Longer dsRNAs Research Articles

DsRNA-induced condensation of antiviral proteins modulates PKR activity

Mammalian cells respond to dsRNA in multiple manners. One key response to dsRNA is the activation of PKR, an eIF2α kinase, which triggers translational arrest and the formation of stress granules. However, the process of PKR activation in cells is not fully understood. In response to increased endogenous or exogenous dsRNA, we observed that PKR forms novel cytosolic condensates, referred to as dsRNA-induced foci (dRIFs). dRIFs contain dsRNA, form in proportion to dsRNA, and are enhanced by longer dsRNAs. dRIFs enrich several other dsRNA-binding proteins, including ADAR1, Stau1, NLRP1, and PACT. Strikingly, dRIFs correlate with and form before translation repression by PKR and localize to regions of cells where PKR activation is initiated. We hypothesize that dRIF formation is a mechanism that cells use to enhance the sensitivity of PKR activation in response to low levels of dsRNA or to overcome viral inhibitors of PKR activation.

Ubiquitin-Dependent and -Independent Roles of E3 Ligase RIPLET in Innate Immunity

The conventional view posits that E3 ligases function primarily through conjugating ubiquitin (Ub) to their substrate molecules. We report here that RIPLET, an essential E3 ligase in antiviral immunity, promotes the antiviral signaling activity of the viral RNA receptor RIG-I through both Ub-dependent and -independent manners. RIPLET uses its dimeric structure and a bivalent binding mode to preferentially recognize and ubiquitinate RIG-I pre-oligomerized on dsRNA. In addition, RIPLET can cross-bridge RIG-I filaments on longer dsRNAs, inducing aggregate-like RIG-I assemblies. The consequent receptor clustering synergizes with the Ub-dependent mechanism to amplify RIG-I-mediated antiviral signaling in an RNA-length dependent manner. These observations show the unexpected role of an E3 ligase as a co-receptor that directly participates in receptor oligomerization and ligand discrimination. It also highlights a previously unrecognized mechanism by which the innate immune system measures foreign nucleic acid length, a common criterion for self versus non-self nucleic acid discrimination.

Engineering active siRNA therapeutics

The discovery of RNA interference (RNAi) has led to a rush to develop short interfering RNAs (siRNAs) for a variety of applications, especially for therapeutics. A particular advantage of RNAi-based therapeutics is that they utilize the existing regulatory machinery of gene expression in eukaryotic cells. For this reason, siRNAs have the potential to modulate cell function with exquisite specificity and limited side effects. Nonetheless, the development of siRNAs as therapeutics will require continued research focused on the critical bottlenecks in the process. To initiate RNAi in vivo, siRNAs are first encapsulated into a delivery vehicle that is typically cationic, to balance charge, and either lipid-like or polymeric in nature. As with other drugs, the vehicle–siRNA complexes are either administered directly to the site of interest (e.g. by injection) to minimize trafficking to nontargeted tissues and maximize local concentration of the drug, or delivered through the bloodstream (intravenous) or gastrointestinal system (oral) to simplify administration. Once delivered to the tissue of interest, the vehicle–siRNA complexes must reach the target cells and cross the plasma membrane to enter the cytoplasm. At this point, the siRNA must be released from the vehicle in order to be recognized by the RNAi machinery. In humans, the primary proteins of the RNAi machinery are Dicer, TAR RNA-binding protein (TRBP) and Argonaute 2 (Ago2). Together with the siRNA, these three proteins form the RNA-induced silencing complex (RISC) and its precursor, the RISC loading complex (RLC). The three proteins bind the siRNA and select which strand of the double-stranded siRNA will become the single-stranded guide strand in RISC. Activated RISC, possessing the single-stranded guide strand, then specifically cleaves an mRNA that contains a sequence complementary to the guide strand. The reduction in mRNA concentration leads to a consequential reduction in protein concentration and the corresponding therapeutic effect. However, many other proteins also recognize double-stranded RNAs (dsRNAs), and many of these proteins are involved in innate immunity and the cellular response to viral infections. Although shorter dsRNAs, like siRNAs, are less immunostimulatory than longer dsRNAs, siRNA therapeutic design must focus on avoiding nonspecific recognition by either cell-surface or cytoplasmic proteins. Understanding how to control potential interactions with nonpathway proteins will greatly expand the therapeutic window for siRNA-based therapeutics. The focus of this minireview series is on the means by which siRNA therapeutics can be generated with the greatest activity and specificity. The three articles focus on different steps in this process: (a) selection of the siRNA sequence most likely to have high activity, (b) in vivo delivery of that siRNA to the tissue and cells of interest, and (c) avoiding side effects associated with the administration of foreign nucleic acids. Current approaches and future challenges are discussed.

Small Non-Coding RNAs as Novel Therapeutics

RNA interference (RNAi), an evolutionarily conserved sequence-specific post-transcriptional gene silencing mechanism, is triggered by double-stranded RNA (dsRNA) that results in the degradation of homologous mRNA or in the inhibition of mRNA translation. The naturally occurring triggers for the RNAi pathway are small regulatory RNAs, including small interfering RNAs (siRNAs), processed from longer dsRNAs by the RNAse III enzyme Dicer, and microRNAs (miRNAs), generated in a regulated multistep process from endogenous primary transcripts (pri-miRNA). These primary transcripts are capped, polyadenylated and spliced, thus resembling conventional mRNAs. It is estimated that miRNAs regulate more than one third of all cellular mRNAs, and bioinformatic data indicate that each miRNA can control hundreds of gene targets. Thus, there are likely to be few biological processes not regulated by miRNAs. Although the biological functions of miRNAs are not completely revealed, there is growing evidence that miRNA pathways are a new mechanism of gene regulation in both normal and diseased conditions. Recent evidence has shown that miRNA mutations or aberrant expression patterns correlate with various diseases, such as cancer, viral infections, cardiovascular or neurodegenerative diseases and indicates that miRNAs can function as tumor suppressors and oncogenes. MiRNAs have not only emerged as a powerful tool for gene regulation studies but also for the development of novel drugs. Since they do not encode proteins, they are not traditional therapeutic targets of small-molecule inhibitors and thus comprise a novel class of therapeutics. This article will focus on the current progress in drug discovery using the miRNA strategy.

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 approximately 42 A A-form helix with approximately 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.

Specific and nontoxic silencing in mammalian cells with expressed long dsRNAs

A number of groups have developed libraries of siRNAs to identify genes through functional genomics. While these studies have validated the approach of making functional RNAi libraries to understand fundamental cellular mechanisms, they require information and knowledge of existing sequences since the RNAi sequences are generated synthetically. An alternative strategy would be to create an RNAi library from cDNA. Unfortunately, the complexity of such a library of siRNAs would make screening difficult. To reduce the complexity, longer dsRNAs could be used; however, concerns of induction of the interferon response and off-target effects of long dsRNAs have prevented their use. As a first step in creating such libraries, long dsRNA was expressed in mammalian cells. The 250 nt dsRNAs were capable of efficiently silencing a luciferase reporter gene that was stably transfected in MDA-MB-231 cells without inducing the interferon response or off-target effects any more than reported for siRNAs. In addition, a long dsRNA expressed in the same cell line was capable of silencing endogenous c-met expression and inhibited cell migration, whereas the dsRNA against luciferase had no effect on c-met or cell migration. The studies suggest that large dsRNA libraries are feasible and that functional selection of genes will be possible.

Tethering RITS to a Nascent Transcript Initiates RNAi- and Heterochromatin-Dependent Gene Silencing

In the fission yeast Schizosaccharomyces pombe, the RNA-Induced Transcriptional Silencing (RITS) complex has been proposed to target the chromosome via siRNA-dependent base-pairing interactions to initiate heterochromatin formation. Here we show that tethering of the RITS subunit, Tas3, to the RNA transcript of the normally active ura4+ gene silences ura4+ expression. This silencing depends on a functional RNAi pathway, requires the heterochromatin proteins, Swi6/HP1, Clr4/Suv39h, and Sir2, and is accompanied by the generation of ura4+ siRNAs, histone H3-lysine 9 methylation, and Swi6 binding. Furthermore, the ability of the newly generated ura4+ siRNAs to silence a second ura4+ allele in trans is strongly inhibited by the conserved siRNA nuclease, Eri1. Surprisingly, silencing of tethered ura4+, or ura4+ inserted within centromeric heterochromatin, or some of the endogenous centromeric repeat promoters, is not associated with changes in RNA polymerase II occupancy. These findings support a model in which targeting of nascent transcripts by RITS mediates chromatin modifications and suggest that cotranscriptional processing events play a primary role in the silencing mechanism.

RNAi expression mediated inhibition of HCV replication

The RNA interference (RNAi) mechanism is a recently observed process in which the introduction of a double-stranded RNA (dsRNA) into a cell causes the specific degradation of an mRNA containing the same sequence. The 21-23 nt guide RNAs, generated by RNase III cleavage from longer dsRNAs, are associated with sequence-specific mRNA degradation. Here, we show that vector derived dsRNA specifically inhibit the replication of HCV RNA in HCV replicon cells. We designed a long dsRNA targeted to the full length HCV IRES region (1-377 nt). Real Time RT-PCR was performed with a TaqMan RT-PCR, to solely amplify and enable quantification of HCV RNA. Our results indicated HCV replication reduction to near background levels in a sequence-specific manner by the long-dsRNAs in the HCV replicon cells. Our results support the potential of using siRNA gene therapy to inhibit HCV replication, which may prove to be valuable in the treatment of hepatitis C.

RNA interference: an exciting new approach for target validation, gene expression analysis and therapeutics

Recent discoveries suggest that nature employs a novel approach for the regulation of gene expression by the silencing of endogenous genes. This phenomenon referred to as RNA interference (RNAi) is a posttranscriptional gene-silencing strategy that involves the intermediacy of double-stranded RNA (dsRNA) that complex with and destroy a target messenger RNA (mRNA) (1-4). The targeting and destruction of mRNA has been demonstrated to be highly specific and more selective compared to the antisense approach. Furthermore, it has also been shown that exogenously administered 21- to 23-mer dsRNA, referred to as small interfering RNA (siRNA), is equally efficient as a gene-silencing tool compared to the much longer dsRNA. These discoveries have led to the exciting possibility of using RNAi not only as a potential tool for elucidating the function of genes and analyzing gene expression, but also as a novel therapeutic modality. This review will highlight various aspects of this rapidly advancing field.

Prevention of HIV-1 infection in human peripheral blood mononuclear cells by specific RNA interference.

The RNA interference (RNAi) phenomenon is a recently observed process in which the introduction of a double-stranded RNA (dsRNA) into a cell causes the specific degradation of a mRNA containing the same sequence. The 21-23 nt guide RNAs, generated by RNase III cleavage from longer dsRNAs, are associated with sequence-specific mRNA degradation. Here, we show that dsRNA specifically suppresses the expression of HIV-1 genes. To study dsRNA-mediated gene interference in HIV-1-infected cells, we have designed six long dsRNAs containing the HIV-1 gag and env genes. HIV-1 replication was totally suppressed in a sequence-specific manner by the dsRNAs in HIV-1-infected cells. Especially, E2 dsRNA containing the major CD4-binding domain sequence of gp120, as the target of the HIV-1 env gene, dramatically inhibited the expression of the HIV-1 p24 antigen in PBMCs for a relatively long time. The dsRNA interference method seems to be a promising new strategy for anti-HIV-1 gene therapeutics.

Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells.

RNA interference (RNAi) is the process of sequence-specific, posttranscriptional gene silencing in animals and plants initiated by double-stranded (ds) RNA that is homologous to the silenced gene. This technology has usually involved injection or transfection of dsRNA in model nonvertebrate organisms. The longer dsRNAs are processed into short (19 25 nucleotides) small interfering RNAs (siRNAs) by a ribonucleotide protein complex that includes an RNAse III related nuclease (Dicer), a helicase family member, and possibly a kinase and an RNA-dependent RNA polymerase (RdRP). In mammalian cells it is known that dsRNA 30 base pairs or longer can trigger interferon responses that are intrinsically sequence-nonspecific, thus limiting the application of RNAi as an experimental and therapeutic agent. Duplexes of 21-nucleotide siRNAs with short 3' overhangs, however, can mediate RNAi in a sequence-specific manner in cultured mammalian cells. One limitation in the use of siRNA as a therapeutic reagent in vertebrate cells is that short, highly defined RNAs need to be delivered to target cells--a feat thus far only accomplished by the use of synthetic, duplex RNAs delivered exogenously to cells. In this report, we describe a mammalian Pol III promoter system capable of expressing functional double-stranded siRNAs following transfection into human cells. In the case of the 293 cells cotransfected with the HIV-1 pNL4-3 proviral DNA and the siRNA-producing constructs, we were able to achieve up to 4 logs of inhibition of expression from the HIV-1 DNA.

Post-transcriptional suppression of gene expression in Xenopus embryos by small interfering RNA.

Double-stranded RNA (dsRNA) induces gene-specific silencing in organisms from fungi to animals, a phenomenon known as RNA interference (RNAi). RNAi represents an evolutionarily conserved system to protect against aberrant expression of genes and a powerful tool for gene manipulation. Despite reports that RNAi can be induced in vertebrates, severe sequence-non-specific effects of long dsRNA have been documented in various systems. It has recently been shown in cultured mammalian cells that small interfering RNAs (siRNAs) of 21-23 nt can mediate RNAi but bypass the non-specific response induced by longer dsRNAs. However, the effectiveness of siRNAs has not been demonstrated in living vertebrates. In addition, the mechanism of siRNA suppression of gene expression in vertebrate cells remains to be elucidated. Here we show that synthetic 21 nt siRNAs can specifically inhibit the expression of exogenously introduced as well as endogenous genes in the embryos of Xenopus laevis. siRNAs significantly reduced the steady-state amount of both the mRNA and protein of the cognate gene target. Moreover, co-injection of siRNA with the target RNA transcript specifically suppressed the activity of the latter. Taken together, our findings establish siRNA-mediated post-transcriptional suppression of gene expression in Xenopus embryos.

Analysis of gene function in somatic mammalian cells using small interfering RNAs

RNA interference (RNAi) is a highly conserved gene silencing mechanism that uses double-stranded RNA (dsRNA) as a signal to trigger the degradation of homologous mRNA. The mediators of sequence-specific mRNA degradation are 21- to 23-nt small interfering RNAs (siRNAs) generated by ribonuclease III cleavage from longer dsRNAs. Twenty-one-nucleotide siRNA duplexes trigger specific gene silencing in mammalian somatic cells without activation of the unspecific interferon response. Here we provide a collection of protocols for siRNA-mediated knockdown of mammalian gene expression. Because of the robustness of the siRNA knockdown technology, genomewide analysis of human gene function in cultured cells has now become possible.

Specific inhibition of HIV-1 gene expression by double-stranded RNA.

RNA interference (RNAi) is phenomenon in which introduced double-stranded RNAs (ds-RNAs) silence gene expression through specific degradation of their cognate mRNAs. RNAi has been observed in a wide variety of organisms and is considered to be a feature of nearly all eukaryotes. The mediators of sequence specific messenger RNA degradation are 21-23 nucleotide small interfering RNAs generated by ribonucleaseIII cleavage from longer dsRNAs. To investigate the potential of dsRNA to interfere with the function of HIV-1 genes, we have designed six longer dsRNAs containing the HIV-1 gag and env genes. Double-stranded RNAs were tested for inhibitory effects using monkey COS cells. In these anti-HIV-1 activity tests, the dsRNAs targeted to the HIV-1 env sequence showed more than 90% anti-HIV-1 efficiency. Our results show that dsRNA interference can be used as a powerful method for the inhibition of HIV-1 gene expression.

Specific double-stranded RNA interference in undifferentiated mouse embryonic stem cells.

Specific mRNA degradation mediated by double-stranded RNA (dsRNA) interference (RNAi) is a powerful way of suppressing gene expression in plants, nematodes, and fungal, insect, and protozoan systems. However, only a few cases of RNAi have been reported in mammalian systems. Here, we investigated the feasibility of the RNAi strategy in several mammalian cells by using the enhanced green fluorescent protein gene as a target, either by in situ production of dsRNA from transient transfection of a plasmid harboring a 547-bp inverted repeat or by direct transfection of dsRNA made by in vitro transcription. Several mammalian cells including differentiated embryonic stem (ES) cells did not exhibit specific RNAi in transient transfection. This long dsRNA, however, was capable of inducing a sequence-specific RNAi for the episomal and chromosomal target gene in undifferentiated ES cells. dsRNA at 8.3 nM decreased the cognate gene expression up to 70%. However, RNAi activity was not permanent because it was more pronounced in early time points and diminished 5 days after transfection. Thus, undifferentiated ES cells may lack the interferon response, similar to mouse embryos and oocytes. Regardless of their apparent RNAi activity, however, cytoplasmic extracts from mammalian cells produced a small RNA of 21 to 22 nucleotides from the long dsRNA. Our results suggest that mammalian cells may possess RNAi activity but nonspecific activation of the interferon response by longer dsRNA may mask the specific RNAi. The findings offer an opportunity to use dsRNA for inhibition of gene expression in ES cells to study differentiation.

Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells.

RNA interference (RNAi) is the process of sequence-specific, post-transcriptional gene silencing in animals and plants, initiated by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced gene. The mediators of sequence-specific messenger RNA degradation are 21- and 22-nucleotide small interfering RNAs (siRNAs) generated by ribonuclease III cleavage from longer dsRNAs. Here we show that 21-nucleotide siRNA duplexes specifically suppress expression of endogenous and heterologous genes in different mammalian cell lines, including human embryonic kidney (293) and HeLa cells. Therefore, 21-nucleotide siRNA duplexes provide a new tool for studying gene function in mammalian cells and may eventually be used as gene-specific therapeutics.