Bioenergetics and gene silencing approaches for unraveling nucleotide recognition by the human EIF2C2/Ago2 PAZ domain.
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.
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.
Mahmoud
M
Kandeel
Department of Physiology, Biochemistry and Pharmacology, Faculty of Veterinary Medicine and Animal Resources, King Faisal University, Alhofuf, Alahsa, Saudi Arabia.
Abdullah
A
Al-Taher
United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, Gifu, Japan.
Remi
R
Nakashima
Department of Biomolecular Science, Faculty of Engineering, Gifu University, Gifu, Japan.
Tomoya
T
Sakaguchi
Department of Biology, Faculty of Sciences and Arts, Alkamil Branch, King Abdul Aziz University, Alkamil, Saudi Arabia; Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt.
Ali
A
Kandeel
Department of Biomolecular Science, Faculty of Engineering, Gifu University, Gifu, Japan.
Yuki
Y
Nagaya
United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, Gifu, Japan.
Yoshiaki
Y
Kitamura
United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, Gifu, Japan; Department of Biomolecular Science, Faculty of Engineering, Gifu University, Gifu, Japan.
Yukio
Y
Kitade
D060565:Argonaute Proteins / Q000737:chemistry* / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D004734:Energy Metabolism; D019277:Entropy; D006367:HeLa Cells; D006358:Hot Temperature; D006801:Humans; D008958:Models, Molecular; D009711:Nucleotides / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D013379:Substrate Specificity
C466422:AGO2 protein, human; D060565:Argonaute Proteins; D009711:Nucleotides; D034741:RNA, Small Interfering