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Studies on human eRF3-PABP interaction reveal the influence of eRF3a N-terminal glycin repeat on eRF3-PABP binding affinity and the lower affinity of eRF3a 12-GGC allele involved in cancer susceptibility.

Jerbi S, Jolles B, Bouceba T, Jean-Jean O - RNA Biol (2016)

Bottom Line: There are five known eRF3a/GSPT1 alleles in the human population, encoding 7, 9, 10, 11 and 12 glycines.We found that the N-terminal glycine repeat of eRF3a influences eRF3a-PABP interaction and that eRF3a 12-GGC allele has a decreased binding affinity for PABP.Our comparative analysis on eRF3a alleles suggests that the presence of eRF3a 12-GGC allele could modify the coupling between translation termination and mRNA deadenylation.

View Article: PubMed Central - PubMed

Affiliation: a Sorbonne Universités, UPMC Univ Paris 06, Institut de Biologie Paris-Seine (IBPS), CNRS-UMR8256 , 7 quai Saint Bernard, Paris , France.

ABSTRACT
The eukaryotic release factor 3 (eRF3) has been involved in the control of mRNA degradation through its association with the cytoplasmic Poly(A) Binding Protein, PABP. In mammals, eRF3 N-terminal domain contains two overlapping PAM2 motifs which specifically recognize the MLLE domain of PABP. In humans, eRF3a/GSPT1 gene contains a stable GGC repeat encoding a repeat of glycine residues in eRF3a N-terminus. There are five known eRF3a/GSPT1 alleles in the human population, encoding 7, 9, 10, 11 and 12 glycines. Several studies have reported that the presence of eRF3a 12-GGC allele is correlated with an increased risk of cancer development. Using surface plasmon resonance, we have studied the interaction of the various allelic forms of eRF3a with PABP alone or poly(A)-bound PABP. We found that the N-terminal glycine repeat of eRF3a influences eRF3a-PABP interaction and that eRF3a 12-GGC allele has a decreased binding affinity for PABP. Our comparative analysis on eRF3a alleles suggests that the presence of eRF3a 12-GGC allele could modify the coupling between translation termination and mRNA deadenylation.

No MeSH data available.


Related in: MedlinePlus

Kinetic analyses of PABP binding to 5′ biotinylated OligoA120 RNA (220 RU) immobilized onto a SA sensor chip surface. (A) Sensorgrams of the binding profiles using a Langmuir 1:1 binding model (top panel) and residuals plots (bottom panel) are shown. Concentrations of PABP injected are indicated on the right of the sensorgrams. Kinetic parameters (ka, kd and KD) and the Chi2 value of the fitting are also indicated. (B) Plot of the response vs. PABP concentration used for the steady-state affinity fitting with the BIAevaluation software (see text for details).
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f0002: Kinetic analyses of PABP binding to 5′ biotinylated OligoA120 RNA (220 RU) immobilized onto a SA sensor chip surface. (A) Sensorgrams of the binding profiles using a Langmuir 1:1 binding model (top panel) and residuals plots (bottom panel) are shown. Concentrations of PABP injected are indicated on the right of the sensorgrams. Kinetic parameters (ka, kd and KD) and the Chi2 value of the fitting are also indicated. (B) Plot of the response vs. PABP concentration used for the steady-state affinity fitting with the BIAevaluation software (see text for details).

Mentions: We next determined the kinetic parameters of PABP-OligoA120 interaction (Fig. 2). A range of PABP (0.75 to 2.5 nM) concentrations was injected for 5 min, followed by a dissociation phase of 8 min over the immobilized OligoA120 (220 RU). Disruption of any complex that remained bound after a 8 min dissociation was achieved using a 1 min injection of 0.1% SDS at 30 µl/min. After blank subtraction (no target RNA was captured on the reference flow cell surface), the analysis of the sensorgrams yielded good fits when a single site interaction model was applied. The fitting to this model was also judged by the low value of the Chi2 and the good quality of the residuals (Fig. 2A). The results from the fittings of the PABP-OligoA120 interaction indicated a moderatly fast association rate (ka) of 1.15 × 104 M−1 s−1, a very slow dissociation rate (kd) of 1.72 × 10−5 s−1, and a resulting KD of 1.5 nM. The values of response at equilibrium obtained for PABP-OligoA120 interaction in the experiments shown in Fig. 2A and Fig. S5, which were performed on the same immobilized OligoA120 surface, were also employed to calculate the equilibrium constant (KD) of PABP-OligoA120 interaction. These values were processed using the BIAevalution software for the steady-state affinity fitting. The curve of the response at equilibrium versus PABP concentration confirmed the KD value of 1.5 nM (Fig. 2B).Figure 2.


Studies on human eRF3-PABP interaction reveal the influence of eRF3a N-terminal glycin repeat on eRF3-PABP binding affinity and the lower affinity of eRF3a 12-GGC allele involved in cancer susceptibility.

Jerbi S, Jolles B, Bouceba T, Jean-Jean O - RNA Biol (2016)

Kinetic analyses of PABP binding to 5′ biotinylated OligoA120 RNA (220 RU) immobilized onto a SA sensor chip surface. (A) Sensorgrams of the binding profiles using a Langmuir 1:1 binding model (top panel) and residuals plots (bottom panel) are shown. Concentrations of PABP injected are indicated on the right of the sensorgrams. Kinetic parameters (ka, kd and KD) and the Chi2 value of the fitting are also indicated. (B) Plot of the response vs. PABP concentration used for the steady-state affinity fitting with the BIAevaluation software (see text for details).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4829321&req=5

f0002: Kinetic analyses of PABP binding to 5′ biotinylated OligoA120 RNA (220 RU) immobilized onto a SA sensor chip surface. (A) Sensorgrams of the binding profiles using a Langmuir 1:1 binding model (top panel) and residuals plots (bottom panel) are shown. Concentrations of PABP injected are indicated on the right of the sensorgrams. Kinetic parameters (ka, kd and KD) and the Chi2 value of the fitting are also indicated. (B) Plot of the response vs. PABP concentration used for the steady-state affinity fitting with the BIAevaluation software (see text for details).
Mentions: We next determined the kinetic parameters of PABP-OligoA120 interaction (Fig. 2). A range of PABP (0.75 to 2.5 nM) concentrations was injected for 5 min, followed by a dissociation phase of 8 min over the immobilized OligoA120 (220 RU). Disruption of any complex that remained bound after a 8 min dissociation was achieved using a 1 min injection of 0.1% SDS at 30 µl/min. After blank subtraction (no target RNA was captured on the reference flow cell surface), the analysis of the sensorgrams yielded good fits when a single site interaction model was applied. The fitting to this model was also judged by the low value of the Chi2 and the good quality of the residuals (Fig. 2A). The results from the fittings of the PABP-OligoA120 interaction indicated a moderatly fast association rate (ka) of 1.15 × 104 M−1 s−1, a very slow dissociation rate (kd) of 1.72 × 10−5 s−1, and a resulting KD of 1.5 nM. The values of response at equilibrium obtained for PABP-OligoA120 interaction in the experiments shown in Fig. 2A and Fig. S5, which were performed on the same immobilized OligoA120 surface, were also employed to calculate the equilibrium constant (KD) of PABP-OligoA120 interaction. These values were processed using the BIAevalution software for the steady-state affinity fitting. The curve of the response at equilibrium versus PABP concentration confirmed the KD value of 1.5 nM (Fig. 2B).Figure 2.

Bottom Line: There are five known eRF3a/GSPT1 alleles in the human population, encoding 7, 9, 10, 11 and 12 glycines.We found that the N-terminal glycine repeat of eRF3a influences eRF3a-PABP interaction and that eRF3a 12-GGC allele has a decreased binding affinity for PABP.Our comparative analysis on eRF3a alleles suggests that the presence of eRF3a 12-GGC allele could modify the coupling between translation termination and mRNA deadenylation.

View Article: PubMed Central - PubMed

Affiliation: a Sorbonne Universités, UPMC Univ Paris 06, Institut de Biologie Paris-Seine (IBPS), CNRS-UMR8256 , 7 quai Saint Bernard, Paris , France.

ABSTRACT
The eukaryotic release factor 3 (eRF3) has been involved in the control of mRNA degradation through its association with the cytoplasmic Poly(A) Binding Protein, PABP. In mammals, eRF3 N-terminal domain contains two overlapping PAM2 motifs which specifically recognize the MLLE domain of PABP. In humans, eRF3a/GSPT1 gene contains a stable GGC repeat encoding a repeat of glycine residues in eRF3a N-terminus. There are five known eRF3a/GSPT1 alleles in the human population, encoding 7, 9, 10, 11 and 12 glycines. Several studies have reported that the presence of eRF3a 12-GGC allele is correlated with an increased risk of cancer development. Using surface plasmon resonance, we have studied the interaction of the various allelic forms of eRF3a with PABP alone or poly(A)-bound PABP. We found that the N-terminal glycine repeat of eRF3a influences eRF3a-PABP interaction and that eRF3a 12-GGC allele has a decreased binding affinity for PABP. Our comparative analysis on eRF3a alleles suggests that the presence of eRF3a 12-GGC allele could modify the coupling between translation termination and mRNA deadenylation.

No MeSH data available.


Related in: MedlinePlus