Bioenergetics and Gene Silencing Approaches for Unraveling Nucleotide Recognition by the Human EIF2C2/Ago2 PAZ Domain

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.

Label‐Free Electrophoretic Mobility Shift Assay (EMSA) for Measuring Dissociation Constants of Protein‐RNA Complexes

“…1A), but published data reveal somewhat divergent val-ues for the stability of the complex. Thus, the K d values for the interactions between Ago2 PAZ and mono-(e.g., UMP) and dinucleotides (e.g., UpUMP) varied between 10 and 60 μM based on ITC (Kandeel et al, 2014). In contrast, the K d of the interaction between Ago1 PAZ and an 11mer/13mer RNA duplex with a 3 -terminal dinucleotide overhang ( Fig.…”

“…1). The identities of the 3 -overhanging nucleotides in the siRNA guide strand and their chemical modification affect the interaction with Ago2 PAZ and silencing activity (Alagia et al, 2018;Kandeel et al, 2014). We relied on the EMSA protocol presented here to determine equilibrium dissociation constants K d of the interactions between native and chemically modified RNAs and the human Ago2 PAZ domain.…”

Regulation of insulin resistance and type II diabetes by hepatitis C virus infection: A driver function of circulating miRNAs

“…This trimming process is only for AGO2‐bound miRNA but not for non‐cleaving AGO1/3/4‐associated miRNAs, suggesting a potential bias for AGO proteins towards certain nucleotide sequences . miRNA with 3′‐end adenine nucleotides shows weak interaction with PAZ domain, whereas this interaction becomes stabilized when U/G/C nucleotides (U>G>C>A) are present at the 3′‐end . As miR‐122's 3′‐end nucleotide is G and all 6 nucleotides in the 3′ tail are U or G, miR‐122 likely exhibits strong binding to the AGO2 PAZ domain, thus protecting it from cellular nuclease cleavage (Figure B).…”

Molecular analysis of some camel cytochrome P450 enzymes reveals lower evolution and drug-binding properties

“…By the advent of new genome technologies, it became easier to map genetic variations and changes in genes by comparison of domesticated and wild animals. Furthermore, phylogenetic and other computational tools improved the understanding of biological functions, disease control and discovery of drugs (Kandeel et al, 2014;Kandeel and Kitade, 2013a, b). The selection pressure for rapid evolution, slow evolution or loss of certain gene can be analyzed and compared between the Arabian camel and wild camels.…”