The poly(A)-binding protein and an mRNA stability protein jointly regulate an endoribonuclease activity

Abstract

We previously identified a sequence-specific erythroid cell-enriched endoribonuclease (ErEN) activity involved in the turnover of the stable alpha-globin mRNA. We now demonstrate that ErEN activity is regulated by the poly(A) tail. The unadenylated alpha-globin 3' untranslated region (3'UTR) was an efficient substrate for ErEN cleavage, while the polyadenylated 3'UTR was inefficiently cleaved in an in vitro decay assay. The influence of the poly(A) tail was mediated through the poly(A)-binding protein (PABP) bound to the poly(A) tail, which can inhibit ErEN activity. ErEN cleavage of an adenylated alpha-globin 3'UTR was accentuated upon depletion of PABP from the cytosolic extract, while addition of recombinant PABP reestablished the inhibition of endoribonuclease cleavage. PABP inhibited ErEN activity indirectly through an interaction with the alphaCP mRNA stability protein. Sequestration of alphaCP resulted in an increase of ErEN cleavage activity, regardless of the polyadenylation state of the RNA. Using electrophoretic mobility shift assays, PABP was shown to enhance the binding efficiency of alphaCP to the alpha-globin 3'UTR, which in turn protected the ErEN target sequence. Conversely, the binding of PABP to the poly(A) tail was also augmented by alphaCP, implying that a stable higher-order structural network is involved in stabilization of the alpha-globin mRNA. Upon deadenylation, the interaction of PABP with alphaCP would be disrupted, rendering the alpha-globin 3'UTR more susceptible to endoribonuclease cleavage. The data demonstrated a specific role for PABP in protecting the body of an mRNA in addition to demonstrating PABP's well-characterized effect of stabilizing the poly(A) tail.

FIG. 1

ErEN cleavage activity is poly(A) tail dependent. An in vitro decay assay was carried out with the 5′-end-³²P-labeled and capped α-globin 3′UTR (α^wt) and incubated with MEL cell S130 cytosolic extract. Reactions were carried out at room temperature for 5 to 60 min as indicated above the lanes with the unadenylated (α^wt) or adenylated (α^wtA⁺) 3′UTR. The location of the 5′ intermediate fragment (5′-Int) generated by the initial ErEN cleavage is shown. The smaller bands which accumulate with increasing incubation time in lanes 5 and 6 are a consequence of subsequent 3′-to-5′ exoribonuclease activities present within the extract (51). The RNA substrates used are shown schematically at the bottom of the figure. The filled circle denotes the 5′ m⁷G cap, the asterisks represent the position of the ³²P labeling, and A_80–90 signifies the 80 to 90 adenosine residues in the poly(A) tail. The relative position and size of the 5′-Int are shown. Single-stranded DNA size markers are shown on the right in nucleotides.

FIG. 2

ErEN activity is inhibited by PABP bound to the poly(A) tail. 3′-end-labeled α^wtA⁺ RNA containing 80 to 90 adenosines (A_80–90) was incubated for 15 min with either MEL cell S130 extract (lane 2) or with S130 depleted with poly(A) agarose beads (lanes 3 to 5) in an in vitro decay assay. One microgram of PABP or the hnRNP U RNA-binding domain (RBD) was included as indicated (lane 4 or 5, respectively). The schematic of the RNA is shown at the bottom and is as described in the legend to Fig. 1, as are the size markers. The migration of the 3′ intermediate fragment is indicated on the left and shown schematically on the bottom.

FIG. 3

ErEN can cleave polyadenylated α^wt upon sequestration of αCP. ErEN activity on polyadenylated α^wt RNA was determined in the in vitro decay assays by using oligo(dC), which is an efficient competitor for the sequestration of αCP, or by using poly(C)-depleted extract, which is devoid of αCP (51). (A) An in vitro RNA decay reaction was carried out with 5′-end-labeled α^wt containing a poly(A) tail of approximately 80 to 90 residues as described in the legend to Fig. 1, except that 10 pmol of an oligo(dC) competitor was included in lanes 2 to 5 or poly(C)-depleted extract was used in lanes 6 to 9 for the indicated times. The reaction with complete S130 extract at the 60-min time point is shown in lane 10. (B) In vitro decay reactions were carried out as described for panel A except 5 mM EDTA was included in the reaction mixtures to minimize deadenylation and exoribonuclease activity. α^wtA⁺ uniformly labeled with ³²P was used in lanes 1 to 5, and 3′-end-labeled α^wtA⁺ was used in lanes 6 to 10. Labeling is as described in the legend to Fig. 1.

FIG. 4

PABP enhances the binding of αCP1 to the α^wtA⁺ RNA. EMSA were carried out to determine the effect of PABP on the binding of αCP1 to the α^wtA⁺ RNA. (A) Binding of the αCP1 protein to uniformly labeled α^wtA₆₀ was carried out in the presence of increasing amounts of PABP as indicated. The RNase T₁-resistant complex was resolved on a 5% native polyacrylamide gel. Migration of the bound αCP1 complex is shown on the left. Addition of PABP increases the binding of αCP1 (lanes 4 to 7) to the 3′UTR, while addition of an unrelated protein had no effect (lanes 8 to 11). A schematic of the uniformly ³²P-labeled α^wtA₆₀ is shown at the bottom. (B) Quantitation of the results of the binding experiments presented in panel A are plotted as the relative levels of binding of αCP1 in the presence of PABP derived from three independent experiments. The vertical bars represent standard deviations. (C) An EMSA reaction mixture with uniformly labeled α^wtA₆₀ was incubated with 10 pmol of the indicated proteins. PABP-NT can also stimulate αCP1 binding, while PABP-CT or the hnRNP U RNA-binding domain (RBD) cannot. The schematic of the RNA is as described for panel A. (D) An EMSA similar to that described for panel C was carried out with uniformly ³²P-labeled α^wt RNA lacking a poly(A) tail. There was no detectable enhancement of αCP1 binding to the α^wt RNA upon the addition of PABP when the RNA lacked a poly(A) tail. A schematic of uniformly ³²P-labeled α^wt is shown at the bottom.

FIG. 4

PABP enhances the binding of αCP1 to the α^wtA⁺ RNA. EMSA were carried out to determine the effect of PABP on the binding of αCP1 to the α^wtA⁺ RNA. (A) Binding of the αCP1 protein to uniformly labeled α^wtA₆₀ was carried out in the presence of increasing amounts of PABP as indicated. The RNase T₁-resistant complex was resolved on a 5% native polyacrylamide gel. Migration of the bound αCP1 complex is shown on the left. Addition of PABP increases the binding of αCP1 (lanes 4 to 7) to the 3′UTR, while addition of an unrelated protein had no effect (lanes 8 to 11). A schematic of the uniformly ³²P-labeled α^wtA₆₀ is shown at the bottom. (B) Quantitation of the results of the binding experiments presented in panel A are plotted as the relative levels of binding of αCP1 in the presence of PABP derived from three independent experiments. The vertical bars represent standard deviations. (C) An EMSA reaction mixture with uniformly labeled α^wtA₆₀ was incubated with 10 pmol of the indicated proteins. PABP-NT can also stimulate αCP1 binding, while PABP-CT or the hnRNP U RNA-binding domain (RBD) cannot. The schematic of the RNA is as described for panel A. (D) An EMSA similar to that described for panel C was carried out with uniformly ³²P-labeled α^wt RNA lacking a poly(A) tail. There was no detectable enhancement of αCP1 binding to the α^wt RNA upon the addition of PABP when the RNA lacked a poly(A) tail. A schematic of uniformly ³²P-labeled α^wt is shown at the bottom.

FIG. 5

αCP1 enhances the binding of PABP to the poly(A) tail. (A) An α^wtA⁺ RNA containing a ³²P-labeled poly(A) tail was used in EMSA reaction mixtures to detect the binding of PABP to the poly(A) tail. Where indicated, 10 pmol of PABP or PABP-NT was used in the binding reaction mixtures. Lanes 4, 7, and 9 contain 10 pmol of αCP1, and lanes 5, 8, and 10 contain 10 pmol of the hnRNP U RNA-binding domain (RBD). The binding of both PABP and PABP-NT are enhanced by αCP1. The PABP-poly(A) tail and the PABP-NT–poly(A) tail complexes are indicated. The migrations of α^wtA₆₀ and the released poly(A) tail (A₆₀) are indicated on the left of the figure. A schematic of α^wtA₆₀ labeled with ³²P on the poly(A) tail is shown at the bottom. (B) The relative levels of binding of PABP to the poly(A) tail in the presence of increasing amounts of αCP1 derived from three independent experiments are plotted. The vertical bars denote standard deviations.