA. The region of each mRNA that is actively translated is indicated by an arrow.
FIG. 8
FIG. 8
Translation antitermination mutations reduce the stability of hβ-globin mRNA in intact erythroid cells. (A) Representative two-probe RPA. RNA prepared from bone marrow progenitors (B) and peripheral blood reticulocytes (P) obtained from representative mice containing the βWT transgene (lanes 3 and 4) or the βRT transgene (lanes 5 and 6) was analyzed using 32P-labeled probes complementary to hβ-globin (hβ) and mα-globin (mα) mRNAs. The migration of the protected mα and hβ probe fragments is indicated by control reactions utilizing either of the probes alone (lanes 1 and 2). The composition of each reaction and the migration of the protected fragments are shown above and to the left of the autoradiograph, respectively. Control lanes indicating functional probe excess, performed for every experiment, were cropped from the figure to maintain clarity. (B) Stabilities of βWT and βRT mRNAs in multiple transgenic lines. The average stabilities of βWT and βRT mRNAs in each of five and one independent line, respectively, are plotted (●). For each line, a minimum of two mice were studied on at least two separate occasions using the two-probe (hβ and mα) RPA. The stability of each mRNA, averaged across all lines, is indicated as a bar; the stability of mα-globin mRNA, defined as 1.0, is denoted by a dashed line.
FIG. 9
FIG. 9
β-Globin mRNAs containing deletion or replacement of PRE-2 are destabilized in vivo. (A) Structures of wild-type and variant β-globin mRNA 3′ UTRs. Transgenes encoding variant β-globin mRNAs were constructed from the βWT transgene by exchanging β PRE-2 for an equal-length purine-rich sequence (βSUB) or deleting it in its entirety (βDEL). The relevant segments of the βWT, βDEL, and βSUB 3′ UTRs have been aligned for comparison. The β μLCR (LCR), promoter (P), and 3′ enhancer elements (E) are indicated. (B) Relative stabilities of βWT, βSUB, and βDEL mRNAs in representative transgenic mice. RNAs recovered from bone marrow (B) and peripheral blood (P) erythroid cells from mice containing the βWT, βSUB, and βDEL transgenes were analyzed by two-probe RPA. The positions of the protected [32P]-labeled mα and hβ RNA fragments are indicated on the left. (C) Stabilities of βSUB and βDEL mRNAs in multiple transgenic lines. The average stabilities of βSUB and βDEL mRNAs in each of two and three independent transgenic lines, respectively, are plotted (●). For each line, a minimum of two mice was studied on at least two separate occasions using two-probe (hβ and mα) RPA. The stability of each mRNA, averaged across all lines, is indicated as a bar; the stability of mα-globin mRNA, defined as 1.0, is denoted by a dashed line. A bar indicating the average stability of transgenic human βWT mRNA is reproduced from Fig. 8 for comparison.

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