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Bioenergetics and gene silencing approaches for unraveling nucleotide recognition by the human EIF2C2/Ago2 PAZ domain.

Kandeel M, Al-Taher A, Nakashima R, Sakaguchi T, Kandeel A, Nagaya Y, Kitamura Y, Kitade Y - PLoS ONE (2014)

Bottom Line: While a considerable amount of research has focused on RNA interference and its associated mechanisms, the nature and mechanisms of nucleotide recognition by the PAZ domain of EIF2C2/Ago2 have not yet been characterized.Additionally, higher in vivo gene silencing efficacy was correlated with the stronger PAZ domain binders.These findings provide new insights into the nature of the interactions of the EIF2C2/Ago2 PAZ domain.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Biochemistry and Pharmacology, Faculty of Veterinary Medicine and Animal Resources, King Faisal University, Alhofuf, Alahsa, Saudi Arabia; Department of Pharmacology, Faculty of Veterinary Medicine, Kafrelshikh University, Kafrelshikh, Egypt.

ABSTRACT
Gene silencing and RNA interference are major cellular processes that control gene expression via the cleavage of target mRNA. Eukaryotic translation initiation factor 2C2 (EIF2C2, Argonaute protein 2, Ago2) is considered to be the major player of RNAi as it is the core component of RISC complexes. While a considerable amount of research has focused on RNA interference and its associated mechanisms, the nature and mechanisms of nucleotide recognition by the PAZ domain of EIF2C2/Ago2 have not yet been characterized. Here, we demonstrate that the EIF2C2/Ago2 PAZ domain has an inherent lack of binding to adenine nucleotides, a feature that highlights the poor binding of 3'-adenylated RNAs with the PAZ domain as well as the selective high trimming of the 3'-ends of miRNA containing adenine nucleotides. We further show that the PAZ domain selectively binds all ribonucleotides (except adenosine), whereas it poorly recognizes deoxyribonucleotides. In this context, the modification of dTMP to its ribonucleotide analogue gave a drastic improvement of binding enthalpy and, hence, binding affinity. Additionally, higher in vivo gene silencing efficacy was correlated with the stronger PAZ domain binders. These findings provide new insights into the nature of the interactions of the EIF2C2/Ago2 PAZ domain.

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ITC profiles of the binding of nucleotides with the PAZ domain bound with various nucleotides.
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pone-0094538-g002: ITC profiles of the binding of nucleotides with the PAZ domain bound with various nucleotides.

Mentions: In a mutual competitive binding experiment, we assumed that there was residual binding of the ribonucleotide AMP and the deoxyribonucleotides dAMP and dCMP or uridine nucleoside with the PAZ domain. Thus, two sequential experiments were performed. The first experiment included the titration of the first (low or no affinity) ligand followed by titration with UMP. If binding of AMP, dAMP, dCMP, or uridine is likely to occur, the binding of UMP could be minimized by the degree of binding of the other ligand. As a control, the same experiments were carried out in the presence of stronger binding ligands such as GMP. Figure 2 shows the results of the competitive binding experiments. Titration of the EIF2C2/Apo2 PAZ domain with UMP gave the characteristic figure shown as (UMP). Titration with UMP into a preformed complex of the PAZ domain with GMP (GMP-UMP) or CMP (CMP-UMP) showed very weak signal without measurable thermal parameters, corresponding to a lack of UMP binding. Furthermore, in the presence of the medium affinity binding ligand dGMP (dGMP-UMP) or TMP (TMP-UMP), UMP showed a corresponding decreased access to the binding site as evidenced by lower exothermic peaks compared with the control (UMP). Finally, there was a slight decrease in the affinity of UMP in the presence of other ligands (dCMP, dAMP, and uridine). The calculated values of the weak binding ligands are given in Table 3. The estimated values of such low affinity ligands were 72–159 M−1, while uridine was not expected to bind with the PAZ domain.


Bioenergetics and gene silencing approaches for unraveling nucleotide recognition by the human EIF2C2/Ago2 PAZ domain.

Kandeel M, Al-Taher A, Nakashima R, Sakaguchi T, Kandeel A, Nagaya Y, Kitamura Y, Kitade Y - PLoS ONE (2014)

ITC profiles of the binding of nucleotides with the PAZ domain bound with various nucleotides.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4008379&req=5

pone-0094538-g002: ITC profiles of the binding of nucleotides with the PAZ domain bound with various nucleotides.
Mentions: In a mutual competitive binding experiment, we assumed that there was residual binding of the ribonucleotide AMP and the deoxyribonucleotides dAMP and dCMP or uridine nucleoside with the PAZ domain. Thus, two sequential experiments were performed. The first experiment included the titration of the first (low or no affinity) ligand followed by titration with UMP. If binding of AMP, dAMP, dCMP, or uridine is likely to occur, the binding of UMP could be minimized by the degree of binding of the other ligand. As a control, the same experiments were carried out in the presence of stronger binding ligands such as GMP. Figure 2 shows the results of the competitive binding experiments. Titration of the EIF2C2/Apo2 PAZ domain with UMP gave the characteristic figure shown as (UMP). Titration with UMP into a preformed complex of the PAZ domain with GMP (GMP-UMP) or CMP (CMP-UMP) showed very weak signal without measurable thermal parameters, corresponding to a lack of UMP binding. Furthermore, in the presence of the medium affinity binding ligand dGMP (dGMP-UMP) or TMP (TMP-UMP), UMP showed a corresponding decreased access to the binding site as evidenced by lower exothermic peaks compared with the control (UMP). Finally, there was a slight decrease in the affinity of UMP in the presence of other ligands (dCMP, dAMP, and uridine). The calculated values of the weak binding ligands are given in Table 3. The estimated values of such low affinity ligands were 72–159 M−1, while uridine was not expected to bind with the PAZ domain.

Bottom Line: While a considerable amount of research has focused on RNA interference and its associated mechanisms, the nature and mechanisms of nucleotide recognition by the PAZ domain of EIF2C2/Ago2 have not yet been characterized.Additionally, higher in vivo gene silencing efficacy was correlated with the stronger PAZ domain binders.These findings provide new insights into the nature of the interactions of the EIF2C2/Ago2 PAZ domain.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Biochemistry and Pharmacology, Faculty of Veterinary Medicine and Animal Resources, King Faisal University, Alhofuf, Alahsa, Saudi Arabia; Department of Pharmacology, Faculty of Veterinary Medicine, Kafrelshikh University, Kafrelshikh, Egypt.

ABSTRACT
Gene silencing and RNA interference are major cellular processes that control gene expression via the cleavage of target mRNA. Eukaryotic translation initiation factor 2C2 (EIF2C2, Argonaute protein 2, Ago2) is considered to be the major player of RNAi as it is the core component of RISC complexes. While a considerable amount of research has focused on RNA interference and its associated mechanisms, the nature and mechanisms of nucleotide recognition by the PAZ domain of EIF2C2/Ago2 have not yet been characterized. Here, we demonstrate that the EIF2C2/Ago2 PAZ domain has an inherent lack of binding to adenine nucleotides, a feature that highlights the poor binding of 3'-adenylated RNAs with the PAZ domain as well as the selective high trimming of the 3'-ends of miRNA containing adenine nucleotides. We further show that the PAZ domain selectively binds all ribonucleotides (except adenosine), whereas it poorly recognizes deoxyribonucleotides. In this context, the modification of dTMP to its ribonucleotide analogue gave a drastic improvement of binding enthalpy and, hence, binding affinity. Additionally, higher in vivo gene silencing efficacy was correlated with the stronger PAZ domain binders. These findings provide new insights into the nature of the interactions of the EIF2C2/Ago2 PAZ domain.

Show MeSH