In vivo association of the stability control protein alphaCP with actively translating mRNAs - PubMed (original) (raw)

In vivo association of the stability control protein alphaCP with actively translating mRNAs

Xinjun Ji et al. Mol Cell Biol. 2003 Feb.

Abstract

Posttranscriptional controls play a major role in eucaryotic gene expression. These controls are mediated by sequence-specific interactions of cis-acting determinants in target mRNAs with one or more protein factors. The positioning of a subset of these mRNA-protein (RNP) complexes within the 3' untranslated region (3' UTR) may allow them to remain associated with the mRNA during active translation. Robust expression of human alpha-globin mRNA during erythroid differentiation has been linked to formation of a binary complex between a KH-domain protein, alphaCP, and a 3' UTR C-rich motif. Detection of this "alpha-complex" has been limited to in vitro studies, and the functional state of the alpha-globin mRNA targeted by alphaCP has not been defined. In the present study we demonstrate that a significant fraction of alphaCP is associated with polysomal mRNA. Targeted analysis of the polysomal RNP complexes revealed that alphaCP is specifically bound to actively translating alpha-globin mRNA. The bound alphaCP is restricted to the poly(C)-rich 3' UTR motif and is dislodged when ribosomes are allowed to enter this region. These data validate the general importance of the 3' UTR as a sheltered site for RNP complexes and support a specific model in which the stabilizing function of alphaCP is mediated on actively translating target mRNAs.

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Figures

FIG. 1.

FIG. 1.

hα-globin mRNA localizes to the polysome fraction of K562 cells. A clarified (S20) cytoplasmic extract from log-growth K562 was pelleted through a 30% sucrose cushion to separate prepolysome supernatant (S130) and polysomal (P) pellet fractions. hα-globin and GAPDH mRNAs were detected by RPA. The positions of the protected bands corresponding to GAPDH and α-globin are noted. The band above α-globin corresponds in size to the primary α-globin transcript and may be detecting contaminant genomic DNA. A 25-nt DNA ladder is shown (M).

FIG. 2.

FIG. 2.

Distribution of hα- and ζ-globin mRNAs across a K562 cell polysome gradient. (A) Sucrose gradient profile of K562 cytosolic extract. The absorbance profile (OD254) of the gradient is shown. The top of the gradient is to the left; the positions of absorbance peaks corresponding to preribosomal RNPs, 40S, 60S, and 80S, and polysomes (2-, 3-, 4-, 5-, 6-, 7-, and 8-somes) are indicated. The 18 fractions (Fx #) collected for subsequent analysis are identified below the tracing. (B) Agarose gel electrophoresis of RNA extracted from the polysome gradient fractions. A 2-μg RNA sample from each fraction (in panel A) was applied to the gel and electrophoresed, and the abundant 28S, 18S, and 5S rRNAs were directly visualized by ethidium bromide staining. The distributions of these RNAs were consistent with the OD peak assignment (in panel A). (C) Detection of globin mRNAs across the K562 polysome gradient. Each gradient fraction was assessed for hα-globin and hζ-globin mRNAs by RPA with corresponding 32P-labeled probes. hα-globin and hζ-globin mRNAs protected probe fragments of 132 and 150 bp, respectively. (D) Distribution of globin mRNAs across the K562 polysome gradient. The contents of hα-globin and ζ-globin mRNAs in each fraction (in panel C) were quantified by PhosphorImager analysis. The amount of each mRNA species in each fraction is depicted as a percentage (ordinate) of the total for the corresponding species across the gradient.

FIG. 3.

FIG. 3.

αCP proteins are ribosome associated. A clarified K562 cell cytoplasmic extract (S20) was fractionated into prepolysomal (S130) and polysomal (P) fractions by sedimentation through a 30% sucrose cushion. Each of the three preparations was resolved on an SDS-PAGE gel; equal aliquots of S20 and S130 fractions and fivefold-concentrated aliquots of the polysome fraction were separated by SDS-PAGE. αCP2/KL and ribosomal L7a proteins were detected by Western blotting with corresponding antisera. The band at 37 kDa represents an isoform of αCP2 (αCP2KL). The positions of the molecular mass markers (not shown) are indicated on the left.

FIG. 4.

FIG. 4.

Characterization of the αCP-polysome interaction. Polysome aliquots (as in Fig. 3) were treated in the indicated manners and analyzed on 10-to-50% sucrose gradients. (A) No additional treatment. The OD254 is indicated (upper panel; Polysome Profile). Proteins in each fraction were precipitated, separated by SDS-12% PAGE, transferred to membranes, and probed with the indicated antibodies (Western blots). See the legend to Fig. 3 for details. (B) EDTA treatment. The polysome sample was resuspended in 20 mM EDTA (final concentration) prior to sucrose gradient fractionation. This treatment dissociates polysomes into 40S and 60S ribosome subunits. The splitting of the αCP signal seen in this Western blot is occasionally observed. (C) Treatment with 0.5 M KCl. The polysome fraction was brought to 0.5 M KCl (final concentration) prior to sucrose gradient fractionation. This treatment removes proteins loosely associated with the polysomes. (D) Treatment with 0.8 M KCl. The polysome fraction was supplemented with 0.8 M KCl (final concentration) prior to sucrose gradient fractionation. This treatment removes almost all proteins from the polysomes that are not intrinsic ribosomal proteins.

FIG. 5.

FIG. 5.

Association of αCP with polysomes is RNA dependent. The polysome fraction was prepared as detailed in the legend for Fig. 3, and an aliquot was treated with RNase A prior to sucrose gradient fractionation. Details of the analysis are as in the legend to Fig. 4.

FIG. 6.

FIG. 6.

In vivo association of αCP with polysome-bound hα-globin mRNA. Equal amounts of the prepolysomal (S130) and polysomal (P) fractions of K562 cytoplasmic extracts were individually immunoprecipitated with an antibody specific to the αCP2 and αCP2-KL isoforms. RNA extracted from the immunoprecipitates was assessed for specific mRNA content by RPA. This study was carried out independently four times with consistent results. (A) RPA using probes to hα-globin and GAPDH mRNAs. (B) RPA using probes to hα-globin and γ-globin mRNAs.

FIG. 7.

FIG. 7.

Interaction of αCP2 with α-globin mRNA is uniquely dependent on the 3′ UTR poly(C)-rich region and is sensitive to displacement by an antiterminated 80S. (A) Expression of hα-globin mRNAs with distinct 3′ UTRs (WT, Neu, and CS) in MEL/tTA cells. The structures of the three encoded mRNAs under the control of a tet promoter are shown. The positions of the translation start site (AUG), termination site (UAA), αCP binding site (protected region [PR]), and the antitermination mutation in the αCS (UAA → CAA) are shown. The cross-hatched box represents the substitution of a neutral sequence for the PR motif. (B) αCP binding on hα-globin mRNA is restricted to the C-rich 3′ UTR stability motif. Each of the indicated α-globin mRNAs was expressed in transfected cells from a corresponding plasmid. After 24 h of induction of expression in TET-deficient medium, the cells were lysed and the clarified cytoplasmic (S20) extracts were layered onto a 30% sucrose cushion. The isolated prepolysomal (S130) and polysomal (P) fractions were separately immunoprecipitated with antibody to αCP2 and αCP2-KL or with preimmune serum (PI). RNA was extracted from the starting material and from each immunoprecipitate. hα-globin and GAPDH mRNAs were detected by RPA. The origin of each sample is indicated above its respective lane. The positions of the RPA probes are indicated to the right of the gel. (C) Selective dissociation of αCP from the antiterminated αCS mRNA. MEL/tTA cells were separately transfected with pTet-WT and pTet-CS plasmids. The transfected genes were transcriptionally induced for 24 h in TET-deficient medium. TET was then added back to the medium at a concentration of 500 ng/ml for an additional 2 h. The cells were subsequently lysed and clarified (S10), and RNP complexes were immunoprecipitated with anti-αCP2 sera. mRNA content in the precipitate was analyzed by RPA as described for panel B. These studies were carried out independently three times with consistent results.

FIG. 7.

FIG. 7.

Interaction of αCP2 with α-globin mRNA is uniquely dependent on the 3′ UTR poly(C)-rich region and is sensitive to displacement by an antiterminated 80S. (A) Expression of hα-globin mRNAs with distinct 3′ UTRs (WT, Neu, and CS) in MEL/tTA cells. The structures of the three encoded mRNAs under the control of a tet promoter are shown. The positions of the translation start site (AUG), termination site (UAA), αCP binding site (protected region [PR]), and the antitermination mutation in the αCS (UAA → CAA) are shown. The cross-hatched box represents the substitution of a neutral sequence for the PR motif. (B) αCP binding on hα-globin mRNA is restricted to the C-rich 3′ UTR stability motif. Each of the indicated α-globin mRNAs was expressed in transfected cells from a corresponding plasmid. After 24 h of induction of expression in TET-deficient medium, the cells were lysed and the clarified cytoplasmic (S20) extracts were layered onto a 30% sucrose cushion. The isolated prepolysomal (S130) and polysomal (P) fractions were separately immunoprecipitated with antibody to αCP2 and αCP2-KL or with preimmune serum (PI). RNA was extracted from the starting material and from each immunoprecipitate. hα-globin and GAPDH mRNAs were detected by RPA. The origin of each sample is indicated above its respective lane. The positions of the RPA probes are indicated to the right of the gel. (C) Selective dissociation of αCP from the antiterminated αCS mRNA. MEL/tTA cells were separately transfected with pTet-WT and pTet-CS plasmids. The transfected genes were transcriptionally induced for 24 h in TET-deficient medium. TET was then added back to the medium at a concentration of 500 ng/ml for an additional 2 h. The cells were subsequently lysed and clarified (S10), and RNP complexes were immunoprecipitated with anti-αCP2 sera. mRNA content in the precipitate was analyzed by RPA as described for panel B. These studies were carried out independently three times with consistent results.

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