Focusing on RISC assembly in mammalian cells

RISC (RNA-induced silencing complex) is a central protein complex in RNAi, into which a siRNA strand is assembled to become effective in gene silencing. By using an in vitro RNAi reaction based on Drosophila embryo extract, an asymmetric model was recently proposed for RISC assembly of siRNA strands, suggesting that the strand that is more loosely paired at its 5′ end is selectively assembled into RISC and results in target gene silencing. However, in the present study, we were unable to establish such a correlation in cell-based RNAi assays, as well as in large-scale RNAi data analyses. This suggests that the thermodynamic stability of siRNA is not a major determinant of gene silencing in mammalian cells. Further studies on fork siRNAs showed that mismatch at the 5′ end of the siRNA sense strand decreased RISC assembly of the antisense strand, but surprisingly did not increase RISC assembly of the sense strand. More interestingly, measurements of melting temperature showed that the terminal stability of fork siRNAs correlated with the positions of the mismatches, but not gene silencing efficacy. In summary, our data demonstrate that there is no definite correlation between siRNA stability and gene silencing in mammalian cells, which suggests that instead of thermodynamic stability, other features of the siRNA duplex contribute to RISC assembly in RNAi.     

Short interfering RNA strand selection is independent of dsRNA processing polarity during RNAi in Drosophila

Short interfering RNAs (siRNAs) guide mRNA cleavage during RNA interference (RNAi). Only one siRNA strand assembles into the RNA-induced silencing complex (RISC), with preference given to the strand whose 5' terminus has lower base-pairing stability. In Drosophila, Dcr-2/R2D2 processes siRNAs from longer double-stranded RNAs (dsRNAs) and also nucleates RISC assembly, suggesting that nascent siRNAs could remain bound to Dcr-2/R2D2. In vitro, Dcr-2/R2D2 senses base-pairing asymmetry of synthetic siRNAs and dictates strand selection by asymmetric binding to the duplex ends. During dsRNA processing, Dicer (Dcr) liberates siRNAs from dsRNA ends in a manner dictated by asymmetric enzyme-substrate interactions. Because Dcr-2/R2D2 is unlikely to sense base-pairing asymmetry of an siRNA that is embedded within a precursor, it is not clear whether processed siRNAs strictly follow the thermodynamic asymmetry rules or whether processing polarity can affect strand selection. We use a Drosophila in vitro system in which defined siRNAs with known asymmetry can be generated from longer dsRNA precursors. These dsRNAs permit processing specifically from either the 5' or the 3' end of the thermodynamically favored strand of the incipient siRNA. Combined dsRNA-processing/mRNA-cleavage assays indicate that siRNA strand selection is independent of dsRNA processing polarity during Drosophila RISC assembly in vitro.     

Effect of asymmetric terminal structures of short RNA duplexes on the RNA interference activity and strand selection

Short interfering RNAs (siRNAs) are valuable reagents for sequence-specific inhibition of gene expression via the RNA interference (RNAi) pathway. Although it has been proposed that the relative thermodynamic stability at the 5'-ends of siRNAs plays a crucial role in siRNA strand selection, we demonstrate here that a character of the 2-nt 3'-overhang of siRNAs is the predominant determinant of which strand participates in the RNAi pathway. We show that siRNAs with a unilateral 2-nt 3'-overhang on the antisense strand are more effective than siRNAs with 3'-overhangs at both ends, due to preferential loading of the antisense strand into the RNA-induced silencing complex (RISC). Regardless of the relative thermodynamic stabilities at the ends of siRNAs, overhang-containing strands are predominantly selected as the guide strand; whereas, relative stability markedly influences opposite strand selection. Moreover, we show that sense strand modifications, such as deletions or DNA substitutions, of siRNAs with unilateral overhang on the antisense strand have no negative effect on the antisense strand selection, but may improve RNAi potency. Our findings provide useful guidelines for the design of potent siRNAs and contribute to understanding the crucial factors in determining strand selection in mammalian cells.     

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.     

Potent RNAi by short RNA triggers

RNA interference (RNAi) is a gene-silencing mechanism by which a ribonucleoprotein complex, the RNA-induced silencing complex (RISC) and a double-stranded (ds) short-interfering RNA (siRNA), targets a complementary mRNA for site-specific cleavage and subsequent degradation. While longer dsRNA are endogenously processed into 21- to 24-nucleotide (nt) siRNAs or miRNAs to induce gene silencing, RNAi studies in human cells typically use synthetic 19- to 20-nt siRNA duplexes with 2-nt overhangs at the 3′-end of both strands. Here, we report that systematic synthesis and analysis of siRNAs with deletions at the passenger and/or guide strand revealed a short RNAi trigger, 16-nt siRNA, which induces potent RNAi in human cells. Our results indicate that the minimal requirement for dsRNA to trigger RNAi is an ~42 Å A-form helix with ~1.5 helical turns. The 16-nt siRNA more effectively knocked down mRNA and protein levels than 19-nt siRNA when targeting the endogenous CDK9 gene, suggesting that 16-nt siRNA is a more potent RNAi trigger. In vitro kinetic analysis of RNA-induced silencing complex (RISC) programmed in HeLa cells indicates that 16-nt siRNA has a higher RISC-loading capacity than 19-nt siRNA. These results suggest that RISC assembly and activation during RNAi does not necessarily require a 19-nt duplex siRNA and that 16-nt duplexes can be designed as more potent triggers to induce RNAi.     

Effect of target secondary structure on RNAi efficiency

RNA interference (RNAi) mediated by small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) has become a powerful tool for gene knockdown studies. However, the levels of knockdown vary greatly. Here, we examine the effect of target disruption energy, a novel measure of target accessibility, along with other parameters that may affect RNAi efficiency. Based on target secondary structures predicted by the Sfold program, the target disruption energy represents the free energy cost for local alteration of the target structure to allow target binding by the siRNA guide strand. In analyses of 100 siRNAs and 101 shRNAs targeted to 103 endogenous human genes, we find that the disruption energy is an important determinant of RNAi activity and the asymmetry of siRNA duplex asymmetry is important for facilitating the assembly of the RNA-induced silencing complex (RISC). We estimate that target accessibility and duplex asymmetry can improve the target knockdown level significantly by nearly 40% and 26%, respectively. In the RNAi pathway, RISC assembly precedes target binding by the siRNA guide strand. Thus, our findings suggest that duplex asymmetry has significant upstream effect on RISC assembly and target accessibility has strong downstream effect on target recognition. The results of the analyses suggest criteria for improving the design of siRNAs and shRNAs.     

RNA interference tolerates 2'-fluoro modifications at the Argonaute2 cleavage site

Short interfering RNA (siRNA) molecules with good gene-silencing properties are needed for drug development based on RNA interference (RNAi). An initial step in RNAi is the activation of the RNA-induced silencing complex RISC, which requires degradation of the sense strand of the siRNA duplex. Although various chemical modifications have been introduced to the antisense strand, modifications to the Argonaute2 (Ago2) cleavage site in the sense strand have, so far, not been described in detail. In this work, novel 2'-F-purine modifications were introduced to siRNAs, and their biological efficacies were tested in cells stably expressing human tartrate-resistant acid phosphatase (TRACP). A validated siRNA that contains both purine and pyrimidine nucleotides at the putative Ago2 cleavage site was chemically modified to contain all possible combinations of 2'-fluorinated 2'-deoxypurines and/or 2'-deoxypyrimidines in the antisense and/or sense strands. The capacity of 2'-F-modified siRNAs to knock down their target mRNA and protein was studied, together with monitoring siRNA toxicity. All 2'-F-modified siRNAs resulted in target knockdown at nanomolar concentrations, despite their high thermal stability. These experiments provide the first evidence that RISC activation not only allows 2'-F modifications at the sense-strand cleavage site, but also increase the biological efficacy of modified siRNAs in vitro.     

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.     

The impact of target site accessibility on the design of effective siRNAs

Small-interfering RNAs (siRNAs) assemble into RISC, the RNA-induced silencing complex, which cleaves complementary mRNAs. Despite their fluctuating efficacy, siRNAs are widely used to assess gene function. Although this limitation could be ascribed, in part, to variations in the assembly and activation of RISC, downstream events in the RNA interference (RNAi) pathway, such as target site accessibility, have so far not been investigated extensively. In this study we present a comprehensive analysis of target RNA structure effects on RNAi by computing the accessibility of the target site for interaction with the siRNA. Based on our observations, we developed a novel siRNA design tool, RNAxs, by combining known siRNA functionality criteria with target site accessibility. We calibrated our method on two data sets comprising 573 siRNAs for 38 genes, and tested it on an independent set of 360 siRNAs targeting four additional genes. Overall, RNAxs proves to be a robust siRNA selection tool that substantially improves the prediction of highly efficient siRNAs.     

Fluorescence cross-correlation spectroscopy reveals mechanistic insights into the effect of 2'-O-methyl modified siRNAs in living cells

RNA interference (RNAi) offers a powerful tool to specifically direct the degradation of complementary RNAs, and thus has great therapeutic potential for targeting diseases. Despite the reported preferences of RNAi, there is still a need for new techniques that will allow for a detailed mechanistic characterization of RNA-induced silencing complex (RISC) assembly and activity to further improve the biocompatibility of modified siRNAs. In contrast to previous reports, we investigated the effects of 2′-O-methyl (2′OMe) modifications introduced at specific positions within the siRNA at the early and late stages of RISC assembly, as well as their influence on target recognition and cleavage directly in living cells. We found that six to 10 2′OMe nucleotides on the 3′-end inhibit passenger-strand release as well as target-RNA cleavage without changing the affinity, strand asymmetry, or target recognition. 2′OMe modifications introduced at the 5′-end reduced activated RISC stability, whereas incorporations at the cleavage site showed only minor effects on passenger-strand release when present on the passenger strand. Our new fluorescence cross-correlation spectroscopy assays resolve different steps and stages of RISC assembly and target recognition with heretofore unresolved detail in living cells, which is needed to develop therapeutic siRNAs with optimized in vivo properties.     

Increased siRNA duplex stability correlates with reduced off-target and elevated on-target effects

Argonaute (Ago) proteins form the core of RNA-induced silencing complexes (RISCs) and mediate small RNA-guided gene silencing. In RNAi, short interfering RNAs (siRNAs) guide RISCs to complementary target RNAs, leading to cleavage by the endonuclease Ago2. Noncatalytic Ago proteins, however, contribute to RNAi as well but cannot cleave target RNA and often generate off-target effects. Here we show that synthetic siRNA duplexes interact with all Ago proteins, but a functional RISC rapidly assembles only around Ago2. By stabilizing the siRNA duplex, we show that the noncatalytic Ago proteins Ago1, -3, and -4 can be selectively blocked and do not form functional RISCs. In addition, stabilized siRNAs form an Ago2-RISC more efficiently, leading to increased silencing activity. Our data suggest novel parameters for the design of siRNAs with selective activation of the endonuclease Ago2.     

Asymmetry in the assembly of the RNAi enzyme complex

A key step in RNA interference (RNAi) is assembly of the RISC, the protein-siRNA complex that mediates target RNA cleavage. Here, we show that the two strands of an siRNA duplex are not equally eligible for assembly into RISC. Rather, both the absolute and relative stabilities of the base pairs at the 5' ends of the two siRNA strands determine the degree to which each strand participates in the RNAi pathway. siRNA duplexes can be functionally asymmetric, with only one of the two strands able to trigger RNAi. Asymmetry is the hallmark of a related class of small, single-stranded, noncoding RNAs, microRNAs (miRNAs). We suggest that single-stranded miRNAs are initially generated as siRNA-like duplexes whose structures predestine one strand to enter the RISC and the other strand to be destroyed. Thus, the common step of RISC assembly is an unexpected source of asymmetry for both siRNA function and miRNA biogenesis.     

Conversion of pre-RISC to holo-RISC by Ago2 during assembly of RNAi complexes

In the Drosophila RNA interference (RNAi) pathway, small interfering RNAs (siRNAs) direct Argonaute2 (Ago2), an endonuclease, within the RNA-induced silencing complex (RISC) to cleave complementary mRNA targets. In vitro studies have shown that, for each siRNA duplex, RISC retains only one strand, the guide, and releases the other, the passenger, to form a holo-RISC complex. Here, we have isolated a new Ago2 mutant allele and provide, for the first time, in vivo evidence that endogenous Ago2 slicer activity is important to mount an RNAi response in Drosophila. We demonstrate in vivo that efficient removal of the passenger strand from RISC requires the cleavage activity of Ago2. We have also identified a new intermediate complex in the RISC assembly pathway, pre-RISC, in which Ago2 is stably bound to double-stranded siRNA.     

Cleavage of the siRNA passenger strand during RISC assembly in human cells

A crucial step in the RNA interference (RNAi) pathway involves the assembly of RISC, the RNA-induced silencing complex. RISC initially recognizes a double-stranded short interfering RNA (siRNA), but only one strand is finally retained in the functional ribonucleoprotein complex. The non-incorporated strand, or 'passenger' strand, is removed during the assembly process and most probably degraded thereafter. In this report, we show that the passenger strand is cleaved during the course of RISC assembly following the same rules established for the siRNA-guided cleavage of a target RNA. Chemical modifications impairing the cleavage of the passenger strand also impair the cleavage of a target RNA in vitro as well as the silencing of a reporter gene in vivo, suggesting that passenger strand removal is facilitated by its cleavage during RISC assembly. Interestingly, target RNA cleavage can be rescued if an otherwise non-cleavable passenger strand shows a nick at the scissile phosphodiester bond, which further indicates that the cleavage event per se is not essential.     

Improvements in siRNA properties mediated by 2'-deoxy-2'-fluoro-beta-D-arabinonucleic acid (FANA)

RNA interference (RNAi) has emerged recently as an efficient mechanism for specific gene silencing. Short double-stranded small interfering RNAs (siRNAs) are now widely used for cellular or drug target validation; however, their use for silencing clinically relevant genes in a therapeutic setting remains problematic because of their unfavourable metabolic stability and pharmacokinetic properties. To address some of these concerns, we have investigated the properties of siRNA modified with 2'-deoxy-2'-fluoro-beta-d-arabinonucleotide units (araF-N or FANA units). Here we provide evidence that these modified siRNAs are compatible with the intracellular RNAi machinery and can mediate specific degradation of target mRNA. We also show that the incorporation of FANA units into siRNA duplexes increases activity and substantially enhances serum stability of the siRNA. A fully modified sense 2'-deoxy-2'-fluoro-beta-D-arabinonucleic acid (FANA) strand when hybridized to an antisense RNA (i.e. FANA/RNA hybrid) was shown to be 4-fold more potent and had longer half-life in serum (approximately 6 h) compared with an unmodified siRNA (<15 min). While incorporation of FANA units is well tolerated throughout the sense strand of the duplex, modifications can also be included at the 5' or 3' ends of the antisense strand, in striking contrast to other commonly used chemical modifications. Taken together, these results offer preliminary evidence of the therapeutic potential of FANA modified siRNAs.     

RNA诱导沉默复合体中的生物大分子及其装配

在RNA干扰机制中,双链RNA诱导同源RNA降解的过程依赖于RNA诱导沉默复合体（RISC）的活性。RISC由Dicer酶,Argonaute蛋白,siRNA等多种生物大分子装配而成,对这些大分子的结构和功能进行深入细致的研究,有助于进一步了解RISC的形成过程、作用方式,以及阐明整个RNAi过程的作用机制。研究表明,RISC中的Dicer具有RNaseIII结构域,在RNAi的起始阶段负责催化siRNA的产生,在RISC装配过程中起稳定RISC中间体结构和功能的作用;Argonaute蛋白是RISC中的核心蛋白,有PAZ和PIWI两个主要的结构域,前者为siRNA的传递提供结合位点,后者是RISC中的酶切割活性中心;siRNA是RISC完成特异性切割作用的向导,在成熟的RISC中虽然只包含siRNA的一条链,但siRNA在RISC形成过程中的双链结构是保证RNAi效应的决定因素。尽管RISC中还存在其他一些功能未知的蛋白质,但在RISC组分结构及功能研究方面取得的进展为建立一个可能的RISC装配模型提供了理论基础。