Local and global effects of Mg2+ on Ago and miRNA-target interactions

Three magnesium ions (Mg(2+)), named Mg1 (in Mid domain), Mg2 and Mg3 (both in PIWI domain), located at the small RNA binding domain of Argonaute (Ago) protein, are important for sequence-specific miRNA-target interactions. Such conjunction between the Ago protein and miRNA raises the question: How do Mg(2+) ions participate in the recognition process of miRNA by Ago or its target. Furthermore, it is still unclear whether the Mg(2+) ions contribute to the local or global stability of the miRNA complex. In this work, we have performed a series of 16 independent molecular dynamic simulations (MD) to characterize the functions of Mg(2+), hydration patterns and the conformational events involved in the miRNA-target interactions. The cross correlation analysis shows that Mg1 and Mg2 significantly enhance a locally cooperated movement of the PAZ, PIWI and Mid domains with the average correlation coefficient of ～0.65, producing an "open-closed" motion (rotation Angle, 46.5°) between the PAZ and PIWI domains. Binding of Mg3 can globally stabilize the whole Ago protein with the average RMSD of ～0.34 ?, compared with the systems in absence of Mg3 (average RMSD?=?～0.43 ?). Three structural water molecules surrounding the Mg(2+)-binding regions also stabilize these ions, thus facilitating the recognition of miRNA to its target. In addition, the thermodynamic analysis also verifies the positive contribution of all three Mg(2+) to the binding of miRNA to Ago, as well as the importance Mg2 plays in the cleavage of the miRNA targets.     

Dynamic mechanisms for pre-miRNA binding and export by Exportin-5

The biogenesis and function of mature microRNAs (miRNAs) is dependent on the nuclear export of miRNA precursors (pre-miRNA) by Exportin-5 (Exp5). To characterize the molecular mechanisms of how pre-miRNA is recognized and transported by Exp5, we have performed 21 molecular dynamic (MD) simulations of RNA-bound Exp5 (Exp5-RanGTP-premiRNA, Exp5-RanGDP-premiRNA, Exp5-premiRNA), RNA-unbound Exp5 (Exp5-RanGTP, Exp5-RanGDP, apo-Exp5), and pre-miRNA. Our simulations with standard MD, steered molecular dynamics (SMD), and energy analysis have shown that (1) Free Exp5 undergoes extensive opening motion, and in this way facilitates the RanGTP binding. (2) RanGTP efficiently regulates the association/dissociation of pre-miRNA to its complex by inducing conformational changes in the HEAT-repeat helix stacking of Exp5. (3) The GTP hydrolysis prevents Ran from rebinding to Exp5 by regulating the hydrophobic interfaces and salt bridges between Ran and Exp5. (4) The transition from the A'-form to the A-form of the pre-miRNA modulates the structural complementarities between the protein and the pre-miRNA, thus promoting efficient assembly of the complex. (5) The base-flipping process (from the closed to the fully flipped state) of the 2-nt 3' overhang is a prerequisite for the pre-miRNA recognition by Exp5, which occurs in a sequence-independent manner as evidenced by the fact that different 2-nt 3' overhangs bind to Exp5 in essentially the same way. And finally, a plausible mechanism of the pre-miRNA export cycle has been proposed explaining how the protein-protein and protein-RNA interactions are coordinated in physiological conditions.     

Assembly and analysis of eukaryotic Argonaute–RNA complexes in microRNA-target recognition

Experimental studies have uncovered a variety of microRNA (miRNA)–target duplex structures that include perfect, imperfect and seedless duplexes. However, non-canonical binding modes from imperfect/seedless duplexes are not well predicted by computational approaches, which rely primarily on sequence and secondary structural features, nor have their tertiary structures been characterized because solved structures to date are limited to near perfect, straight duplexes in Argonautes (Agos). Here, we use structural modeling to examine the role of Ago dynamics in assembling viable eukaryotic miRNA-induced silencing complexes (miRISCs). We show that combinations of low-frequency, global modes of motion of Ago domains are required to accommodate RNA duplexes in model human and C. elegans Ago structures. Models of viable miRISCs imply that Ago adopts variable conformations at distinct target sites that generate distorted, imperfect miRNA-target duplexes. Ago''s ability to accommodate a duplex is dependent on the region where structural distortions occur: distortions in solvent-exposed seed and 3′-end regions are less likely to produce steric clashes than those in the central duplex region. Energetic analyses of assembled miRISCs indicate that target recognition is also driven by favorable Ago-duplex interactions. Such structural insights into Ago loading and target recognition mechanisms may provide a more accurate assessment of miRNA function.     

Mechanism of MicroRNA-Target Interaction: Molecular Dynamics Simulations and Thermodynamics Analysis

MicroRNAs (miRNAs) are endogenously produced ∼21-nt riboregulators that associate with Argonaute (Ago) proteins to direct mRNA cleavage or repress the translation of complementary RNAs. Capturing the molecular mechanisms of miRNA interacting with its target will not only reinforce the understanding of underlying RNA interference but also fuel the design of more effective small-interfering RNA strands. To address this, in the present work the RNA-bound (Ago-miRNA, Ago-miRNA-target) and RNA-free Ago forms were analyzed by performing both molecular dynamics simulations and thermodynamic analysis. Based on the principal component analysis results of the simulation trajectories as well as the correlation analysis in fluctuations of residues, we discover that: 1) three important (PAZ, Mid and PIWI) domains exist in Argonaute which define the global dynamics of the protein; 2) the interdomain correlated movements are so crucial for the interaction of Ago-RNAs that they not only facilitate the relaxation of the interactions between residues surrounding the RNA binding channel but also induce certain conformational changes; and 3) it is just these conformational changes that expand the cavity of the active site and open putative pathways for both the substrate uptake and product release. In addition, by thermodynamic analysis we also discover that for both the guide RNA 5′-end recognition and the facilitated site-specific cleavage of the target, the presence of two metal ions (of Mg²⁺) plays a predominant role, and this conclusion is consistent with the observed enzyme catalytic cleavage activity in the ternary complex (Ago-miRNA-mRNA). Our results find that it is the set of arginine amino acids concentrated in the nucleotide-binding channel in Ago, instead of the conventionally-deemed seed base-paring, that makes greater contributions in stabilizing the binding of the nucleic acids to Ago.     

RNA editing of microRNA prevents RNA-induced silencing complex recognition of target mRNA

MicroRNAs (miRNAs) integrate with Argonaut (Ago) to create the RNA-induced silencing complex, and regulate gene expression by silencing target mRNAs. RNA editing of miRNA may affect miRNA processing, assembly of the Ago complex and target mRNA binding. However, the function of edited miRNA, assembled within the Ago complex, has not been extensively investigated. In this study, sequence analysis of the Ago complex of Marsupenaeus japonicus shrimp infected with white spot syndrome virus (WSSV) revealed that host ADAR (adenosine deaminase acting on RNA) catalysed A-to-I RNA editing of a viral miRNA (WSSV-miR-N12) at the +16 site. This editing of the non-seed sequence did not affect association of the edited miRNA with the Ago protein, but inhibited interaction between the miRNA and its target gene (wsv399). The WSSV early gene wsv399 inhibited WSSV infection. As a result, the RNA editing of miRNA caused virus latency. Our results highlight a novel example of miRNA editing in the miRNA-induced silencing complex.     

Identification of targets of specific miRNAs by selective amplification of Ago2-associated mRNA

To study the function of a miRNA, it is necessary to identify its target genes. The most common methods to reveal miRNA target genes rely on ectopically expressed tagged Ago2 and nonphysiological overexpression or inhibition of the miRNA of interest. To uncover the natural association between miRNAs and their target genes, we isolated endogenous Ago2 protein followed by a selective strategy, which only amplified target genes of the selected miRNA from the purified RNA-induced silencing complex by miRNA specific primers. This enabled us to identify the mRNAs regulated by miRNAs of interest. Our data demonstrated that this strategy is effective and highly credible. Moreover, our results showed the evidence of efficient miRNA target sites in 5' untranslated regions and open reading frames of target mRNAs.     

Systematic identification of mRNAs recruited to argonaute 2 by specific microRNAs and corresponding changes in transcript abundance

microRNAs (miRNAs) are small non-coding RNAs that regulate mRNA stability and translation through the action of the RNAi-induced silencing complex (RISC). Our current understanding of miRNA function is inferred largely from studies of the effects of miRNAs on steady-state mRNA levels and from seed match conservation and context in putative targets. Here we have taken a more direct approach to these issues by comprehensively assessing the miRNAs and mRNAs that are physically associated with Argonaute 2 (Ago2), which is a core RISC component. We transfected HEK293T cells with epitope-tagged Ago2, immunopurified Ago2 together with any associated miRNAs and mRNAs, and quantitatively determined the levels of these RNAs by microarray analyses. We found that Ago2 immunopurified samples contained a representative repertoire of the cell's miRNAs and a select subset of the cell's total mRNAs. Transfection of the miRNAs miR-1 and miR-124 caused significant changes in the association of scores of mRNAs with Ago2. The mRNAs whose association with Ago2 increased upon miRNA expression were much more likely to contain specific miRNA seed matches and to have their overall mRNA levels decrease in response to the miRNA transfection than expected by chance. Hundreds of mRNAs were recruited to Ago2 by each miRNA via seed sequences in 3'-untranslated regions and coding sequences and a few mRNAs appear to be targeted via seed sequences in 5'-untranslated regions. Microarray analysis of Ago2 immunopurified samples provides a simple, direct method for experimentally identifying the targets of miRNAs and for elucidating roles of miRNAs in cellular regulation.     

Identification of novel argonaute-associated proteins

RNA silencing processes are guided by small RNAs known as siRNAs and microRNAs (miRNAs) . They reside in ribonucleoprotein complexes, which guide the cleavage of complementary mRNAs or affect stability and translation of partial complementary mRNAs . Argonaute (Ago) proteins are at the heart of silencing effector complexes and bind the single-stranded siRNA and miRNA . Our biochemical analysis revealed that Ago2 is present in a pre-miRNA processing complex that is able to transfer the miRNA into a target-mRNA cleaving complex. To gain insight into the function and composition of RNA silencing complexes, we purified Ago1- and Ago2-containing complexes from human cells. Several known Ago1- and/or Ago2-associated proteins including Dicer were identified, but also two novel factors, the putative RNA helicase MOV10, and the RNA recognition motif (RRM)-containing protein TNRC6B/KIAA1093. The new proteins localize, similar to Ago proteins, to mRNA-degrading cytoplasmic P bodies, and they are functionally required to mediate miRNA-guided mRNA cleavage.     

Sequence- and structure-based analysis of proteins involved in miRNA biogenesis

miRNA biogenesis is a multistage process for the generation of a mature miRNA and involves several different proteins. In this work, we have carried out both sequence- and structure-based analysis for crucial proteins involved in miRNA biogenesis, namely Dicer, Drosha, Argonaute (Ago), and Exportin-5 to understand evolution of these proteins in animal kingdom and also to identify key sequence and structural features that are determinants of their function. Our analysis reveals that in animals the miRNA biogenesis pathway first originated in molluscs. The phylogeny of Dicer and Ago indicated evolution through gene duplication followed by sequence divergence that resulted in functional divergence. Our detailed structural analysis also revealed that RIIIDb domains of Drosha and Dicer, share significant similarity in sequence, structure, and substrate-binding pocket. On the other hand, PAZ domains of Dicer and Ago show only conservation of the substrate-binding pockets in the catalytic sites despite significant divergence in sequence and overall structure. Based on a comparative structural analysis of all four human Ago proteins (hAgo1–4) and their known biochemical activity, we have also attempted to identify key residues in Ago2 which are responsible for the unique slicer activity of hAgo2 among all isoforms. We have identified six key residues in N domain of hAgo2, which are located far away from the catalytic pocket, but might be playing a major role in slicer activity of hAgo2 protein because of their involvement in mRNA binding.     

Targeting the microRNA binding domain of argonaute 2: rational inhibitor design and study of mutation effects on protein-ligand interaction

Abstract

Based on the accumulative evidences during recent decades, miRNAs have been found overexpressed in several human cancer types and also in Down syndrome patients, contributing to the neuropathology of Down syndrome. From this point of view, investigations on the structure and dynamic mechanisms related to the Argonaute 2 miRNAs binding in which silencing of the mRNA occurs, have inspired many clinical researchers to target this complex to inhibit the silencing process. In the current research, we have virtually screened the OTAVA_CNS_library to introduce new inhibitor compounds for the Ago2/miRNA complex. Ten hit compounds were obtained, with just one of them nominated as the best compound. Following the interaction analysis, by utilizing molecular dynamics (MD) simulations, effects of two mutations (Thr526 to isoleucine and Gln545 to alanine) on the dynamic properties of Ago2 in the complex with the best inhibitor compound were investigated. RMSD, RMSF and h-bond number beside other analyses, highlighted the importance of the Thr526 and Gln545 mutations for the stability and flexibility of the (Ago2)/ligand complex.

Communicated by Ramaswamy H. Sarma     

A role for both conformational selection and induced fit in ligand binding by the LAO protein

Molecular recognition is determined by the structure and dynamics of both a protein and its ligand, but it is difficult to directly assess the role of each of these players. In this study, we use Markov State Models (MSMs) built from atomistic simulations to elucidate the mechanism by which the Lysine-, Arginine-, Ornithine-binding (LAO) protein binds to its ligand. We show that our model can predict the bound state, binding free energy, and association rate with reasonable accuracy and then use the model to dissect the binding mechanism. In the past, this binding event has often been assumed to occur via an induced fit mechanism because the protein's binding site is completely closed in the bound state, making it impossible for the ligand to enter the binding site after the protein has adopted the closed conformation. More complex mechanisms have also been hypothesized, but these have remained controversial. Here, we are able to directly observe roles for both the conformational selection and induced fit mechanisms in LAO binding. First, the LAO protein tends to form a partially closed encounter complex via conformational selection (that is, the apo protein can sample this state), though the induced fit mechanism can also play a role here. Then, interactions with the ligand can induce a transition to the bound state. Based on these results, we propose that MSMs built from atomistic simulations may be a powerful way of dissecting ligand-binding mechanisms and may eventually facilitate a deeper understanding of allostery as well as the prediction of new protein-ligand interactions, an important step in drug discovery.     

microRNA‐independent recruitment of Argonaute 1 to nanos mRNA through the Smaug RNA‐binding protein

Argonaute (Ago) proteins are typically recruited to target messenger RNAs via an associated small RNA such as a microRNA (miRNA). Here, we describe a new mechanism of Ago recruitment through the Drosophila Smaug RNA‐binding protein. We show that Smaug interacts with the Ago1 protein, and that Ago1 interacts with and is required for the translational repression of the Smaug target, nanos mRNA. The Ago1/nanos mRNA interaction does not require a miRNA, but it does require Smaug. Taken together, our data suggest a model whereby Smaug directly recruits Ago1 to nanos mRNA in a miRNA‐independent manner, thereby repressing translation.     

A combined coarse-grained and all-atom simulation of TRPV1 channel gating and heat activation

The transient receptor potential (TRP) channels act as key sensors of various chemical and physical stimuli in eukaryotic cells. Despite years of study, the molecular mechanisms of TRP channel activation remain unclear. To elucidate the structural, dynamic, and energetic basis of gating in TRPV1 (a founding member of the TRPV subfamily), we performed coarse-grained modeling and all-atom molecular dynamics (MD) simulation based on the recently solved high resolution structures of the open and closed form of TRPV1. Our coarse-grained normal mode analysis captures two key modes of collective motions involved in the TRPV1 gating transition, featuring a quaternary twist motion of the transmembrane domains (TMDs) relative to the intracellular domains (ICDs). Our transition pathway modeling predicts a sequence of structural movements that propagate from the ICDs to the TMDs via key interface domains (including the membrane proximal domain and the C-terminal domain), leading to sequential opening of the selectivity filter followed by the lower gate in the channel pore (confirmed by modeling conformational changes induced by the activation of ICDs). The above findings of coarse-grained modeling are robust to perturbation by lipids. Finally, our MD simulation of the ICD identifies key residues that contribute differently to the nonpolar energy of the open and closed state, and these residues are predicted to control the temperature sensitivity of TRPV1 gating. These computational predictions offer new insights to the mechanism for heat activation of TRPV1 gating, and will guide our future electrophysiology and mutagenesis studies.     

An mRNA m7G cap binding-like motif within human Ago2 represses translation

microRNAs (miRNAs) bind to Argonaute (Ago) proteins and inhibit translation or promote degradation of mRNA targets. Human let-7 miRNA inhibits translation initiation of mRNA targets in an m(7)G cap-dependent manner and also appears to block protein production, but the molecular mechanism(s) involved is unknown and the role of Ago proteins in translational regulation remains elusive. Here we identify a motif (MC) within the Mid domain of Ago proteins, which bears significant similarity to the m(7)G cap-binding domain of eIF4E, an essential translation initiation factor. We identify conserved aromatic residues within the MC motif of human Ago2 that are required for binding to the m(7)G cap and for translational repression but do not affect the assembly of Ago2 with miRNA or its catalytic activity. We propose that Ago2 represses the initiation of mRNA translation by binding to the m(7)G cap of mRNA targets, thus likely precluding the recruitment of eIF4E.     

Features of molecular recognition of intrinsically disordered proteins via coupled folding and binding

The sequence–structure–function paradigm of proteins has been revolutionized by the discovery of intrinsically disordered proteins (IDPs) or intrinsically disordered regions (IDRs). In contrast to traditional ordered proteins, IDPs/IDRs are unstructured under physiological conditions. The absence of well‐defined three‐dimensional structures in the free state of IDPs/IDRs is fundamental to their function. Folding upon binding is an important mode of molecular recognition for IDPs/IDRs. While great efforts have been devoted to investigating the complex structures and binding kinetics and affinities, our knowledge on the binding mechanisms of IDPs/IDRs remains very limited. Here, we review recent advances on the binding mechanisms of IDPs/IDRs. The structures and kinetic parameters of IDPs/IDRs can vary greatly, and the binding mechanisms can be highly dependent on the structural properties of IDPs/IDRs. IDPs/IDRs can employ various combinations of conformational selection and induced fit in a binding process, which can be templated by the target and/or encoded by the IDP/IDR. Further studies should provide deeper insights into the molecular recognition of IDPs/IDRs and enable the rational design of IDP/IDR binding mechanisms in the future.