Improved siRNA/shRNA functionality by mismatched duplex

siRNA (small interfering RNA) and shRNA (small hairpin RNA) are powerful and commonly used tools in biomedical research. Currently, siRNAs are generally designed as two 21 nt strands of RNA that include a 19 nt completely complementary part and a 2 nt overhang. However, since the si/shRNAs use the endogenous miRNA machinery for gene silencing and the miRNAs are generally 22 nt in length and contain multiple internal mismatches, we tested if the functionality can be increased by designing the si/shRNAs to mimic a miRNA structure. We systematically investigated the effect of single or multiple mismatches introduced in the passenger strand at different positions on siRNA functionality. Mismatches at certain positions could significantly increase the functionality of siRNAs and also, in some cases decreased the unwanted passenger strand functionality. The same strategy could also be used to design shRNAs. Finally, we showed that both si and miRNA structured oligos (siRNA with or without mismatches in the passenger strand) can repress targets in all individual Ago containing cells, suggesting that the Ago proteins do not differentiate between si/miRNA-based structure for silencing activity.     

Translation repression in human cells by microRNA-induced gene silencing requires RCK/p54

RNA interference is triggered by double-stranded RNA that is processed into small interfering RNAs (siRNAs) by Dicer enzyme. Endogenously, RNA interference triggers are created from small noncoding RNAs called microRNAs (miRNAs). RNA-induced silencing complexes (RISC) in human cells can be programmed by exogenously introduced siRNA or endogenously expressed miRNA. siRNA-programmed RISC (siRISC) silences expression by cleaving a perfectly complementary target mRNA, whereas miRNA-induced silencing complexes (miRISC) inhibits translation by binding imperfectly matched sequences in the 3′ UTR of target mRNA. Both RISCs contain Argonaute2 (Ago2), which catalyzes target mRNA cleavage by siRISC and localizes to cytoplasmic mRNA processing bodies (P-bodies). Here, we show that RCK/p54, a DEAD box helicase, interacts with argonaute proteins, Ago1 and Ago2, in affinity-purified active siRISC or miRISC from human cells; directly interacts with Ago1 and Ago2 in vivo, facilitates formation of P-bodies, and is a general repressor of translation. Disrupting P-bodies by depleting Lsm1 did not affect RCK/p54 interactions with argonaute proteins and its function in miRNA-mediated translation repression. Depletion of RCK/p54 disrupted P-bodies and dispersed Ago2 throughout the cytoplasm but did not significantly affect siRNA-mediated RNA functions of RISC. Depleting RCK/p54 released general, miRNA-induced, and let-7-mediated translational repression. Therefore, we propose that translation repression is mediated by miRISC via RCK/p54 and its specificity is dictated by the miRNA sequence binding multiple copies of miRISC to complementary 3′ UTR sites in the target mRNA. These studies also suggest that translation suppression by miRISC does not require P-body structures, and location of miRISC to P-bodies is the consequence of translation repression.     

Dual regulation of hepatitis C viral RNA by cellular RNAi requires partitioning of Ago2 to lipid droplets and P-bodies

The antiviral role of RNA interference (RNAi) in humans remains to be better understood. In RNAi, Ago2 proteins and microRNAs (miRNAs) or small interfering RNAs (siRNAs) form endonucleolytically active complexes which down-regulate expression of target mRNAs. P-bodies, cytoplasmic centers of mRNA decay, are involved in these pathways. Evidence exists that hepatitis C virus (HCV) utilizes host cellular RNAi machinery, including miRNA-122, Ago1-4, and Dicer proteins for replication and viral genome translation in Huh7 cells by, so far, nebulous mechanisms. Conversely, synthetic siRNAs have been used to suppress HCV replication. Here, using a combination of biochemical, transfection, confocal imaging, and digital image analysis approaches, we reveal that replication of HCV RNA depends on recruitment of Ago2 and miRNA-122 to lipid droplets, while suppression of HCV RNA by siRNA and Ago2 involves interaction with P-bodies. Such partitioning of Ago2 proteins into different complexes and separate subcellular domains likely results in modulation of their activity by different reaction partners. We propose a model in which partitioning of host RNAi and viral factors into physically and functionally distinct subcellular compartments emerges as a mechanism regulating the dual interaction of cellular RNAi with HCV RNA.     

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.     

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.     

Off-target and a portion of target-specific siRNA mediated mRNA degradation is Ago2 ‘Slicer’ independent and can be mediated by Ago1

It is known that siRNAs are capable of reducing expression of non-target genes due to the interaction of the siRNA guide strand with a partially complementary site on the ‘off-target’ mRNA. In the current study, we show that reduction of cellular Ago2 levels has no effect on off-target reduction of endogenous genes and that off-target degradation of mRNA can occur even in an Ago2 knockout cell line. Using antisense mediated reduction of Ago proteins and chemically modified cleavage- and binding-deficient siRNAs, we demonstrate that siRNA mediated off-target reduction is Ago2 cleavage independent, but does require siRNA interaction with either Ago1 or Ago2 and the RISC-loading complex. We also show that depletion of P-body associated proteins results in a reduction of off-target siRNA-mediated degradation of mRNA. Finally, we present data suggesting that a significant portion of on-target siRNA activity is also Ago2 cleavage independent, however, this activity does not appear to be P-body associated.     

Sorting of Drosophila small silencing RNAs

In Drosophila, small interfering RNAs (siRNAs), which direct RNA interference through the Argonaute protein Ago2, are produced by a biogenesis pathway distinct from microRNAs (miRNAs), which regulate endogenous mRNA expression as guides for Ago1. Here, we report that siRNAs and miRNAs are sorted into Ago1 and Ago2 by pathways independent from the processes that produce these two classes of small RNAs. Such small-RNA sorting reflects the structure of the double-stranded assembly intermediates--the miRNA/miRNA( *) and siRNA duplexes--from which Argonaute proteins are loaded. We find that the Dcr-2/R2D2 heterodimer acts as a gatekeeper for the assembly of Ago2 complexes, promoting the incorporation of siRNAs and disfavoring miRNAs as loading substrates for Drosophila Ago2. A separate mechanism acts in parallel to favor miRNA/miRNA( *) duplexes and exclude siRNAs from assembly into Ago1 complexes. Thus, in flies small-RNA duplexes are actively sorted into Argonaute-containing complexes according to their intrinsic structures.     

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.     

Human Ago2 is required for efficient microRNA 122 regulation of hepatitis C virus RNA accumulation and translation

MicroRNA 122 (miR-122) increases the accumulation and translation of hepatitis C virus (HCV) RNA in infected cells through direct interactions with homologous sequences in the 5' untranslated region (UTR) of the HCV genome. Argonaute 2 (Ago2) is a component of the RNA-induced silencing complex (RISC) and mediates small interfering RNA (siRNA)-directed mRNA cleavage and microRNA translational suppression. We investigated the function of Ago2 in HCV replication to determine whether it plays a role in enhancing the synthesis and translation of HCV RNA that is associated with miR-122. siRNA-mediated depletion of Ago2 in human hepatoma cells reduced HCV RNA accumulation in transient HCV replication assays. The treatment did not adversely affect cell viability, as assessed by cell proliferation, capped translation, and interferon assays. These data are consistent with complementary roles for Ago2 and miR-122 in enhancing HCV RNA amplification. By using a transient HCV replication assay that is dependent on an exogenously provided mutant miR-122, we determined that Ago2 depletion still reduced luciferase expression and HCV RNA accumulation, independently of miR-122 biogenesis. miR-122 has previously been found to stimulate HCV translation. Similarly, Ago2 knockdown also reduced HCV translation, and its depletion reduced the ability of miR-122 to stimulate viral translation. These data suggest a direct role for Ago2 in miR-122-mediated translation. Finally, Ago2 was also necessary for efficient miR-122 enhancement of HCV RNA accumulation. These data support a model in which miR-122 functions within an Ago2-containing protein complex to augment both HCV RNA accumulation and translation.     

The Cricket Paralysis Virus Suppressor Inhibits microRNA Silencing Mediated by the Drosophila Argonaute-2 Protein

Small RNAs are potent regulators of gene expression. They also act in defense pathways against invading nucleic acids such as transposable elements or viruses. To counteract these defenses, viruses have evolved viral suppressors of RNA silencing (VSRs). Plant viruses encoded VSRs interfere with siRNAs or miRNAs by targeting common mediators of these two pathways. In contrast, VSRs identified in insect viruses to date only interfere with the siRNA pathway whose effector Argonaute protein is Argonaute-2 (Ago-2). Although a majority of Drosophila miRNAs exerts their silencing activity through their loading into the Argonaute-1 protein, recent studies highlighted that a fraction of miRNAs can be loaded into Ago-2, thus acting as siRNAs. In light of these recent findings, we re-examined the role of insect VSRs on Ago-2-mediated miRNA silencing in Drosophila melanogaster. Using specific reporter systems in cultured Schneider-2 cells and transgenic flies, we showed here that the Cricket Paralysis virus VSR CrPV1-A but not the Flock House virus B2 VSR abolishes silencing by miRNAs loaded into the Ago-2 protein. Thus, our results provide the first evidence that insect VSR have the potential to directly interfere with the miRNA silencing pathway.     

Inhibition of 3' modification of small RNAs in virus-infected plants require spatial and temporal co-expression of small RNAs and viral silencing-suppressor proteins

Plant viruses are inducers and targets of RNA silencing. Viruses counteract with RNA silencing by expressing silencing-suppressor proteins. Many of the identified proteins bind siRNAs, which prevents assembly of silencing effector complexes, and also interfere with their 3' methylation, which protects them against degradation. Here, we investigated the 3' modification of silencing-related small RNAs in Nicotiana benthamiana plants infected with viruses expressing RNA silencing suppressors, the p19 protein of Carnation Italian ringspot virus (CIRV) and HC-Pro of Tobacco etch virus (TEV). We found that CIRV had only a slight effect on viral siRNA 3' modification, but TEV significantly inhibited the 3' modification of si/miRNAs. We also found that p19 and HC-Pro were able to bind both 3' modified and non-modified small RNAs in vivo. The findings suggest that the 3' modification of viral siRNAs occurs in the cytoplasm, though miRNA 3' modification likely takes place in the nucleus as well. Both silencing suppressors inhibited the 3' modification of si/miRNAs when they and small RNAs were transiently co-expressed, suggesting that the inhibition of si/miRNA 3' modification requires spatial and temporal co-expression. Finally, our data revealed that a HEN1-like methyltransferase might account for the small RNA modification at the their 3'-terminal nucleotide in N. benthamiana.     

Identification of the suppressive factors for human immunodeficiency virus type-1 replication using the siRNA mini-library directed against host cellular genes

We performed the screening to find the novel host factors affecting human immunodeficiency virus type-1 (HIV-1) replication using the siRNA mini-library consisted with 257 siRNAs directed against cellular genes. J111 cells, a human acute monocytic leukemia cell line, were transfected with individual siRNA, followed by either infected or transfected with the HIV-1 molecular clone with luciferase reporter gene in 96-well plate format. The results showed that six siRNAs significantly enhanced the HIV-1 replication in J111 cells, indicating that the target cellular genes of those siRNAs may negatively regulate HIV-1 replication in normal cell culture condition. We also discuss the possible mechanisms by which those cellular proteins regulate viral replication.     

高致病性PRRSVNsp2基因RNA干扰对病毒复制的影响

旨在构建针对猪繁殖与呼吸综合征病毒(PRRSV)TJ株Nsp2基因的siRNA的表达载体,研究Nsp2基因的表达抑制对PRRSV复制的影响。设计并合成针对PRRSV TJ株Nsp2基因的3对优势小发卡RNA(shRNA),连接到pRNAT-U6.1/Neo,构建shRNA表达载体,将鉴定正确的载体质粒转染Marc-145细胞,6 h后接毒,每日观察CPE;在接毒PRRSV TJ株后不同时间点取上清样品,测定样品TCID50;并以β-actin为内参,RT-PCR对PRRSV Nsp2 mRNA进行半定量。结果表明,选取的3条Nsp2基因的优势siRNA均能够抑制PRRSV TJ株在Marc-145细胞上增殖,其中shRNA载体S-1和S-2抑制效果明显,与空载体相比较差异极显著。本研究构建的PRRSV Nsp2基因特异性shRNA载体能够有效抑制PRRSV的复制,可使病毒滴度TCID50最多降低103.38。     

Localization of double-stranded small interfering RNA to cytoplasmic processing bodies is Ago2 dependent and results in up-regulation of GW182 and Argonaute-2

Processing bodies (P-bodies) are cytoplasmic foci implicated in the regulation of mRNA translation, storage, and degradation. Key effectors of microRNA (miRNA)-mediated RNA interference (RNAi), such as Argonaute-2 (Ago2), miRNAs, and their cognate mRNAs, are localized to these structures; however, the precise role that P-bodies and their component proteins play in small interfering RNA (siRNA)-mediated RNAi remains unclear. Here, we investigate the relationship between siRNA-mediated RNAi, RNAi machinery proteins, and P-bodies. We show that upon transfection into cells, siRNAs rapidly localize to P-bodies in their native double-stranded conformation, as indicated by fluorescence resonance energy transfer imaging and that Ago2 is at least in part responsible for this siRNA localization pattern, indicating RISC involvement. Furthermore, siRNA transfection induces up-regulated expression of both GW182, a key P-body component, and Ago2, indicating that P-body localization and interaction with GW182 and Ago2 are important in siRNA-mediated RNAi. By virtue of being centers where these proteins and siRNAs aggregate, we propose that the P-body microenvironment, whether as microscopically visible foci or submicroscopic protein complexes, facilitates siRNA processing and siRNA-mediated silencing through the action of its component proteins.