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.     

Biaryl modification of the 5'-terminus of one strand of a microRNA duplex induces strand specificity

MicroRNAs (miRNAs) are single-stranded non-coding RNAs composed of 20-23 nucleotides. They are initially transcribed in the nucleus as pri-miRNAs. After processing, one strand from the miRNA duplex (miR-5p/miR-3p duplex) is loaded onto the RNA-induced silencing complex (RISC) to produce a functional, mature miRNA that inhibits the expression of multiple target genes. In the case of some miRNAs, both strands can be equally incorporated into the RISC as single strands, and both strands can function as mature miRNAs. Thus, a technique for selective expression of miR-5p and miR-3p strands is required to identify distinct targets of miRNAs. In this Letter, we report the synthesis and properties of miRNA duplexes carrying biaryl units at the 5'-terminus of one strand. We found that incorporation of biaryl units at the 5'-terminus of one strand of miRNA duplexes induced strand specificity in these duplexes. Further, we succeeded in identifying endogenous mRNA targets for each strand of the duplex by using the biaryl-modified miRNA duplexes.     

Strand-specific 5'-O-methylation of siRNA duplexes controls guide strand selection and targeting specificity

Small interfering RNAs (siRNAs) and microRNAs (miRNAs) guide catalytic sequence-specific cleavage of fully or nearly fully complementary target mRNAs or control translation and/or stability of many mRNAs that share 6-8 nucleotides (nt) of complementarity to the siRNA and miRNA 5' end. siRNA- and miRNA-containing ribonucleoprotein silencing complexes are assembled from double-stranded 21- to 23-nt RNase III processing intermediates that carry 5' phosphates and 2-nt overhangs with free 3' hydroxyl groups. Despite the structural symmetry of a duplex siRNA, the nucleotide sequence asymmetry can generate a bias for preferred loading of one of the two duplex-forming strands into the RNA-induced silencing complex (RISC). Here we show that the 5'-phosphorylation status of the siRNA strands also acts as an important determinant for strand selection. 5'-O-methylated siRNA duplexes refractory to 5' phosphorylation were examined for their biases in siRNA strand selection. Asymmetric, single methylation of siRNA duplexes reduced the occupancy of the silencing complex by the methylated strand with concomitant elimination of its off-targeting signature and enhanced off-targeting signature of the phosphorylated strand. Methylation of both siRNA strands reduced but did not completely abolish RNA silencing, without affecting strand selection relative to that of the unmodified siRNA. We conclude that asymmetric 5' modification of siRNA duplexes can be useful for controlling targeting specificity.     

Molecular requirements for RNA-induced silencing complex assembly in the Drosophila RNA interference pathway

Complexes in the Drosophila RNA-induced silencing complex (RISC) assembly pathway can be resolved using native gel electrophoresis, revealing an initiator called R1, an intermediate called R2, and an effector called R3 (now referred to as holo-RISC). Here we show that R1 forms when the Dicer-2/R2D2 heterodimer binds short interfering RNA (siRNA) duplexes. The heterodimer alone can initiate RISC assembly, indicating that other factors are dispensable for initiation. During assembly, R2 requires Argonaute 2 to convert into holo-RISC. This requirement is reminiscent of the RISC-loading complex, which also requires Argonaute 2 for assembly into RISC. We have compared R2 to the RISC-loading complex and show that the two complexes are similar in their sensitivities to ATP and to chemical modifications on siRNA duplexes, indicating that they are likely to be identical. We have examined the requirements for RISC formation and show that the siRNA 5'-termini are repeatedly monitored during RISC assembly, first by the Dcr-2/R2D2 heterodimer and again after R2 formation, before siRNA unwinding. The 2'-position of the 5'-terminal nucleotide also affects RISC assembly, because an siRNA strand bearing a 2'-deoxyribose at this position can inhibit the cognate strand from entering holo-RISC; in contrast, the 2'-deoxyribose-modified strand has enhanced activity in the RNA interference pathway.     

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.     

The Drosophila RNA methyltransferase, DmHen1, modifies germline piRNAs and single-stranded siRNAs in RISC

Small silencing RNAs repress gene expression by a set of related mechanisms collectively called RNA-silencing pathways [1, 2]. In the RNA interference (RNAi) pathway [3], small interfering mRNA (siRNAs) defend cells from invasion by foreign nucleic acids, such as those produced by viruses. In contrast, microRNAs (miRNAs) sculpt endogenous mRNA expression [4]. A third class of small RNAs, Piwi-interacting RNAs (piRNAs), defends the genome from transposons [5-9]. Here, we report that Drosophila piRNAs contain a 2'-O-methyl group on their 3' termini; this is a modification previously reported for plant miRNAs and siRNAs [10] and mouse and rat piRNAs [11, 12, 13]. Plant small-RNA methylation is catalyzed by the protein HEN1 [10, 14, 15]. We find that DmHen1, the Drosophila homolog of HEN1, methylates the termini of siRNAs and piRNAs. Without DmHen1, the length and abundance of piRNAs are decreased, and piRNA function is perturbed. Unlike plant HEN1, DmHen1 acts on single strands, not duplexes, explaining how it can use as substrates both siRNAs-which derive from double-stranded precursors-and piRNAs-which do not [8, 13]. 2'-O-methylation of siRNAs may be the final step in assembly of the RNAi-enzyme complex, RISC, occurring after an Argonaute-bound siRNA duplex is converted to single-stranded RNA.     

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.     

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。     

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.     

Rational siRNA design for RNA interference

Short-interfering RNAs suppress gene expression through a highly regulated enzyme-mediated process called RNA interference (RNAi). RNAi involves multiple RNA-protein interactions characterized by four major steps: assembly of siRNA with the RNA-induced silencing complex (RISC), activation of the RISC, target recognition and target cleavage. These interactions may bias strand selection during siRNA-RISC assembly and activation, and contribute to the overall efficiency of RNAi. To identify siRNA-specific features likely to contribute to efficient processing at each step, we performed a systematic analysis of 180 siRNAs targeting the mRNA of two genes. Eight characteristics associated with siRNA functionality were identified: low G/C content, a bias towards low internal stability at the sense strand 3'-terminus, lack of inverted repeats, and sense strand base preferences (positions 3, 10, 13 and 19). Further analyses revealed that application of an algorithm incorporating all eight criteria significantly improves potent siRNA selection. This highlights the utility of rational design for selecting potent siRNAs and facilitating functional gene knockdown studies.     

Suppression of RNA interference by adenovirus virus-associated RNA

We show that human adenovirus inhibits RNA interference (RNAi) at late times of infection by suppressing the activity of two key enzyme systems involved, Dicer and RNA-induced silencing complex (RISC). To define the mechanisms by which adenovirus blocks RNAi, we used a panel of mutant adenoviruses defective in virus-associated (VA) RNA expression. The results show that the virus-associated RNAs, VA RNAI and VA RNAII, function as suppressors of RNAi by interfering with the activity of Dicer. The VA RNAs bind Dicer and function as competitive substrates squelching Dicer. Further, we show that VA RNAI and VA RNAII are processed by Dicer, both in vitro and during a lytic infection, and that the resulting short interfering RNAs (siRNAs) are incorporated into active RISC. Dicer cleaves the terminal stem of both VA RNAI and VA RNAII. However, whereas both strands of the VA RNAI-specific siRNA are incorporated into RISC, the 3' strand of the VA RNAII-specific siRNA is selectively incorporated during a lytic infection. In summary, our work shows that adenovirus suppresses RNAi during a lytic infection and gives insight into the mechanisms of RNAi suppression by VA RNA.     

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.     

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.     

Small interfering RNAs containing full 2'-O-methylribonucleotide-modified sense strands display Argonaute2/eIF2C2-dependent activity

RNA interference (RNAi) is a process by which short interfering RNAs (siRNAs) direct the degradation of complementary single-strand RNAs. In this study, we investigated the effects of full-strand phosphorothioate (PS) backbone and 2'-O-methyl (2'-OMe) sugar modifications on RNAi-mediated silencing. In contrast to previous reports, we have identified active siRNA duplexes containing full 2'-OMe-modified sense strands that display comparable activity to the unmodified analog of similar sequence. The structure of these modified siRNAs is the predominant determinant of their activity, with sequence and backbone composition being secondary. We further show, by using biotin-tagged siRNAs and affinity-tagged hAgo2/eIF2C2, that activity of siRNA duplexes containing full 2'-OMe substitutions in the sense strand is mediated by the RNA-induced silencing complex (RISC) and that strand-specific loading (or binding) to hAgo2 may be modulated through selective incorporation of these modifications.     

siRNA function in RNAi: a chemical modification analysis

Various chemical modifications were created in short-interfering RNAs (siRNAs) to determine the biochemical properties required for RNA interference (RNAi). Remarkably, modifications at the 2'-position of pentose sugars in siRNAs showed the 2'-OHs were not required for RNAi, indicating that RNAi machinery does not require the 2'-OH for recognition of siRNAs and catalytic ribonuclease activity of RNA-induced silencing complexes (RISCs) does not involve the 2'-OH of guide antisense RNA. In addition, 2' modifications predicted to stabilize siRNA increased the persistence of RNAi as compared with wild-type siRNAs. RNAi was also induced with chemical modifications that stabilized interactions between A-U base pairs, demonstrating that these types of modifications may enhance mRNA targeting efficiency in allele-specific RNAi. Modifications altering the structure of the A-form major groove of antisense siRNA-mRNA duplexes abolished RNAi, suggesting that the major groove of these duplexes was required for recognition by activated RISC*. Comparative analysis of the stability and RNAi activities of chemically modified single-stranded antisense RNA and duplex siRNA suggested that some catalytic mechanism(s) other than siRNA stability were linked to RNAi efficiency. Modified or mismatched ribonucleotides incorporated at internal positions in the 5' or 3' half of the siRNA duplex, as defined by the antisense strand, indicated that the integrity of the 5' and not the 3' half of the siRNA structure was important for RNAi, highlighting the asymmetric nature of siRNA recognition for initiation of unwinding. Collectively, this study defines the mechanisms of RNAi in human cells and provides new rules for designing effective and stable siRNAs for RNAi-mediated gene-silencing applications.     

Single-stranded antisense siRNAs guide target RNA cleavage in RNAi

Small interfering RNAs (siRNAs) are the mediators of mRNA degradation in the process of RNA interference (RNAi). Here, we describe a human biochemical system that recapitulates siRNA-mediated target RNA degradation. By using affinity-tagged siRNAs, we demonstrate that a single-stranded siRNA resides in the RNA-induced silencing complex (RISC) together with eIF2C1 and/or eIF2C2 (human GERp95) Argonaute proteins. RISC is rapidly formed in HeLa cell cytoplasmic extract supplemented with 21 nt siRNA duplexes, but also by adding single-stranded antisense RNAs, which range in size between 19 and 29 nucleotides. Single-stranded antisense siRNAs are also effectively silencing genes in HeLa cells, especially when 5'-phosphorylated, and expand the repertoire of RNA reagents suitable for gene targeting.