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.
Highlights
Gene silencing and RNA interference (RNAi) are cellular processes that control gene expression via the cleavage of target mRNA by either a cellular or synthesized small RNA [1,2]
Which of the known nucleosides can bind with the PAZ domain? Second, what are the determinants of nucleotide recognition by the PAZ domain? Lastly, what is the relationship between strong and weak binders and their in vivo RNAi efficacy? Here, we demonstrate that the EIF2C2/Ago2 PAZ domain has a selective binding affinity for ribonucleotides over deoxyribonucleotides
The initial isothermal titration calorimetry (ITC) experiments showed that some nucleotides (UMP, GMP, CMP, TMP, and dGMP) bound to the PAZ domain with considerable affinity
Summary
Gene silencing and RNA interference (RNAi) are cellular processes that control gene expression via the cleavage of target mRNA by either a cellular or synthesized small RNA [1,2]. A small interfering RNA (siRNA) is loaded into an RNA-induced silencing complex (RISC) that guides the RISC complex to bind with the complementary mRNA sequence followed by endonucleolytic cleavage of the target mRNA, preventing gene expression [3,4,5]. Ago proteins have a conserved structure that is composed of two binding domains (PAZ and MID) and one catalytic (PIWI) domain [19]. The PIWI domain contains an RNase H fold and is considered as the catalytic RNA cleavage center of Ago proteins. The function of the PAZ domain extends beyond a simple binding event to include significant effects on RNA loading and the slicer (endonuclease) activity of the RISC complex. Within the Ago proteins of humans, EIF2C2/Ago is the only member with recognized slicer activity and is considered as the catalytic engine of the RNAi process. Cells lacking EIF2C2/Ago are unable to process siRNA [29]
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