Mg2+ Effect on Argonaute and RNA Duplex by Molecular Dynamics and Bioinformatics Implications

Abstract

RNA interference (RNAi), mediated by small non-coding RNAs (e.g., miRNAs, siRNAs), influences diverse cellular functions. Highly complementary miRNA-target RNA (or siRNA-target RNA) duplexes are recognized by an Argonaute family protein (Ago2), and recent observations indicate that the concentration of Mg2+ ions influences miRNA targeting of specific mRNAs, thereby modulating miRNA-mRNA networks. In the present report, we studied the thermodynamic effects of differential [Mg2+] on slicing (RNA silencing cycle) through molecular dynamics simulation analysis, and its subsequent statistical analysis. Those analyses revealed different structural conformations of the RNA duplex in Ago2, depending on Mg2+ concentration. We also demonstrate that cation effects on Ago2 structural flexibility are critical to its catalytic/functional activity, with low [Mg2+] favoring greater Ago2 flexibility (e.g., greater entropy) and less miRNA/mRNA duplex stability, thus favoring slicing. The latter finding was supported by a negative correlation between expression of an Mg2+ influx channel, TRPM7, and one miRNA’s (miR-378) ability to downregulate its mRNA target, TMEM245. These results imply that thermodynamics could be applied to siRNA-based therapeutic strategies, using highly complementary binding targets, because Ago2 is also involved in RNAi slicing by exogenous siRNAs. However, the efficacy of a siRNA-based approach will differ, to some extent, based on the Mg2+ concentration even within the same disease type; therefore, different siRNA-based approaches might be considered for patient-to-patient needs.

Literature review of baseline information to support the risk assessment of RNAi‐based GM plants

“…This yielded a minimal mechanistic model describing fundamental steps during RNAi, which is consistent with a "two-state" model of RISC action (Deerberg et al, 2013). Finally, it was found that Mg2+ concentration, influences AGO2 structural flexibility and is important for its catalytic/functional activity, with low [Mg2+] favoring greater Ago2 flexibility (e.g., greater entropy) and less miRNA/mRNA duplex stability, thus favoring slicing (Nam et al, 2014b).…”

Exploring the RNA‐bound and RNA‐free human Argonaute‐2 by molecular dynamics simulation method

“…Nam et al. investigated the Mg 2+ ion effects on Argonaute and RNA duplex by molecular dynamics study based on Thermus thermophilus Argonaute protein structure . It showed that low Mg 2+ concentration induced greater Ago2 flexibility and less RNA duplex stability, facilitating slicing process.…”

Why Is a High Temperature Needed by Thermus thermophilus Argonaute During mRNA Silencing: A Theoretical Study

“…Two structures, namely, PDB ID 3F73 and PDB ID 3HM9, whose substrates have different lengths (Wang et al, 2008a , b ), suggested that the PAZ domain undergo a series of motions during a catalytic cycle. They also reveal the PAZ domain disassociates from the 3′ end of the guide upon target binding (Xia et al, 2012 ; Wang et al, 2013 ; Nam et al, 2014 ; Swarts et al, 2014 ; Jiang et al, 2015 ).…”

“…But how temperature affects TtAgo activity, it has not been clarified so far. A few molecular dynamics (MD) studies on siRNA–TtAgo and siRNA–hAGO2 complexes have been conducted (Wang et al, 2013 ; Nam et al, 2014 ; Hanlun et al, 2015 ). Jiang et al constructed from MD simulations and found that MSMs can elucidate the conformational dynamics of AGO at biologically relevant timescales (Hanlun et al, 2015 ).…”

“…Jiang et al constructed from MD simulations and found that MSMs can elucidate the conformational dynamics of AGO at biologically relevant timescales (Hanlun et al, 2015 ). Jinhyuk Lee pointed out that they revealed different structural conformations of the RNA duplex in Ago2, depending on Mg 2+ concentration and demonstrated that cation effects on Ago2 structural flexibility are critical to its catalytic/functional activity, with low Mg 2+ favoring greater Ago2 flexibility and less miRNA/mRNA duplex stability, thus favoring slicing (Nam et al, 2014 ). Wang et al discovered three findings during MD simulations: (1) three important (PAZ, Mid and PIWI) domains existed in Argonaute which defined the global dynamics of the protein; (2) the interdomain correlated movements were so crucial for the interaction of Ago-RNAs that they not only facilitated the relaxation of the interactions between residues surrounding the RNA binding channel but also induced certain conformational changes; and (3) it was just these conformational changes that expand the cavity of the active site and open putative pathways for both the substrate uptake and product release (Wang et al, 2013 ).…”