An mRNA stability complex functions with poly(A)-binding protein to stabilize mRNA in vitro

Abstract

The stable globin mRNAs provide an ideal system for studying the mechanism governing mammalian mRNA turnover. alpha-Globin mRNA stability is dictated by sequences in the 3' untranslated region (3'UTR) which form a specific ribonucleoprotein complex (alpha-complex) whose presence correlates with mRNA stability. One of the major protein components within this complex is a family of two polycytidylate-binding proteins, alphaCP1 and alphaCP2. Using an in vitro-transcribed and polyadenylated alpha-globin 3'UTR, we have devised an in vitro mRNA decay assay which reproduces the alpha-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 alpha-complex by sequestration of alphaCP1 and alphaCP2 enhances deadenylation and decay of the mRNA, while reconstitution of the alpha-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 alphaCP1 and alphaCP2 with PABP suggests that the alpha-complex can directly interact with PABP. Therefore, the alpha-complex is an mRNA stability complex in vitro which could function at least in part by interacting with PABP.

FIG. 1

Differential stability of α-globin mRNA in an in vitro mRNA decay assay. In vitro mRNA decay reactions were carried out with 5′-end-labeled α^wtA⁺ or α^ΔmtA⁺. (A) Decay reactions were carried out in the presence of K562 S130 extract at 37°C. Positions of migration of the unadenylated 3′UTRs are labeled “α^wtA⁻” and “α^ΔmtA⁻” in lanes 1 and 7, respectively, and the input adenylated 3′UTRs are shown in lanes 2 and 8. Incubation times ranged from 1 h to 6 h as indicated. Reactions were stopped with ULB; RNA was isolated and resolved on an 8% polyacrylamide–7 M urea gel and visualized by autoradiography (see Materials and Methods). The bands designated “Internal control” are derived from a labeled oligonucleotide which was included in ULB to normalize for RNA extractions and gel loading. Positions of DNA size markers in nucleotides are indicated on the right. (B) Quantitation of the decay of α^wtA⁺ and α^ΔmtA⁺ RNAs. The half-life is indicated with a dotted line at 50% RNA remaining. The half-life of the wild-type α^wtA⁺ is approximately 5 h; that of the mutant α^ΔmtA⁺ is approximately 1.5 h. The values were derived from an average of three independent experiments; standard deviations are shown for each time point. (C) Quantitation demonstrating the amount of full-length adenylated α^wtA⁺ and α^ΔmtA⁺ RNAs remaining. These data provide a comparison for the initial deadenylation rate of the fully adenylated RNAs. The values were derived from an average of three independent experiments; standard deviations are shown.

FIG. 2

Deadenylation of wild-type α-globin 3′UTR is sensitive to oligo(dC) competition. (A) An in vitro mRNA decay reaction was carried out with uniformly labeled α^wtA⁺ in the presence of 28 pmol of oligo(dN) or oligo(dC) at 37°C for 1 h (lanes 3 and 5) or 2 h (lanes 4 and 6). A trace amount of poly(A) competitor (0.1 pmol) was also included to partially sequester soluble poly(A)-binding activity in the extract (5, 16). Migration of the unadenylated RNA (α^wtA⁻) is shown. Termination of the reactions, RNA analysis, gel conditions, internal control, and markers are as described in the legend to Fig. 1. (B) The uniformly labeled and polyadenylated deletion mutant α^ΔmtA⁺ was used in an in vitro decay reaction in the presence of oligo(dN) or oligo(dC) competitor as describe above except that poly(A) was omitted. Migration of the unadenylated α^ΔmtA⁻ is indicated.

FIG. 3

αCP1 and αCP2 can promote stabilization of mRNA. (A) Western analysis of K562 S130 extract (lane 1) or poly(C)-depleted S130 extract (lane 2), using αCP-specific antibodies. Positions of the αCP band and molecular size markers are indicated on the left and right, respectively. (B) An in vitro mRNA decay reaction was carried out for 30 min in the presence of K562 S130 extract depleted of poly(C)-binding activity. Complete S130 extract was used in lane 3; poly(C)-depleted extract was used in lanes 4 to 10. RNA decay reactions were carried out in the presence of purified αCP1 and αCP2 from K562 cells (lanes 5 to 7) or the hnRNP U protein RBD (lanes 8 to 10). The amount of protein added ranged from 25 ng (lanes 5 and 8) to 100 ng (lanes 7 and 10). The RNA was isolated following a 30-min incubation, resolved on an 8% polyacrylamide–7 M urea gel, and detected by autoradiography. The position of the unadenylated probe (α^wtA⁻) is indicated; numbers on the right represent nucleotide size markers.

FIG. 4

PABP contributes to the stabilization of polyadenylated mRNA. (A) Western analysis detecting PABP in K562 S130 extract (lane 1) and poly(A)-depleted S130 extract (lane 2), using the PABP-specific monoclonal antibody 10E10. Positions of migration of PABP and molecular weight markers are shown. (B) A 2-h in vitro mRNA decay reaction using 5′-end-labeled α^wtA⁺ was carried out with poly(A)-depleted K562 S130 extract. Complete S130 extract was used in lane 3; poly(A)-depleted extract was used in lanes 4 through 11. The amount of recombinant PABP or RBD added is indicated. “α^wtA⁻” denotes migration of the unadenylated α^wt RNA. Resolution of the RNA and molecular size markers (indicated in nucleotides) are as described in the legend to Fig. 1.

FIG. 5

αCP1 and αCP2 can interact with PABP. (A) Combinations of test proteins (fusion protein containing the Gal4 DNA-binding domain [left column] and fusion protein containing the Gal4 transcription activation domain [right column]) used in the yeast two-hybrid analysis. αCP1, αCP2, AUF1, and PABP, test proteins fused to the indicated Gal4 domain; RBD, the hnRNP U protein RBD fused to the indicated Gal4 domain. (B) Relative extent of protein-protein interactions as determined by β-galactosidase (β-GAL) assays, ranging from (−) no interaction to (++) very strong interaction. (C) Tenfold serial dilutions of cells, harboring plasmids expressing the indicated fusion test proteins. Growth of the cells spotted results from protein-protein interactions occurring between the test proteins.

FIG. 6

The interaction of αCP1 and αCP2 with PABP is RNA dependent. (A) Bacterially expressed GST-αCP1, GST-αCP2, and GST alone bound to glutathione-Sepharose beads were incubated with [³⁵S]methionine-labeled PABP without (lanes 1 to 4) or with RNases (40 ng of RNase A and 4 U of RNase T₁; lanes 5 to 8). Copurified proteins were isolated following extensive washes and resolved by SDS-PAGE (12.5% gel) followed by autoradiography. An aliquot of the total translation product is shown in lanes 1 and 5, and the interacting protein is shown in the remaining lanes. (B) Interactions with PABP (lanes 2 to 4) or U1A (lanes 6 to 8) were carried out in the presence of 20 pmol of oligo(dC) with 1 μg of RNase A and 150 U RNase T₁. On average, approximately 40% of the total input [³⁵S]methionine-labeled PABP efficiently interacted with GST-αCP1 or GST-αCP2. Positions of migration of full-length PABP and U1A (arrows) and the molecular size markers are indicated.

FIG. 7

Model of the α-complex function during α-globin mRNA deadenylation. The α-globin mRNA is denoted by a line, the filled circle represents the 5′ cap, and the translation start (AUG) and stop (UAA) sites with the traversing ribosomes shown within the coding region. The α-complex indicated by the large oval on the 3′UTR includes the four unknown proteins p58, p55, p50, and p28 along with AUF1 and an αCP (28). The poly(A) tail is shown on the 3′ end bound by two PABPs. A potential interaction between the αCP in the α-complex and PABP on the poly(A) tail is shown. A putative deadenylase is indicated. The interaction between the α-complex and PABP is postulated to stabilize the poly(A) tail by rendering it resistant to deadenylation.