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. 2001 Feb 15;20(4):880-90.
doi: 10.1093/emboj/20.4.880.

The role of Upf proteins in modulating the translation read-through of nonsense-containing transcripts

W Wang  1 , K Czaplinski, Y Rao, S W Peltz
Affiliations

The role of Upf proteins in modulating the translation read-through of nonsense-containing transcripts

W Wang et al. EMBO J. .

Abstract

The yeast UPF1, UPF2 and UPF3 genes encode trans-acting factors of the nonsense-mediated mRNA decay pathway. In addition, the upf1Delta strain demonstrates a nonsense suppression phenotype and Upf1p has been shown to interact with the release factors eRF1 and eRF3. In this report, we show that both upf2Delta and upf3Delta strains demonstrate a nonsense suppression phenotype independent of their effect on mRNA turnover. We also demonstrate that Upf2p and Upf3p interact with eRF3, and that their ability to bind eRF3 correlates with their ability to complement the nonsense suppression phenotype. In vitro experiments demonstrate that Upf2p, Upf3p and eRF1 compete with each other for interacting with eRF3. Con versely, Upf1p binds to a different region of eRF3 and can form a complex with these factors. These results suggest a sequential surveillance complex assembly pathway, which occurs during the premature translation termination process. We propose that the observed nonsense suppression phenotype in the upfDelta strains can be attributed to a defect in the surveillance complex assembly.

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Figures

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Fig. 1. The upf2Δ and upf3Δ strains demonstrate a nonsense suppression phenotype. The UPF1, UPF2 and UPF3 genes in a wild-type yeast strain (KC2) were disrupted individually. Ten-fold serial dilutions of mid-log phase cells were plated on synthetic complete (SC) (right panel) and –tyr (left panel) plates, and their growth at 30°C was monitored.
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Fig. 2. (A) The abundance of wild-type and UGA-containing LacZ transcripts in the wild-type and upfΔ strains. Total RNAs isolated from the specified strains were separated on a 1% agarose gel and probed with 32P-labeled LacZ and U3 probes. (B) The nonsense suppression activity in wild-type and upfΔ strains was assessed quantitatively using a β-gal reporter system. The indicated yeast strains were transformed with either a wild-type LacZ gene or a LacZ gene containing the specified nonsense codon. The assays were performed as described in Materials and methods. (C) Nonsense suppression activity in wild type and strains harboring multiple UPF deletions.
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Fig. 3. Upf2p and Upf3p interact with the release factor eRF3. (A) Co-immunoprecipitation. Cells were transformed with either vector alone (lane 1) or vector expressing FLAG-tagged Upf1p, Upf2p or Upf3p (lanes 2–4). Cytoplasmic extracts were prepared and immuno precipitated with an anti-FLAG antibody. The immunoprecipitates were resolved by SDS–PAGE and subjected to immunoblotting with an anti-eRF3 polyclonal antibody. (B) Coomassie Blue staining of the purified fusion proteins. The protein molecular weight markers are as indicated. (C) GST pull-down experiment. Purified GST, GST–eRF1 or GST–eRF3 (1.0 µg each) was combined with glutathione–Sepharose beads and FLAG-Upf1p (1.0 µg), Upf2p (1.0 µg) or Upf3p (0.5 µg). Following incubation and extensive washing, the proteins remaining associated with the beads were analyzed by SDS–PAGE and immunoblotting with an anti-FLAG antibody.
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Fig. 4. The ability of Upf2p to complement the nonsense suppression phenotype in a upf2Δ strain correlates with its ability to interact with eRF3. (A) Co-immunoprecipitation. Cells were transformed with either vector alone or vector expressing the specified FLAG-Upf2p. Cytoplasmic extracts were prepared and immunoprecipitated with an anti-FLAG antibody. The immunoprecipitates were separated by SDS–PAGE and immunoblotted using the anti-eRF3 antibody. (B) GST pull-down experiment. Purified wild-type and mutant FLAG-Upf2 proteins (1.0 µg each) were combined with purified GST–eRF3 (1.0 µg) and glutathione–Sepharose beads. Following incubation and extensive washing, the proteins remaining associated with the beads were separated by SDS–PAGE and detected by the anti-FLAG antibody. (C) The ability of upf2 mutants to complement the nonsense suppression phenotype in a upf2Δ strain. Cells harboring either wild type or a UGA-containing LacZ gene were transformed with either vector alone or the vector expressing the specified upf2 gene. The assays were performed as described in Materials and methods.
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Fig. 5. The ability of Upf3p to complement the nonsense suppression phenotype in a upf3Δ strain correlates with its ability to interact with eRF3. (A) Co-immunoprecipitation. Cells were transformed with either vector alone (lane 5) or a vector expressing the specified FLAG-Upf3p. Cytoplasmic extracts were prepared and immunoprecipitated with an anti-FLAG antibody, and the immunoprecipitates were subjected to SDS–PAGE and immunoblotted by the anti-eRF3 antibody. (B) GST pull-down experiment. Purified wild-type and mutant FLAG-Upf3 proteins (0.5 µg each) were combined with purified GST–eRF3 (1.0 µg) and glutathione–Sepharose beads. Following incubation and extensive washing, the proteins remaining associated with the beads were separated by SDS–PAGE and detected by the anti-FLAG antibody. (C) The ability of upf3 mutants to complement the nonsense suppression phenotype in a upf3Δ strain. Cells harboring either wild-type or a UGA-containing LacZ gene were transformed with either the vector expressing the specified upf3 gene (lanes 1–4) or vector alone (lane 5). The assays were performed as described in Materials and methods.
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Fig. 6. The Upf proteins and eRF1 interact with the essential GTPase domain of eRF3. (A) Schematic diagram of the domain structure of the yeast release factor eRF3 (Sup35p). (B) GST pull-down experiment. Purified GST–eRF3 (1.0 µg, lanes 1–4), eRF3-N254Δ (1.0 µg, lanes 5–8) or eRF3-N465 (1.0 µg, lanes 9–12) was combined with FLAG-Upf1p (1.0 µg, lanes 1, 5 and 9), -Upf2p (1.0 µg, lanes 2, 6 and 10), -Upf3p (0.5 µg, lanes 3, 7 and 11) or -eRF1 (0.5 µg, lanes 4, 8 and 12). Following incubation and extensive washing, the proteins remaining associated with the beads were resolved on 12% SDS–PAGE and detected by the anti-FLAG antibody.
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Fig. 7. Upf2p, Upf3p and eRF1 competed with each other, but not with Upf1p, for binding to eRF3. (A) Analysis of the interactions between FLAG-Upf1p (1.0 µg), -Upf2p (1.0 µg), -Upf3p (0.5 µg) and GST–eRF3 (0.5 µg) in the absence or presence of FLAG-eRF1 (0.5 µg) by GST pull-down experiments. (B) Analysis of the interactions between FLAG-Upf2p (1.0 µg), -Upf3p (0.5 µg) and GST–eRF3 (0.5 µg) in the absence or presence of FLAG-Upf1p (1.0 µg) by GST pull-down experiments. (C) Analysis of the interactions between FLAG-Upf2p (1.0 µg) and GST–eRF3 (0.5 µg) in the absence (lane 1) or presence of increasing amounts of FLAG-Upf3p (lanes 2–4, 0.5, 1.0 and 2.0 µg) by GST pull-down experiments.
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Fig. 8. Model for the sequential surveillance complex assembly pathway. (1) The translating ribosome pauses at a premature termination codon and signals the eRF1–eRF3 complex to bind to its A site. The Upf1p becomes associated with the eRF1–eRF3 complex during the termination process. (2) After hydrolysis of the peptidyl-tRNA bond, eRF1 dissociates from the ribosome. Dissociation of eRF1 allows either Upf2p or Upf3p to bind the eRF3–Upf1p complex. (3) Rearrangement of the complex: Upf3p (or Upf2p) joins the complex and displaces eRF3 to form the mature surveillance complex.
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