Convergent Transmission of RNAi Guide-Target Mismatch Information across Argonaute Internal Allosteric Network

In RNA interference, a guide strand derived from a short dsRNA such as a microRNA (miRNA) is loaded into Argonaute, the central protein in the RNA Induced Silencing Complex (RISC) that silences messenger RNAs on a sequence-specific basis. The positions of any mismatched base pairs in an miRNA determine which Argonaute subtype is used. Subsequently, the Argonaute-guide complex binds and silences complementary target mRNAs; certain Argonautes cleave the target. Mismatches between guide strand and the target mRNA decrease cleavage efficiency. Thus, loading and silencing both require that signals about the presence of a mismatched base pair are communicated from the mismatch site to effector sites. These effector sites include the active site, to prevent target cleavage; the binding groove, to modify nucleic acid binding affinity; and surface allosteric sites, to control recruitment of additional proteins to form the RISC. To examine how such signals may be propagated, we analyzed the network of internal allosteric pathways in Argonaute exhibited through correlations of residue-residue interactions. The emerging network can be described as a set of pathways emanating from the core of the protein near the active site, distributed into the bulk of the protein, and converging upon a distributed cluster of surface residues. Nucleotides in the guide strand “seed region” have a stronger relationship with the protein than other nucleotides, concordant with their importance in sequence selectivity. Finally, any of several seed region guide-target mismatches cause certain Argonaute residues to have modified correlations with the rest of the protein. This arises from the aggregation of relatively small interaction correlation changes distributed across a large subset of residues. These residues are in effector sites: the active site, binding groove, and surface, implying that direct functional consequences of guide-target mismatches are mediated through the cumulative effects of a large number of internal allosteric pathways.     

Interactions between the non-seed region of siRNA and RNA-binding RLC/RISC proteins,Ago and TRBP,in mammalian cells

Small interfering RNA (siRNA)-based RNA interference (RNAi) is widely used for target gene silencing in various organisms. We previously showed that 8-nt-long 5′ proximal nucleotides, which include seed sequence (positions 2–8 from the 5′ end of guide strand), and the complementary sequence of the passenger strand are capable of being simultaneously replaced with cognate deoxyribonucleotides without any substantial loss of gene silencing. In the present study, examination was made of RNA requirements in the non-seed region of siRNA. The non-seed region of siRNA was found to be subdivided into four domains, in which two nucleotide pairs (positions 13 and 14) were replaceable with cognate deoxyribonucleotides without reducing RNAi activity. However, RNA sequences at positions 9-12 and 15-18 were essential for effective gene silencing, and these two double-stranded RNA cores are required for binding of the trans-activation response RNA-binding protein (TRBP). The terminal RNA (positions 19–21) provided Argonaute protein binding sites. Argonaute binding was enhanced by the presence of RNAs at positions 15–18. Knockdown experiments showed that, unlike Argonaute and TRBP, Dicer was dispensable for RNAi. Based on these observations, we discuss possible RNA/protein and protein/protein interactions in RNA-induced silencing complex formation.     

Two molecular features contribute to the Argonaute specificity for the microRNA and RNAi pathways in C. elegans

In Caenorhabditis elegans, specific Argonaute proteins are dedicated to the RNAi and microRNA pathways. To uncover how the precise Argonaute selection occurs, we designed dsRNA triggers containing both miRNA and siRNA sequences. While dsRNA carrying nucleotides mismatches can only enter the miRNA pathway, a fully complementary dsRNA successfully rescues let-7 miRNA function and initiates silencing by RNAi. We demonstrated that RDE-1 is essential for RNAi induced by the perfectly paired trigger, yet is not required for silencing by the let-7 miRNA. In contrast, ALG-1/ALG-2 are required for the miRNA function, but not for the siRNA-directed gene silencing. Finally, a dsRNA containing a bulged miRNA and a perfectly paired siRNA can enter both pathways suggesting that the sorting of small RNAs occurs after that the dsRNA trigger has been processed by Dicer. Thus, our data suggest that the selection of Argonaute proteins is affected by two molecular features: (1) the structure of the small RNA duplex; and (2) the Argonautes specific characteristics.     

Single-molecule fluorescence measurements reveal the reaction mechanisms of the core-RISC,composed of human Argonaute 2 and a guide RNA

In eukaryotes, small RNAs play important roles in both gene regulation and resistance to viral infection. Argonaute proteins have been identified as a key component of the effector complexes of various RNA-silencing pathways, but the mechanistic roles of Argonaute proteins in these pathways are not clearly understood. To address this question, we performed single-molecule fluorescence experiments using an RNA-induced silencing complex (core-RISC) composed of a small RNA and human Argonaute 2. We found that target binding of core-RISC starts at the seed region of the guide RNA. After target binding, four distinct reactions followed: target cleavage, transient binding, stable binding, and Argonaute unloading. Target cleavage required extensive sequence complementarity and accelerated core-RISC dissociation for recycling. In contrast, the stable binding of core-RISC to target RNAs required seed-match only, suggesting a potential explanation for the seed-match rule of microRNA (miRNA) target selection. [BMB Reports 2015; 48(12): 643-644]     

Human RISC couples microRNA biogenesis and posttranscriptional gene silencing

RNA interference is implemented through the action of the RNA-induced silencing complex (RISC). Although Argonaute2 has been identified as the catalytic center of RISC, the RISC polypeptide composition and assembly using short interfering RNA (siRNA) duplexes has remained elusive. Here we show that RISC is composed of Dicer, the double-stranded RNA binding protein TRBP, and Argonaute2. We demonstrate that this complex can cleave target RNA using precursor microRNA (pre-miRNA) hairpin as the source of siRNA. Although RISC can also utilize duplex siRNA, it displays a nearly 10-fold greater activity using the pre-miRNA Dicer substrate. RISC distinguishes the guide strand of the siRNA from the passenger strand and specifically incorporates the guide strand. Importantly, ATP is not required for miRNA processing, RISC assembly, or multiple rounds of target-RNA cleavage. These results define the composition of RISC and demonstrate that miRNA processing and target-RNA cleavage are coupled.     

Both Strands of siRNA Have Potential to Guide Posttranscriptional Gene Silencing in Mammalian Cells

Despite the widespread application of RNA interference (RNAi) as a research tool for diverse purposes, the key step of strand selection of siRNAs during the formation of RNA-induced silencing complex (RISC) remains poorly understood. Here, using siRNAs targeted to the complementary region of Survivin and the effector protease receptor 1 (EPR-1), we show that both strands of the siRNA duplex can find their target mRNA and are equally eligible for assembly into Argonaute 2 (Ago2) of RISC in HEK293 cells. Transfection of the synthetic siRNA duplexes with different thermodynamic profiles or short hairpin RNA (shRNA) vectors that generate double-stranded RNAs (dsRNAs), permitting processing specifically from either the 5′ or 3′ end of the incipient siRNA, results in the degradation of the respective target mRNAs of either strand of the siRNA duplex with comparable efficiencies. Thus, while most RNAi reactions may follow the thermodynamic asymmetry rule in strand selection, our study suggests an exceptional mode for certain siRNAs in which both strands of the duplex are competent in sponsoring RNAi, and implies additional factors that might dictate the RNAi targets.     

Persistence of seed-based activity following segmentation of a microRNA guide strand

microRNAs are ～ 22 nucleotide regulatory RNAs that are processed into duplexes from hairpin structures and incorporated into Argonaute proteins. Here, we show that a nick in the middle of the guide strand of an miRNA sequence allows for seed-based targeting characteristic of miRNA activity. Insertion of an inverted abasic, a dye, or a small gap between the two segments still permits target knockdown. While activity from the seed region of the segmented miRNA is apparent, activity from the 3' half of the guide strand is impaired, suggesting that an intact guide backbone is required for contribution from the 3' half. miRNA activity was also observed following nicking of a miRNA precursor. These results illustrate a structural flexibility in miRNA duplexes and may have applications in the design of miRNA mimetics.     

Molecular basis for improved gene silencing by Dicer substrate interfering RNA compared with other siRNA variants

The canonical exogenous trigger of RNA interference (RNAi) in mammals is small interfering RNA (siRNA). One promising application of RNAi is siRNA-based therapeutics, and therefore the optimization of siRNA efficacy is an important consideration. To reduce unfavorable properties of canonical 21mer siRNAs, structural and chemical variations to canonical siRNA have been reported. Several of these siRNA variants demonstrate increased potency in downstream readout-based assays, but the molecular mechanism underlying the increased potency is not clear. Here, we tested the performance of canonical siRNAs and several sequence-matched variants in parallel in gene silencing, RNA-induced silencing complex (RISC) assembly, stability and Argonaute (Ago) loading assays. The commonly used 19mer with two deoxythymidine overhangs (19merTT) variant performed similarly to canonical 21mer siRNA. A shorter 16mer variant (16merTT) did not perform comparably in our assays. Dicer substrate interfering RNA (dsiRNA) demonstrated better gene silencing by the guide strand (target complementary strand), better RISC assembly, persistence of the guide strand and relatively more loading of the guide strand into Ago. Hence, we demonstrate the advantageous properties of dsiRNAs at upstream, intermediate and downstream molecular steps of the RNAi pathway.     

Towards Antiviral shRNAs Based on the AgoshRNA Design

RNA interference (RNAi) can be induced by intracellular expression of a short hairpin RNA (shRNA). Processing of the shRNA requires the RNaseIII-like Dicer enzyme to remove the loop and to release the biologically active small interfering RNA (siRNA). Dicer is also involved in microRNA (miRNA) processing to liberate the mature miRNA duplex, but recent studies indicate that miR-451 is not processed by Dicer. Instead, this miRNA is processed by the Argonaute 2 (Ago2) protein, which also executes the subsequent cleavage of a complementary mRNA target. Interestingly, shRNAs that structurally resemble miR-451 can also be processed by Ago2 instead of Dicer. The key determinant of these “AgoshRNA” molecules is a relatively short basepaired stem, which avoids Dicer recognition and consequently allows alternative processing by Ago2. AgoshRNA processing yields a single active RNA strand, whereas standard shRNAs produce a duplex with guide and passenger strands and the latter may cause adverse off-target effects. In this study, we converted previously tested active anti-HIV-1 shRNA molecules into AgoshRNA. We tested several designs that could potentially improve AgoshRNA activity, including extension of the complementarity between the guide strand and the mRNA target and reduction of the thermodynamic stability of the hairpins. We demonstrate that active AgoshRNAs can be generated. However, the RNAi activity is reduced compared to the matching shRNAs. Despite reduced RNAi activity, comparison of an active AgoshRNA and the matching shRNA in a sensitive cell toxicity assay revealed that the AgoshRNA is much less toxic.     

A systematic analysis of the silencing effects of an active siRNA at all single-nucleotide mismatched target sites

The specificity of small interfering RNA (siRNA)-mediated gene silencing is a critical consideration for the application of RNA interference (RNAi). While the discovery of potential off-target effects by siRNAs is of concern, no systematic analysis has been conducted to explore the specificity of RNAi. Here, we present a study where a functionally validated siRNA (siCD46) was examined for silencing specificity on all possible 57 permutated target sites, each carrying a single-nucleotide mutation that would generate a mismatch when paired with siRNA antisense strand. We found that it was not only the position of the mismatched base pair, but also the identity of the nucleotides forming the mismatch that influenced silencing. Surprisingly, mismatches formed between adenine (A) and cytosine (C), in addition to the G:U wobble base pair, were well tolerated and target sites containing such mismatches were silenced almost as efficiently as its fully matched counterpart by siCD46. Northern blots showed that the silencing of fusion genes harboring the mutated target sites involved target mRNA degradation. This study provides direct evidence that the target recognition of siRNA is far more degenerative than previously considered. This finding is instrumental in the understanding of RNAi specificity and may aid the computational prediction of RNA secondary structure.     

Structural features of small RNA precursors determine Argonaute loading in Caenorhabditis elegans

In C. elegans, DCR-1 is required for the maturation of both short interfering RNAs (siRNAs) and microRNAs (miRNAs), which are subsequently loaded into different Argonaute proteins to mediate silencing via distinct mechanisms. We used in vivo analyses to show that precursors of small RNAs contain structural features that direct the small RNAs into the RNA interference (RNAi) pathway or the miRNA-processing pathway. Nucleotide changes in the pre-let-7 miRNA precursor that make its stem fully complementary cause the resulting small RNA to be recognized as siRNA and induce binding to RDE-1, which leads to RNAi. Mismatches of 1 to 3 nucleotides at various positions in the stem of the precursor restore direction into the miRNA pathway, as the largest portion of such small RNA variants is associated with ALG-1. The Argonaute proteins to which the small RNAs are bound determine the silencing mode, and no functional overlap between RDE-1 and ALG-1 was detected.     

Functional dissection of siRNA sequence by systematic DNA substitution: modified siRNA with a DNA seed arm is a powerful tool for mammalian gene silencing with significantly reduced off-target effect

Short interfering RNA (siRNA)-based RNA interference (RNAi) is widely used for target gene knockdown in mammalian cells. To clarify the position-dependent functions of ribonucleotides in siRNA, siRNAs with various DNA substitutions were constructed. The following could be simultaneously replaced with DNA without substantial loss of gene-silencing activity: the seed arm, which occupies positions 2–8 from the 5′end of the guide strand; its complementary sequence; the 5′end of the guide strand and the 3′overhang of the passenger strand. However, most part of the 3′ two-thirds of the guide strand could not be replaced with DNA, possibly due to binding of RNA-recognition proteins such as TRBP2 and Ago2. The passenger strand with DNA in the 3′end proximal region was incapable of inducing off-target effect. Owing to lesser stability of DNA–RNA hybrid than RNA duplex, modified siRNAs with DNA substitution in the seed region were, in most cases, incapable to exert unintended gene silencing due to seed sequence homology. Thus, it may be possible to design DNA–RNA chimeras which effectively silence mammalian target genes without silencing unintended genes.     

Ancestral Roles of Small RNAs: An Ago-Centric Perspective

RNAi has existed at least since the divergence of prokaryotes and eukaryotes. This collection of pathways responds to a diversity of “abberant” RNAs and generally silences or eliminates genes sharing sequence content with the silencing trigger. In the canonical pathway, double-stranded RNAs are processed into small RNAs, which guide effector complexes to their targets by complementary base pairing. Many alternative routes from silencing trigger to small RNA are continuously being uncovered. Though the triggers of the pathway and the mechanisms of small RNA production are many, all RNAi-related mechanisms share Argonaute proteins as the heart of their effector complexes. These can act as self-contained silencing machines, binding directly to small RNAs, carrying out homology-based target recognition, and in some cases cleaving targets using an endogenous nuclease domain. Here, we discuss the diversity of Argonaute proteins from a structural and functional perspective.     

Structure of Aquifex aeolicus argonaute highlights conformational flexibility of the PAZ domain as a potential regulator of RNA-induced silencing complex function

Gene silencing mediated by RNA interference requires the sequence-specific recognition of target mRNA by the endonuclease Argonaute, the primary enzymatic component of the RNA-induced silencing complex. We report the crystal structure of Aquifex aeolicus Argonaute, refined at 3.2A resolution. Relative to recent Argonaute structures, a 24 degrees reorientation of the PAZ domain in our structure opens a basic cleft between the N-terminal and PAZ domains, exposing the guide strand binding pocket of PAZ. This rearrangement leads to a branched, Y-shaped system of grooves that extends through the molecule and merges in a central channel containing the catalytic residues. A 5.5-ns molecular dynamics simulation of Argonaute shows a strong tendency of the PAZ and N-terminal domains to be mobile. Binding of single-stranded DNA to Argonaute monitored by total internal reflection fluorescence spectroscopy shows biphasic kinetics, also indicative of domain rearrangement upon DNA binding. Conformational rearrangement of the PAZ domain may therefore be critical for the catalytic cycle of Argonaute and the RNA-induced silencing complex.