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, 40 (3), 1160-73

Interaction of PABPC1 With the Translation Initiation Complex Is Critical to the NMD Resistance of AUG-proximal Nonsense Mutations

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Interaction of PABPC1 With the Translation Initiation Complex Is Critical to the NMD Resistance of AUG-proximal Nonsense Mutations

Isabel Peixeiro et al. Nucleic Acids Res.

Abstract

Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and rapidly degrades mRNAs containing premature termination codons (PTC). The strength of the NMD response appears to reflect multiple determinants on a target mRNA. We have previously reported that mRNAs containing PTCs in close proximity to the translation initiation codon (AUG-proximal PTCs) can substantially evade NMD. Here, we explore the mechanistic basis for this NMD resistance. We demonstrate that translation termination at an AUG-proximal PTC lacks the ribosome stalling that is evident in an NMD-sensitive PTC. This difference is associated with demonstrated interactions of the cytoplasmic poly(A)-binding protein 1, PABPC1, with the cap-binding complex subunit, eIF4G and the 40S recruitment factor eIF3 as well as the ribosome release factor, eRF3. These interactions, in combination, underlie critical 3'-5' linkage of translation initiation with efficient termination at the AUG-proximal PTC and contribute to an NMD-resistant PTC definition at an early phase of translation elongation.

Figures

Figure 1.
Figure 1.
Translation termination at the AUG-proximal β15 PTC occurs in the absence of the ribosome stalling evident at the more distal β39 PTC. (A) Diagram of the human β-globin mRNA showing positions of initiation and termination codons [native (N) or premature (at position 15 or 39)]. The arrow indicates the position and orientation of the #17 primer (Table 1) used in the primer-extension (toeprinting) assays. The lines below the mRNA diagram represent the length in nucleotides of the synthesized cDNA from the #17 primer to the 5′-end of the mRNA, the initiation AUG codon, or to the βN, β15 and β39 translation termination codons. (B) Electropherograms of βN, β15 and β39 toeprint assays using the fluorescently labeled #17 primer. The toeprint reactions were performed in the presence or absence of cycloheximide (+CHX or −CHX, respectively). A control reaction was carried out in the absence of added rabbit reticulocyte lysate (−RRL). Primer extension products were suspended in a mix containing ROX 2500 molecular weight marker (Applied Biosystems) and subjected to capillary electrophoresis. Size standard peaks are shown below and sequence position is indicated in nucleotides. The toeprint peaks at 626 nt represent the full-length transcript; the peaks at 620 nt correspond to the cap-binding complex-bound full-length transcript; the peaks at 555-nt map 18-nt downstream the AUG codon (AUG TP). The peak at 440-nt maps to a position 16 nt downstream the β39 stop codon (PTC TP). (C) The arrow below the mRNA diagram represents position and orientation of #18 primer (Table 1) used in the second, high-resolution toeprinting assays. The lines below the mRNA diagram represent the length in nucleotides of the primer-extended cDNA to the 5′-end of the mRNA, to the initiation AUG, as well as to the translation termination codons at positions 15 or 39. (D) Electropherograms of β15 and β39 toeprint assays using the fluorescently labeled #18 primer. A parallel sequencing reaction on the βN cDNA, performed with the same primer is shown on the top. Underlined sequences indicate codon position: 0 (AUG), 15 and 39. The toeprint reactions were performed in the presence or absence of cycloheximide (+CHX or −CHX, respectively). A control reaction lacking −RRL is shown. Primer extension and sequencing products were resuspended in a mix containing ROX 500 (Applied Biosystems) molecular weight standard and separated by capillary electrophoresis. Sequence position is indicated in nucleotides. Peaks at 230 nt correspond to the initiation AUG toeprint that maps 18-nt downstream the AUG codon. The peak at 115 nt, present uniquely in the β39 analysis, maps 16-nt downstream of the PTC.
Figure 1.
Figure 1.
Translation termination at the AUG-proximal β15 PTC occurs in the absence of the ribosome stalling evident at the more distal β39 PTC. (A) Diagram of the human β-globin mRNA showing positions of initiation and termination codons [native (N) or premature (at position 15 or 39)]. The arrow indicates the position and orientation of the #17 primer (Table 1) used in the primer-extension (toeprinting) assays. The lines below the mRNA diagram represent the length in nucleotides of the synthesized cDNA from the #17 primer to the 5′-end of the mRNA, the initiation AUG codon, or to the βN, β15 and β39 translation termination codons. (B) Electropherograms of βN, β15 and β39 toeprint assays using the fluorescently labeled #17 primer. The toeprint reactions were performed in the presence or absence of cycloheximide (+CHX or −CHX, respectively). A control reaction was carried out in the absence of added rabbit reticulocyte lysate (−RRL). Primer extension products were suspended in a mix containing ROX 2500 molecular weight marker (Applied Biosystems) and subjected to capillary electrophoresis. Size standard peaks are shown below and sequence position is indicated in nucleotides. The toeprint peaks at 626 nt represent the full-length transcript; the peaks at 620 nt correspond to the cap-binding complex-bound full-length transcript; the peaks at 555-nt map 18-nt downstream the AUG codon (AUG TP). The peak at 440-nt maps to a position 16 nt downstream the β39 stop codon (PTC TP). (C) The arrow below the mRNA diagram represents position and orientation of #18 primer (Table 1) used in the second, high-resolution toeprinting assays. The lines below the mRNA diagram represent the length in nucleotides of the primer-extended cDNA to the 5′-end of the mRNA, to the initiation AUG, as well as to the translation termination codons at positions 15 or 39. (D) Electropherograms of β15 and β39 toeprint assays using the fluorescently labeled #18 primer. A parallel sequencing reaction on the βN cDNA, performed with the same primer is shown on the top. Underlined sequences indicate codon position: 0 (AUG), 15 and 39. The toeprint reactions were performed in the presence or absence of cycloheximide (+CHX or −CHX, respectively). A control reaction lacking −RRL is shown. Primer extension and sequencing products were resuspended in a mix containing ROX 500 (Applied Biosystems) molecular weight standard and separated by capillary electrophoresis. Sequence position is indicated in nucleotides. Peaks at 230 nt correspond to the initiation AUG toeprint that maps 18-nt downstream the AUG codon. The peak at 115 nt, present uniquely in the β39 analysis, maps 16-nt downstream of the PTC.
Figure 2.
Figure 2.
PABPC1 plays an essential role in NMD resistance of an AUG-proximal PTC. (A) Diagram representing βN, the NMD-resistant β-globin mRNA with an AUG-proximal nonsense mutation at codon 15 (UGA; β15) and the NMD-sensitive β39 mRNA. The positions of initiation (AUG) and termination (native and premature) codons are indicated. (B) Representative western blot analysis of HeLa cells extracts transfected with human PABPC1 siRNA (lanes 4–6) or with a control Luciferase siRNA target (LUC siRNA; lanes 1–3). Twenty-four hours after siRNA treatment, cells were transfected with the β-globin reporter constructs (βN, β15 or β39) with or without a plasmid expressing PABPdelC mutant protein (pDESTPABPC1delC plasmid); lanes 4–6 or 1–3, respectively. Twenty-four hours post-transfection, protein and RNA were isolated from the cells for analysis. Immunoblotting to confirm PABPC1 knockdown was carried out with anti-PABPC1 (specific for the N-terminal domain) and with anti-α-tubulin antibodies as a loading control (lanes 4–6 versus lanes 1–3). Identification of each band is on the right. (C) Depletion of endogenous PABPC1 in conjunction with expression of exogenous PABPdelC protein represses β15 mRNA levels. Relative β-globin mRNA levels under control conditions (LUC siRNA-treated HeLa cells; dark bars) and at conditions of PABPdelC expression in PABPC1-depleted HeLa cells (PABPC1 siRNA + PABPdelC overexpression; light bars), normalized to the levels of puromycin resistance mRNA (Puror; plasmids carrying the reporter β-globin gene also contain the Puror gene), were determined by quantitative RT–qPCR and compared to the corresponding βN mRNA levels (defined as 100%). Average and standard deviation (SD) of three independent experiments corresponding to three independent transfections are shown in the histogram. (D) Representative western blot analysis of HeLa cells extracts transfected with control Luciferase siRNA (LUC siRNA; lanes 1–3) or with siRNA targeting the human PABPC1 3′-UTR (PABPC1 3′-UTR siRNA; lanes 4–9). After siRNA treatment, cells were transfected with the plasmids expressing βN, β15 or β39 mRNAs (lanes 1–3) or cotransfected with these plasmids in combination with a plasmid expressing PABPdelC mutant protein [pDESTPABPC1delC plasmid as above in (B); lanes 4–6], or with a plasmid expressing wild-type PABPC1 (encoded by an mRNA with an heterologous 3′-UTR resistant to the PABPC1 siRNA; lanes 7–9). Protein levels present in the cell extracts were analyzed by western blot for PABPC1 and α-tubulin (loading control) as in (B), to monitor endogenous PABPC1 knockdown (lanes 4–9 versus lanes 1–3) and exogenous expression of mutant PABPdelC protein (lanes 4–6) or PABPC1 expression rescue (lanes 7–9). Identification of each band is indicated to the right of the gel image. The histogram below the immunoblot shows the average and SD values of three independent experiments for quantification of relative PABPC1 protein levels. (E) Expression of wild-type PABPC, but not PABPdelC, restores β15 expression in PABPC1 depleted cells. Relative β-globin mRNA levels for the control conditions (LUC siRNA-treated cells; dark bars), at conditions of PABPdelC expression in PABPC1-depleted HeLa cells (PABPC1 siRNA + PABPdelC overexpression; light bars), and at conditions of PABPC1 rescue (PABPC1 siRNA-treated cells plus expression of exogenous PABPC1; light bars) were determined by RT–qPCR and compared to the corresponding βN mRNA levels as in (C). Average values and SD of three independent experiments corresponding to three independent transfections are shown in the histogram. All values are represented as a percentage (%) of the corresponding βN mRNA (defined as 100%).
Figure 3.
Figure 3.
The N-terminal domain of eRF3 is critical to NMD evasion by an AUG-proximal nonsense-mutated mRNA. (A) Western blot analysis of HeLa cells extracts transfected with human eRF3 siRNA (lanes 4–6) or with a control Luciferase siRNA target (LUC siRNA; lanes 1–3). After siRNA treatment, cells were transfected with plasmids expressing βN, β15 or β39 mRNAs with or without a plasmid expressing eRF3delN mutant protein (pcDNAeRF3delN plasmid); lanes 4–6 or 1–3, respectively. Twenty-four hours post-transfection, protein and RNA were isolated from the cells. Immunoblotting was carried out with anti-eRF3 to monitor endogenous eRF3 knockdown (lanes 4–6 versus lanes 1–3) and expression of mutant eRF3delN protein (lanes 4–6). Detection of α-tubulin served as a loading control. Identification of each band is on the right. (B) Depletion of endogenous eRF3 in combination with expression of exogenous eRF3delN represses β15 mRNA levels. Relative levels of β-globin mRNA under control conditions (LUC siRNA-treated cells; dark bars) and in eRF3-depleted cells expressing exogenous eRF3delN protein (eRF3 siRNA + eRF3delN overexpression; light bars), normalized to the levels of Puror mRNA expressed from the β-globin plasmids, were determined by quantitative RT–PCR and compared to the corresponding βN mRNA levels (defined as 100%). Average and SD values of four independent experiments corresponding to three independent transfections are shown in the histogram.
Figure 4.
Figure 4.
Overexpression of PAIP2 induces NMD sensitivity in the β15 nonsense-mutated mRNA. (A) Western blot analysis of HeLa cells treated with siRNAs specific to Luciferase (LUC) (lanes 1–6), or siRNA targeting UPF1 (lanes 7–9). After siRNA treatment, cells were transfected with plasmids expressing βN, β15 or β39 mRNAs with or without a plasmid expressing PAIP2 protein (pDEST26PAIP2 plasmid); lanes 4–9 or 1–3, respectively. Twenty-four hours post-transfection, protein and RNA were isolated from the cells for analysis. Immunoblotting was carried out with anti-UPF1, anti-PAIP2 and anti-α-tubulin antibodies. Detection of α-tubulin served as a loading control. Identification of each band is indicated to the right of the gel image. (B) Overexpression of PAIP2 represses β15 mRNA levels in a UPF1-sensitive manner. Relative β-globin mRNA levels under control conditions (dark bars), PAIP2 overexpression (dark grey bars), and in conditions of PAIP2 overexpression co-existing with UPF1 depletion (light bars) are shown. All values determined by RT–qPCR are normalized to the mRNA levels of Puror, and compared to the corresponding βN mRNA levels. Average values and SD of four independent experiments corresponding to four independent transfections are shown. All values are represented as a percentage (%) of the corresponding βN mRNA (defined as 100%).
Figure 5.
Figure 5.
The translation initiation factors eIF3h and eIF3f are required for the NMD-resistant phenotype of the β15 nonsense-mutated mRNA. (A) Representation of the predicted organization of the mammalian eIF3 subunits [adapted from (40–42)]. Free eIF3 complex can be assigned into three stable modules. One module consists of eIF3a, b, g and i, and it interacts with a second module composed by eIF3c, d, e, l and k. eIF3b functions as a scaffold protein connecting eIF3a, c, i and g subunits. The third subcomplex is composed by eIF3f, h and m. Subunits eIF3f and m bind to the subcomplex eIF3 c:d:e:l:k through subunit eIF3h. The remaining subunit eIF3j, a labile subunit, attaches to the complex via eIF3b (40–42). (B–D) Representative RT–PCR analyses of RNAs extracted from untreated (Ctl; lane 1), Luciferase (LUC; lane 2) or eIF3h, eIF3f and eIF3e (lane 3) siRNAs-treated HeLa cells, respectively, in panels (B–D). RT–PCRs were carried out with eIF3h, eIF3f or eIF3e mRNA specific primers to monitor endogenous eIF3h, eIF3f or eIF3e knockdown, respectively (lanes 3 versus lanes 1–2). The eIF3h, eIF3f and eIF3e mRNA levels were normalized to those of histone deacetylase 1 (HDAC1) mRNA level. In each panel, the right three lanes correspond to serial dilutions of RNA, demonstrating semiquantitative conditions used for RT–PCR. (E) Representative western blot analysis of HeLa cells extracts, untreated (Ctl, lane 1) or treated with Luciferase (LUC; lane 2), eIF3h (lane 3), eIF3f (lane 4) or eIF3e (lane 5) specific siRNAs (see ‘Material and Methods’ section). After siRNA treatment, cells were transiently transfected with the pEGFP plasmid (BD Biosciences) expressing green fluorescent protein (GFP). Protein lysates were analyzed by immunoblotting using anti-GFP and anti-α-tubulin (loading control) antibodies to monitor GFP protein expression. Identification of each band is indicated to the right of the gel image. (F, H and J) Semiquantitative RT–PCR analyses of RNAs extracted from HeLa cells transfected with a Luciferase (LUC) siRNA target (lanes 1–3) or human eIF3h, eIF3f or eIF3e siRNAs, respectively, at panels (F, H or J) (lanes 4–6 of each panel), using the same experimental settings as in (B). Twenty-four hours after siRNA-treatment, cells were transfected with plasmids expressing βN, β15 or β39 mRNAs. Twenty-four hours after constructs transfection, total RNA was isolated from the cells. RT–PCR was carried out as in (B) with eIF3h, eIF3f or eIF3e mRNA specific primers, respectively, to monitor endogenous eIF3h, eIF3f or eIF3e knockdown (lanes 4–6 versus lanes 1–3). (G) Knockdown of eIF3h represses β15 mRNA levels. Relative β-globin mRNA levels for the control conditions (LUC siRNA; dark bars) and at conditions of eIF3h depletion (eIF3h siRNA; light bars), normalized to the levels of puromycin resistance mRNA encoded from the β-globin gene plasmid, were determined by quantitative RT–PCR (RT–qPCR) and compared to the corresponding βN mRNA levels (defined as 100%). (I) Knockdown of eIF3f represses β15 mRNA levels. Relative β-globin mRNA levels for the control conditions (LUC siRNA; dark bars) and at conditions of eIF3f depletion (eIF3f siRNA; light bars), normalized to the levels of puromycin resistance mRNA were determined by RT–qPCR and compared to the corresponding βN mRNA levels (defined as 100%). (K) Knockdown of eIF3e fails to repress β15 mRNA expression. Relative β-globin mRNA levels for the control conditions (LUC siRNA; dark bars) and at conditions of eIF3e depletion (eIF3e siRNA; light bars), normalized to the levels of puromycin resistance mRNA were determined by RT–qPCR as in (G) and compared to the corresponding βN mRNA levels (defined as 100%). (G, I and K) histograms show average and SD values of three independent experiments corresponding to three independent transfections.
Figure 5.
Figure 5.
The translation initiation factors eIF3h and eIF3f are required for the NMD-resistant phenotype of the β15 nonsense-mutated mRNA. (A) Representation of the predicted organization of the mammalian eIF3 subunits [adapted from (40–42)]. Free eIF3 complex can be assigned into three stable modules. One module consists of eIF3a, b, g and i, and it interacts with a second module composed by eIF3c, d, e, l and k. eIF3b functions as a scaffold protein connecting eIF3a, c, i and g subunits. The third subcomplex is composed by eIF3f, h and m. Subunits eIF3f and m bind to the subcomplex eIF3 c:d:e:l:k through subunit eIF3h. The remaining subunit eIF3j, a labile subunit, attaches to the complex via eIF3b (40–42). (B–D) Representative RT–PCR analyses of RNAs extracted from untreated (Ctl; lane 1), Luciferase (LUC; lane 2) or eIF3h, eIF3f and eIF3e (lane 3) siRNAs-treated HeLa cells, respectively, in panels (B–D). RT–PCRs were carried out with eIF3h, eIF3f or eIF3e mRNA specific primers to monitor endogenous eIF3h, eIF3f or eIF3e knockdown, respectively (lanes 3 versus lanes 1–2). The eIF3h, eIF3f and eIF3e mRNA levels were normalized to those of histone deacetylase 1 (HDAC1) mRNA level. In each panel, the right three lanes correspond to serial dilutions of RNA, demonstrating semiquantitative conditions used for RT–PCR. (E) Representative western blot analysis of HeLa cells extracts, untreated (Ctl, lane 1) or treated with Luciferase (LUC; lane 2), eIF3h (lane 3), eIF3f (lane 4) or eIF3e (lane 5) specific siRNAs (see ‘Material and Methods’ section). After siRNA treatment, cells were transiently transfected with the pEGFP plasmid (BD Biosciences) expressing green fluorescent protein (GFP). Protein lysates were analyzed by immunoblotting using anti-GFP and anti-α-tubulin (loading control) antibodies to monitor GFP protein expression. Identification of each band is indicated to the right of the gel image. (F, H and J) Semiquantitative RT–PCR analyses of RNAs extracted from HeLa cells transfected with a Luciferase (LUC) siRNA target (lanes 1–3) or human eIF3h, eIF3f or eIF3e siRNAs, respectively, at panels (F, H or J) (lanes 4–6 of each panel), using the same experimental settings as in (B). Twenty-four hours after siRNA-treatment, cells were transfected with plasmids expressing βN, β15 or β39 mRNAs. Twenty-four hours after constructs transfection, total RNA was isolated from the cells. RT–PCR was carried out as in (B) with eIF3h, eIF3f or eIF3e mRNA specific primers, respectively, to monitor endogenous eIF3h, eIF3f or eIF3e knockdown (lanes 4–6 versus lanes 1–3). (G) Knockdown of eIF3h represses β15 mRNA levels. Relative β-globin mRNA levels for the control conditions (LUC siRNA; dark bars) and at conditions of eIF3h depletion (eIF3h siRNA; light bars), normalized to the levels of puromycin resistance mRNA encoded from the β-globin gene plasmid, were determined by quantitative RT–PCR (RT–qPCR) and compared to the corresponding βN mRNA levels (defined as 100%). (I) Knockdown of eIF3f represses β15 mRNA levels. Relative β-globin mRNA levels for the control conditions (LUC siRNA; dark bars) and at conditions of eIF3f depletion (eIF3f siRNA; light bars), normalized to the levels of puromycin resistance mRNA were determined by RT–qPCR and compared to the corresponding βN mRNA levels (defined as 100%). (K) Knockdown of eIF3e fails to repress β15 mRNA expression. Relative β-globin mRNA levels for the control conditions (LUC siRNA; dark bars) and at conditions of eIF3e depletion (eIF3e siRNA; light bars), normalized to the levels of puromycin resistance mRNA were determined by RT–qPCR as in (G) and compared to the corresponding βN mRNA levels (defined as 100%). (G, I and K) histograms show average and SD values of three independent experiments corresponding to three independent transfections.
Figure 6.
Figure 6.
A model for NMD resistance of AUG-proximal nonsense-mutated mRNAs. The current and prior data supports the model shown in this figure. During cap-mediated translation initiation, PABPC1 interacts with the initiation factor eIF4G. This interaction indirectly tethers PABPC1 to the 40S ribosomal subunit via the interaction of eIF4G with eIF3 subunits. The resulting configuration brings PABPC1 into in the vicinity of the AUG initiation codon as a consequence of 43S scanning and the maintenance of eIF4G–PABPC1 association with the 40S during the initial phase of translation elongation brings it into close contact with an AUG-proximal PTC in a transcript where the ORF is quite short. This proximity to the PTC allows PABPC1 to interact with the release factor eRF3 at the termination complex, thus impairing the association of UPF1 to the ribonucleoprotein complex, resulting in efficient translation termination and inhibition of NMD.

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