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. 2002 Jan 15;21(1-2):165-74.
doi: 10.1093/emboj/21.1.165.

The Mammalian Exosome Mediates the Efficient Degradation of mRNAs That Contain AU-rich Elements

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Free PMC article

The Mammalian Exosome Mediates the Efficient Degradation of mRNAs That Contain AU-rich Elements

Devi Mukherjee et al. EMBO J. .
Free PMC article

Abstract

HeLa cytoplasmic extracts contain both 3'-5' and 5'-3' exonuclease activities that may play important roles in mRNA decay. Using an in vitro RNA deadenylation/decay assay, mRNA decay intermediates were trapped using phosphothioate-modified RNAs. These data indicate that 3'-5' exonucleolytic decay is the major pathway of RNA degradation following deadenylation in HeLa cytoplasmic extracts. Immunodepletion using antibodies specific for the exosomal protein PM-Scl75 demonstrated that the human exosome complex is required for efficient 3'-5' exonucleolytic decay. Furthermore, 3'-5' exonucleolytic decay was stimulated dramatically by AU-rich instability elements (AREs), implicating a role for the exosome in the regulation of mRNA turnover. Finally, PM-Scl75 protein was found to interact specifically with AREs. These data suggest that the interaction between the exosome and AREs plays a key role in regulating the efficiency of ARE-containing mRNA turnover.

Figures

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Fig. 1. 5′–3′ exonuclease activity can be identified in HeLa extracts. Polyadenylated SV-A60 RNA containing either a 5′ cap (7meGpppG lanes) or a 5′ monophosphate (pG lanes) were incubated in HeLa S100 cytoplasmic extract for the times indicated. Reaction products were analyzed on a 5% acrylamide gel containing 7 M urea.
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Fig. 2. Phosphothioate-modified RNAs demonstrate that RNAs are degraded by a 3′–5′ exonuclease following deadenylation. A synthetic RNA containing three consecutive phosphothioate substitutions was prepared and ligated to RNA fragments containing a 5′ cap and a 3′ poly(A) tail as described in Materials and methods. Polyadenylated wild-type or phosphothioate-modified variants of GemARE-A60 RNA were incubated in the in vitro deadenylation/decay system using HeLa cytoplasmic extracts for the times indicated. As shown in (A), trapping of an 82-base intermediate would identify a block by the phosphothioate modification to 3′–5′ exonucleases, while trapping a 106-base fragment would be consistent with decay via a 5′–3′ exonucleolytic pathway. (B) Reaction products were analyzed on a 5% acrylamide gel containing 7 M urea. The arrow on the right indicates the 82-base fragment that was trapped specifically by the phosphothioate modifications.
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Fig. 3. Preparation and characterization of recombinant PM-Scl75 protein and antibodies. (A) Purified recombinant His-tagged PM-Scl75 protein was prepared and analyzed by Coomassie Blue staining following electrophoresis on a 10% acrylamide gel containing SDS. The arrow indicates the position of the purified recombinant protein. (B) Polyclonal antibodies were raised against recombinant PM-Scl75 protein in mice and used in a western blot against HeLa cytoplasmic S100 extract to assess their specificity. The blot was developed using chemiluminescence. The arrow indicates the position of PM-Scl75 protein. (C) PM-Scl75 protein is present in both nuclear and cytoplasmic fractions. Fractionated HeLa cell extracts were probed for the presence of PM-Scl75 protein by western blotting using rabbit anti-PM-Scl75 antiserum. Cyto = cytoplasmic fraction; Nuc = nuclear fraction; Rest = membrane and nuclear remainder fraction; and rPMScl-75 = purified recombinant protein. (D) PM-Scl75 antiserum co-immunoprecipitates components of the exosome. Immunoprecipitations of S100 extract were performed using rabbit serum recognizing hRrp46p (lane anti-hRrp46p), PM-Scl75 (lane antiPM-Scl75) or normal rabbit serum (lane NRS). The precipitates were separated by SDS–PAGE and blotted and stained with a patient antiserum recognizing both PMScl-75 and PM-Scl100 proteins (indicated by the arrows on the right).
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Fig. 4. The exosome is required for 3′–5′ exonucleolytic decay of the body of RNA substrates in in vitro deadenylation/decay assays. HeLa S100 cytoplasmic extracts were immunodepleted using anti-PM-Scl75 mouse antiserum or pre-immune serum prior to their use in in vitro RNA deadenylation/decay assays. (A) Western blotting with PM-Scl75 antiserum demonstrates that most of the endogenous PMScl-75 protein (indicated by the arrow) was removed by immunodepletion with anti-PM-Scl75 antiserum (lane α-PM-Scl75) but not by normal mouse serum (control lane) in the extracts used for the assay in (B). (B) Capped and polyadenylated GemARE-A60 RNA was incubated in the in vitro deadenylation/decay assay for 30 min using either untreated S100 extract (lane no antibody), extract that was treated with normal serum (lane normal sera) or extract that had been immunodepleted with antibodies specific for PM-Scl75 protein (lane α-PM-Scl75). Reaction products were analyzed on a 5% acrylamide gel containing 7 M urea. The positions of the polyadenylated input and deadenylated RNAs are indicated on the left. (C and D) Capped and polyadenylated GemARE-A60 RNA was incubated in the in vitro deadenylation/decay assay for the times indicated using either extract that was untreated (control lanes), extract that was treated with normal serum (control depleted lanes) or extract that had been immunodepleted with antibodies specific for PM-Scl75 protein (lanes α-PM-Scl75 depleted), hRrp40 protein (lanes α-hRrp40 depleted) or hRrp46 protein (lanes α-hRrp46 depleted). Reaction products were analyzed on a 5% acrylamide gel containing 7 M urea. The positions of the polyadenylated input and deadenylated RNAs are indicated on the left.
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Fig. 5. 3′–5′ Decay is regulated by AU-rich instability elements. (A) Polyadenylated SV-A60 RNA or a derivative that contains the 34 base ARE from TNF-α (SVARE-A60) was incubated with HeLa S100 cytoplasmic extracts for the times indicated. Reaction products were analyzed on a 5% acrylamide gel containing 7 M urea. The positions of the polyadenylated input RNAs and deadenylated intermediates are indicated on the left. Note that while the rate of deadenylation is only moderately affected, the presence of an ARE greatly stimulates the decay of the SVARE-A60 transcript. (B) Gem-A0 RNA [which lacks a poly(A) tail] or a derivative that contains the 34 base ARE from TNF-α (GemARE-A0) was incubated with HeLa S100 cytoplasmic extracts for the times indicated. Reaction products were analyzed on a 5% acrylamide gel containing 7 M urea. (C) Gem-A0 RNA or a derivative that contains the 51 base ARE from GMCSF (GemGMCSF-A0) was incubated with HeLa S100 cytoplasmic extracts for the times indicated. Reaction products were analyzed on a 5% acrylamide gel containing 7 M urea. (D) GemARE-A0 RNA was incubated in the in vitro decay assay for 30 min using either untreated S100 extract (lane no antibody), extract that was treated with normal serum (lane normal sera) or extract that had been immunodepleted with antibodies specific for PM-Scl75 protein (lane α-PM-Scl75). Reaction products were analyzed on a 5% acrylamide gel containing 7 M urea.
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Fig. 6. PM-Scl75 protein interacts with RNAs that contain AU-rich instability elements. (A) Gem-A0 RNA or a derivative that contains the 34-base AU-rich element from TNF-α (GemARE-A0) was incubated with the indicated amounts of purified recombinant PM-Scl75 protein. Heparin-resistant protein–RNA complexes were resolved on a 5% native acrylamide gel. (B) Gem-A0 RNA or a derivative that contains the 51-base ARE from GMCSF (GemGMCSF-A0) was incubated with the indicated amounts of purified recombinant PM-Scl75 protein. Heparin- and spermidine-resistant protein–RNA complexes were resolved on a 5% native acrylamide gel.
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Fig. 7. The exosomal protein PM-Scl75 specifically binds to AU-rich instability elements. (A) RNAs that contain either the wild-type TNF-α ARE (lanes wt ARE) or a mutated variant (lanes mut ARE) were incubated with the indicated amounts of purified recombinant PM-Scl75 protein. Heparin-resistant protein–RNA complexes were resolved on a 5% native acrylamide gel. The sequence of the wild-type and mutated versions of the TNF-α ARE are shown at the bottom of the figure. (B) GemARE-A0 RNA was incubated with 50 ng of purified PM-Scl75 protein in the presence of the indicated amounts of a synthetic competitor RNA that contained either the TNF-α ARE (lanes ARE RNA competitor) or an unrelated sequence (lanes control RNA competitor). Heparin-resistant protein–RNA complexes were resolved on a 5% native acrylamide gel.
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Fig. 8. A model for the regulation of mammalian mRNA turnover by the exosome. (1) Loading of the exosome complex onto mRNAs is promoted by AREs in the 3′-UTR and may be influenced by known ARE-binding proteins such as HuR, AUF-1/hnRNP D, etc. (2) Once loaded, the exosome may help promote mRNA deadenylation. (3) Following deadenylation, the transcript is handed off very efficiently to the exosome that was loaded onto the 3′-UTR, and is degraded rapidly.

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