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.     

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.     

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.     

Autoantigen La promotes efficient RNAi, antiviral response, and transposon silencing by facilitating multiple-turnover RISC catalysis

The effector of RNA interference (RNAi) is the RNA-induced silencing complex (RISC). C3PO promotes the activation of RISC by degrading the Argonaute2 (Ago2)-nicked passenger strand of duplex siRNA. Active RISC is a multiple-turnover enzyme that uses the guide strand of siRNA to direct the Ago2-mediated sequence-specific cleavage of complementary mRNA. How this effector step of RNAi is regulated is currently unknown. Here, we used the human Ago2 minimal RISC system to purify Sj?gren's syndrome antigen B (SSB)/autoantigen La as an activator of the RISC-mediated mRNA cleavage activity. Our reconstitution studies showed that La could promote multiple-turnover RISC catalysis by facilitating the release of cleaved mRNA from RISC. Moreover, we demonstrated that La was required for efficient RNAi, antiviral defense, and transposon silencing in vivo. Taken together, the findings of C3PO and La reveal a general concept that regulatory factors are required to remove Ago2-cleaved products to assemble or restore active RISC.     

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.     

RNAi: RISC gets loaded

When an siRNA or miRNA proceeds through the RNA-induced silencing complex assembly pathway, only one of the two approximately 21-nucleotide RNA strands survives in the final, active complex. In this issue of Cell, Matranga et al. (2005) and Rand et al. (2005) reveal the fate of the rejected passenger siRNA strand. Additionally, Gregory et al. (2005) define a heterotrimeric complex from humans that appears to execute dsRNA loading, strand selection, and target mRNA cleavage activities.     

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.     

MicroRNA: Biogenesis, Function and Role in Cancer

MicroRNAs are small, highly conserved non-coding RNA molecules involved in the regulation of gene expression. MicroRNAs are transcribed by RNA polymerases II and III, generating precursors that undergo a series of cleavage events to form mature microRNA. The conventional biogenesis pathway consists of two cleavage events, one nuclear and one cytoplasmic. However, alternative biogenesis pathways exist that differ in the number of cleavage events and enzymes responsible. How microRNA precursors are sorted to the different pathways is unclear but appears to be determined by the site of origin of the microRNA, its sequence and thermodynamic stability. The regulatory functions of microRNAs are accomplished through the RNA-induced silencing complex (RISC). MicroRNA assembles into RISC, activating the complex to target messenger RNA (mRNA) specified by the microRNA. Various RISC assembly models have been proposed and research continues to explore the mechanism(s) of RISC loading and activation. The degree and nature of the complementarity between the microRNA and target determine the gene silencing mechanism, slicer-dependent mRNA degradation or slicer-independent translation inhibition. Recent evidence indicates that P-bodies are essential for microRNA-mediated gene silencing and that RISC assembly and silencing occurs primarily within P-bodies. The P-body model outlines microRNA sorting and shuttling between specialized P-body compartments that house enzymes required for slicer -dependent and -independent silencing, addressing the reversibility of these silencing mechanisms. Detailed knowledge of the microRNA pathways is essential for understanding their physiological role and the implications associated with dysfunction and dysregulation.     

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。     

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.     

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.     

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.     

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.     

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.     

Slicer-independent mechanism drives small-RNA strand separation during human RISC assembly

Small RNA silencing is mediated by the effector RNA-induced silencing complex (RISC) that consists of an Argonaute protein (AGOs 1–4 in humans). A fundamental step during RISC assembly involves the separation of two strands of a small RNA duplex, whereby only the guide strand is retained to form the mature RISC, a process not well understood. Despite the widely accepted view that ‘slicer-dependent unwinding’ via passenger-strand cleavage is a prerequisite for the assembly of a highly complementary siRNA into the AGO2-RISC, here we show by careful re-examination that ‘slicer-independent unwinding’ plays a more significant role in human RISC maturation than previously appreciated, not only for a miRNA duplex, but, unexpectedly, for a highly complementary siRNA as well. We discovered that ‘slicer-dependency’ for the unwinding was affected primarily by certain parameters such as temperature and Mg²⁺. We further validate these observations in non-slicer AGOs (1, 3 and 4) that can be programmed with siRNAs at the physiological temperature of humans, suggesting that slicer-independent mechanism is likely a common feature of human AGOs. Our results now clearly explain why both miRNA and siRNA are found in all four human AGOs, which is in striking contrast to the strict small-RNA sorting system in Drosophila.     

Dicer is dispensable for asymmetric RISC loading in mammals

In flies, asymmetric loading of small RNA duplexes into Argonaute2-containing RNA-induced silencing complex (Ago2-RISC) requires Dicer-2/R2D2 heterodimer, which acts as a protein sensor for the thermodynamic stabilities of the ends of small RNA duplexes. However, the mechanism of small RNA asymmetry sensing in mammalian RISC assembly remains obscure. Here, we quantitatively examined RISC assembly and target silencing activity in the presence or absence of Dicer in mammals. Our data show that, unlike the well-characterized fly Ago2-RISC assembly pathway, mammalian Dicer is dispensable for asymmetric RISC loading in vivo and in vitro.     

Argonaute2 cleaves the anti-guide strand of siRNA during RISC activation

The mRNA-cleavage step of RNA interference is mediated by an endonuclease, Argonaute2 (Ago2), within the RNA-induced silencing complex (RISC). Ago2 uses one strand of the small interfering (si) RNA duplex as a guide to find messenger RNAs containing complementary sequences and cleaves the phosphodiester backbone at a specific site measured from the guide strand's 5' end. Here, we show that both strands of siRNA get loaded onto Ago2 protein in Drosophila S2 cell extracts. The anti-guide strand behaves as a RISC substrate and is cleaved by Ago2. This cleavage event is important for the removal of the anti-guide strand from Ago2 protein and activation of RISC.