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At least 775 records · Page 43Linked to original sources
PubMed2004

RNA interference in protozoan parasites.

RNA interference or RNAi is defined as the mechanism through which gene-specific, double-stranded RNA (dsRNA) triggers degradation of homologous transcripts. Besides providing an invaluable tool to downregulate gene expression in a variety of organisms, it is now evident that RNAi extends its tentacles into both the nucleus and the cytoplasm and is involved in a variety of gene silencing phenomena. Here we review the current status of RNAi in protozoan parasites that cause diseases of considerable medical and veterinary importance throughout Africa, Asia and the Americas. RNAi was first discovered in Trypanosoma brucei, a species of the family Trypanosomatidae, and it rapidly became the method of choice to downregulate gene expression in these organisms. At the same time, mechanistic studies exposed a role for RNAi in the control of retroposon transcript abundance. Whereas RNAi is also present in T. congolense, other members of the same family of organisms, namely T. cruzi and Leishmania major, are RNAi-negative. In apicomplexan parasites, there is experimental evidence for RNAi in Plasmodium, but this is not supported by their genetic make up. In contrast, the genome of Toxoplasma gondii harbours gene candidates with convincing similarity to 'classical' RNAi genes. Thus, as previously shown in fungi, protozoan parasites are genetically heterogeneous as far as the RNAi pathway is concerned. Finally, database mining predicts that Entamoeba histolytica and Giardia intestinalis have an RNAi pathway and the presence of RNAi genes in Giardia supports the view that gene silencing by dsRNA appeared very early during evolution of the eukaryotic lineage.

Animals↗
PubMed2005

Dual role of TRBP in HIV replication and RNA interference: viral diversion of a cellular pathway or evasion from antiviral immunity?

Increasing evidence indicates that RNA interference (RNAi) may be used to provide antiviral immunity in mammalian cells. Human micro (mi)RNAs can inhibit the replication of a primate virus, whereas a virally-encoded miRNA from HIV inhibits its own replication. Indirect proof comes from RNAi suppressors encoded by mammalian viruses. Influenza NS1 and Vaccinia E3L proteins can inhibit RNAi in plants, insects and worms. HIV-1 Tat protein and Adenovirus VA RNAs act as RNAi suppressors in mammalian cells. Surprisingly, many RNAi suppressors are also inhibitors of the interferon (IFN)-induced protein kinase R (PKR) but the potential overlap between the RNAi and the IFN pathways remains to be determined. The link between RNAi as an immune response and the IFN pathway may be formed by a cellular protein, TRBP, which has a dual role in HIV replication and RNAi. TRBP has been isolated as an HIV-1 TAR RNA binding protein that increases HIV expression and replication by inhibiting PKR and by increasing translation of structured RNAs. A recent report published in the Journal of Virology shows that the poor replication of HIV in astrocytes is mainly due to a heightened PKR response that can be overcome by supplying TRBP exogenously. In two recent papers published in Nature and EMBO Reports, TRBP is now shown to interact with Dicer and to be required for RNAi mediated by small interfering (si) and micro (mi)RNAs. The apparent discrepancy between TRBP requirement in RNAi and in HIV replication opens the hypotheses that RNAi may be beneficial for HIV-1 replication or that HIV-1 may evade the RNAi restriction by diverting TRBP from Dicer and use it for its own benefit.

HIV↗
PubMed2006

The interplay between virus infection and the cellular RNA interference machinery.

RNA interference (RNAi) plays a pivotal role in the regulation of gene expression to control cell development and differentiation. In plants, insects and nematodes RNAi also functions as an innate defence response against viruses. Similarly, there is accumulating evidence that RNAi functions as an antiviral defence mechanism in mammalian cells. Viruses have evolved highly sophisticated mechanisms for interacting with the host cell machinery, and recent evidence indicates that this also involves RNAi pathways. The cellular RNAi machinery can inhibit virus replication, but viruses may also exploit the RNAi machinery for their own replication. In addition, viruses can encode proteins or RNA molecules that suppress existing RNAi pathways or trigger the silencing of specific host genes. Besides the natural interplay between RNAi and viruses, induced RNAi provides an attractive therapy approach for the fight against human pathogenic viruses. Here, we summarize the latest news on virus-RNAi interactions and RNAi based antiviral therapy.

Animals↗
PubMed2005

RNA interference spreading in C. elegans.

The phenomenon of RNA interference (RNAi) occurs in eukaryotic organisms from across the boundaries of taxonomic kingdoms. In all cases, the basic mechanism of RNAi appears to be conserved--an initial trigger [double-stranded RNA (dsRNA) containing perfect homology over at least 19-21/bp with an endogenous gene] is processed into short interfering RNA (siRNA) molecules and these siRNAs stimulate degradation of the homologous mRNA. In the vast majority of species, RNAi can only be initiated following the deliberate introduction of dsRNA into a cell by microinjection, electroporation, or transfection. However, in the nematode worm Caenorhabditis elegans, RNAi can be simply initiated by supplying dsRNA in the surrounding medium or in the diet. Following uptake, this dsRNA triggers a systemic effect, initiating RNAi against the corresponding target gene in tissues that are not in direct contact with the external milieu. This phenomenon of systemic RNAi, or RNAi spreading, is notably absent from mammalian species, a fact that is likely to prove a substantial barrier to the wider use of RNAi as a clinical therapy. An understanding of the mechanism of systemic RNAi is therefore of considerable importance, and several advances of the last few years have begun to shed light on this process. Here we review our current understanding of systemic RNAi in C. elegans and draw comparisons with systemic RNAi pathways in other organisms.

Animals↗
PubMed2003

RNA interference in the moss Physcomitrella patens.

The moss Physcomitrella patens performs efficient homologous recombination, which allows for the study of individual gene function by generating gene disruptions. Yet, if the gene of study is essential, gene disruptions cannot be isolated in the predominantly haploid P. patens. Additionally, disruption of a gene does not always generate observable phenotypes due to redundant functions from related genes. However, RNA interference (RNAi) can provide mutants for both of these situations. We show that RNAi disrupts gene expression in P. patens, adding a significant tool for the study of plant gene function. To assay for RNAi in moss, we constructed a line (NLS-4) expressing a nuclearly localized green fluorescent protein (GFP):beta-glucuronidase (GUS) fusion reporter protein. We targeted the reporter protein with two RNAi constructs, GUS-RNAi and GFP-RNAi, expressed transiently by particle bombardment. Transformed protonemal cells are marked by cobombardment with dsRed2, which diffuses between the nucleus and cytoplasm. Cells transformed with control constructs have nuclear/cytoplasmic red fluorescence and nuclear green fluorescence. In cells transformed with GUS-RNAi or GFP-RNAi constructs, the nuclear green fluorescence was reduced on average 9-fold as soon as 48 h after transformation. Moreover, isolated lines of NLS-4 stably transformed with GUS-RNAi construct have silenced nuclear GFP, indicating that RNAi is propagated stably. Thus, RNAi adds a powerful tool for functional analysis of plant genes in moss.

Bryopsida↗
PubMed2004

RNA interference and its current application in mammals.

OBJECTIVE: The aim of this review was to assess RNA interference (RNAi) and its possibility as a potential and powerful tool to develop highly specific double-stranded RNA (dsRNA) or small interfering RNA (siRNA) based gene-silencing therapeutics. DATA SOURCES: The data used in this review were obtained from the current RNAi-related research reports. STUDY SELECTION: dsRNA-mediated RNAi has recently emerged as a powerful reverse genetic tool to silence gene expression in multiple organisms. The discovery that synthetic duplexes of 21 nucleotides siRNAs trigger gene-specific silencing in mammalian cells has further expanded the utility of RNAi in to the mammalian system. DATA EXTRACTION: The currently published papers reporting the discovery and mechanism of RNAi phenomena and application of RNAi on gene function in mammalian cells were included. DATA SYNTHESIS: Since the recent development of RNAi technology in the mammalian system, investigators have used RNAi to elucidate gene function, and to develop gene-based therapeutics by delivery exogenous siRNA or siRNA expressing vector. The general and sequence-specific inhibitory effects of RNAi that will be selective, long-term, and systemic to modulate gene targets mentioned in similar reports have caused much concern about its effectiveness in mammals and its eventual use as a therapeutic mordality. CONCLUSIONS: It is certain that the ability of RNAi in mammals to silence specific genes, either when transfected directly as siRNAs or when generated from DNA vectors, will undoubtedly accelerate the study of gene function and might also be used as a potentially useful method to develop highly gene-specific therapeutic methods. It is also expected that RNAi might one day be used to treat human diseases.

Animals↗
PubMed2003

Analysis of protein phosphatase function in Drosophila cells using RNA interference.

Double stranded RNA-mediated RNA interference is an effective method to downregulate the levels of protein phosphatases in Drosophila S2 cells. In many cases, nearly complete ablation of the targeted protein can be achieved. RNAi-mediated knockdown of protein phosphatases is akin to pharmacological inhibition with drugs and can be used to determine the roles of specific protein phosphatases in intact cells. RNAi can avoid the problems associated with less than adequate specificity of phosphatase inhibitors. Although information about the signaling pathways present in Drosophila S2 cells is not as well developed as many mammalian cell lines, the Drosophila system is particularly attractive for the study of oligomeric phosphatases like PP2A. Drosophila has far fewer isoforms for the phosphatases we have examined. This is especially true of the genes for PP2A regulatory subunits where over 50 isoforms are present in mammals but only four are present in Drosophila. Once hypotheses regarding phosphatase function have been generated from RNAi experiments in S2 cells, they can potentially be tested utilizing recent advances in the use of siRNAs to conduct RNAi experiments in mammalian cell lines. RNAi in Drosophila S2 cells has proven to be a powerful technique for identifying physiological functions of signaling proteins. The RNAi method is straightforward and works routinely with almost all proteins. RNAi in S2 cells can be used to assess the role of signaling proteins in specific pathways and as a screening tool to identify new roles for signaling molecules. For example, results from RNAi analysis of PP2A show that regulation of MAP kinase signaling involves the R2/B regulatory subunit and that the R5/B56 subunits play a previously unidentified role in apoptosis. While RNAi in Drosophila S2 cells is a powerful tool for analyzing protein function, the method does have limitations. Foremost, cells may exhibit an RNAi response to any nonspecific dsRNA, even in the absence of interferon. Therefore, physiological processes that respond to nonspecific dsRNA will be difficult to study. A second limitation is the need to produce antibodies that react with Drosophila isoforms. We have found that many antibodies to mammalian protein phosphatases do not cross-react with the corresponding Drosophila proteins. Finally, the physiology and signaling pathways of S2 cells have not been extensively studied. This lack of information limits the number of available readouts that can be used when assessing the effects of protein knockdowns.

Animals↗
PubMed2005

A plant RNA virus suppresses RNA silencing through viral RNA replication.

RNA interference (RNAi) is a post-transcriptional gene-regulatory mechanism that operates in many eukaryotes. RNAi is induced by double-stranded RNA (dsRNA) and is mainly involved in defence against transposons and viruses. To counteract RNAi, viruses have RNAi suppressors. Here we show a novel mechanism of RNAi suppression by a plant virus Red clover necrotic mosaic virus (RCNMV). To suppress RNAi, RCNMV needs multiple viral components, which include viral RNAs and putative RNA replicase proteins. A close relationship between the RNA elements required for negative-strand RNA synthesis and RNAi suppression suggests a strong link between the viral RNA replication machinery and the RNAi machinery. In a transient assay, RCNMV interferes with the accumulation of small-interfering RNA (siRNAs) in RNAi induced by a hairpin dsRNA and it also interferes with microRNA (miRNA) biogenesis. An Arabidopsis dcl1 mutant showed reduced susceptibility to RCNMV infection. Based on these results, we propose a model in which, to replicate, RCNMV deprives the RNAi machinery of Dicer-like enzymes that are involved in both siRNA and miRNA biogenesis.

3' Untranslated Regions↗
PubMed2005

A computational study of off-target effects of RNA interference.

RNA interference (RNAi) is an intracellular mechanism for post-transcriptional gene silencing that is frequently used to study gene function. RNAi is initiated by short interfering RNA (siRNA) of approximately 21 nt in length, either generated from the double-stranded RNA (dsRNA) by using the enzyme Dicer or introduced experimentally. Following association with an RNAi silencing complex, siRNA targets mRNA transcripts that have sequence identity for destruction. A phenotype resulting from this knockdown of expression may inform about the function of the targeted gene. However, 'off-target effects' compromise the specificity of RNAi if sequence identity between siRNA and random mRNA transcripts causes RNAi to knockdown expression of non-targeted genes. The complete off-target effects must be investigated systematically on each gene in a genome by adjusting a group of parameters, which is too expensive to conduct experimentally and motivates a study in silico. This computational study examined the potential for off-target effects of RNAi, employing the genome and transcriptome sequence data of Homo sapiens, Caenorhabditis elegans and Schizosaccharomyces pombe. The chance for RNAi off-target effects proved considerable, ranging from 5 to 80% for each of the organisms, when using as parameter the exact identity between any possible siRNA sequences (arbitrary length ranging from 17 to 28 nt) derived from a dsRNA (range 100-400 nt) representing the coding sequences of target genes and all other siRNAs within the genome. Remarkably, high-sequence specificity and low probability for off-target reactivity were optimally balanced for siRNA of 21 nt, the length observed mostly in vivo. The chance for off-target RNAi increased (although not always significantly) with greater length of the initial dsRNA sequence, inclusion into the analysis of available untranslated region sequences and allowing for mismatches between siRNA and target sequences. siRNA sequences from within 100 nt of the 5' termini of coding sequences had low chances for off-target reactivity. This may be owing to coding constraints for signal peptide-encoding regions of genes relative to regions that encode for mature proteins. Off-target distribution varied along the chromosomes of C.elegans, apparently owing to the use of more unique sequences in gene-dense regions. Finally, biological and thermodynamical descriptors of effective siRNA reduced the number of potential siRNAs compared with those identified by sequence identity alone, but off-target RNAi remained likely, with an off-target error rate of approximately 10%. These results also suggest a direction for future in vivo studies that could both help in calibrating true off-target rates in living organisms and also in contributing evidence toward the debate of whether siRNA efficacy is correlated with, or independent of, the target molecule. In summary, off-target effects present a real but not prohibitive concern that should be considered for RNAi experiments.

Animals↗
PubMed2003

TbAGO1, an argonaute protein required for RNA interference, is involved in mitosis and chromosome segregation in Trypanosoma brucei.

BACKGROUND: RNA silencing processes are widespread in almost all eukaryotic organisms. They have various functions including genome protection, and the control of gene expression, development and heterochromatin formation. RNA interference (RNAi) is the post-transcriptional destruction of RNA, which is mediated by a ribonucleoprotein complex that contains, among several components, RNA helicases and Argonaute proteins. RNAi is functional in trypanosomes, protozoan parasites that separated very early from the main eukaryotic lineage and exhibit several intriguing features in terms of the control of gene expression. In this report, we investigated the functions of RNAi in Trypanosoma brucei. RESULTS: By searching through genome databases, novel Argonaute-like proteins were identified in several protozoa that belong to the kinetoplastid order, a group of organisms that diverged early from the main eukaryotic lineage. T. brucei possesses two Argonaute-like genes termed TbAGO1 and TbPWI1. Dual transient transfection assays suggest that TbAGO1, but not TbPWI1, is involved in RNAi. The entire coding region of TbAGO1 was deleted by double gene knockout. TbAGO1-/- cells turned out to be completely resistant to RNAi generated either by transfected double-stranded RNA or by expression of an inverted repeat. TbAGO1-/- cells were viable but showed a dramatically reduced growth rate. This was probably due to defects in mitosis and abnormal chromosome segregation as revealed by in situ analysis. The RNAi and growth phenotypes were complemented by the inducible expression of a GFP::TbAGO1 fusion protein that revealed the cytoplasmic location of the protein. CONCLUSIONS: The requirement of TbAGO1 for RNAi in trypanosomes demonstrates the evolutionary ancient involvement of Argonaute proteins in RNAi silencing processes. RNAi-deficient TbAGO1-/- cells showed numerous defects in chromosome segregation and mitotic spindle assembly. We propose a working hypothesis in which RNAi would be involved in heterochromatin formation at the centromere and therefore in chromosome segregation.

Animals↗
PubMedDate not supplied

[RNA-interference, induced by transient and continuous expression of hairpin RNA in cells from Drosophila and mammals].

RNA interference (RNAi) may be induced by a plasmid with an inverted repeat (IR) sequence directing transcription of hairpin-type double-stranded RNA (dsRNA). This study examines the effects of changing various parameters of IR constructs on Drosophila and mammalian RNAi, using the dual luciferase system, RNAi activity was found to vary depending on IR length ass well as the length and sequence of the internal loop separating sense and antisense sequences. Both transient and stable RNAi occurred in Drosophila cultured cells. Although transient DNA-mediated RNAi was noted in most mammalian cells, no mammalian cells stably possessing IR sequences and hence RNAi activity could be obtained. In Drosophila, DNA-mediated RNAi was considerably weaker than long-dsRNA-mediated RNAi. The cytological data indicated that this was most probably caused by abortive processing of hairpin RNA produced within cells. DNA-mediated RNAi was examined at the level of Drosophila individuals using extramacrochaetae as a model gene, and the presence of an intron sequence in the single-stranded loop region was shown to be essential for effective RNAi.

Animals↗
PubMed2004

RNA interference: the molecular immune system.

Introduction of double-stranded RNA (dsRNA) into cells expressing a homologous gene triggers RNA interference (RNAi), or RNA-based gene silencing (RBGS). The dsRNA degrades corresponding host mRNA into small interfering RNAs (siRNAs) by a protein complex containing Dicer. siRNAs in turn are incorporated into the RNA-induced silencing complex (RISC) that includes helicase, RecA, and exo- and endo-nucleases as well as other proteins. Following its assembly, the RISC guides the RNA degradation machinery to the target RNAs and cleaves the cognate target RNA in a sequence-specific, siRNA-dependent manner. RNAi has now been documented in a wide variety of organisms, including plants, fungi, flies, worms, and more recently, higher mammals. In eukaryotes, dsRNA directed against a range of viruses (i.e., HIV-1, RSV, HPV, poliovirus and others) and endogenous genes can induce sequence-specific inhibition of gene expression. In invertebrates, RNAi can be efficiently triggered by either long dsRNAs or 21- to 23-nt-long siRNAs. However, in jawed vertebrates, dsRNA longer than 30 bp can induce interferon and thus trigger undesirable side effects instead of initiating RNAi. siRNAs have been shown to act as potent inducers of RNAi in cultured mammalian cells. Many investigators have suggested that siRNAs may have evolved as a normal defense against endogenous and exogenous transposons and retroelements. Through a combination of genetic and biochemical approaches, some of the mechanisms underlying RNAi have been described. Recent data in C. elegans shows that two homologs of siRNAs, microRNAs (miRNAs) and tiny noncoding RNAs (tncRNAs) are endogenously expressed. However, many aspects of RNAi-induced gene silencing, including its origins and the selective pressures which maintain it, remain undefined. Its evolutionary history may pass through the more primitive immune functions of prokaryotes involving restriction enzymes that degrade plasmid DNA molecules that enter bacterial cells. RNAi has evolved further among eukaryotes, in which its wide distribution suggests early origins. RNAi seems to be involved in a variety of regulatory and immune functions that may differ among various kingdoms and phyla. We present here proposed mechanisms by which RBGS protects the host against endogenous and exogenous transposons and retroelements. The potential for therapeutic application of RBGS technology in treating viral infections such as HIV is also discussed.

AIDS Vaccines↗
PubMed2007

Generation of secondary small interfering RNA in cell-autonomous and non-cell autonomous RNA silencing in tobacco.

Small interfering RNA (siRNA) species with 21-25 nucleotides in length guide mRNA cleavage, translational arrest, and heterochromatin formation in RNA interference (RNAi). To delineate the target region of RNAi, a construct harboring a transcriptional fusion between parts of the target mRNA and the beta-glucuronidase gene was biolistically delivered into tobacco leaves showing an RNAi phenotype and the assay sequence was transiently expressed. The RNAi effect was monitored by amplification of this chimeric transcript. By using this assay method, we addressed the transitive RNA silencing of a tobacco endoplasmic reticulum omega-3 fatty acid desaturase gene (NtFAD3). In the NtFAD3 RNAi plants, the target region of RNAi was restricted in the inducer region corresponding to a stem sequence of the hairpin double-stranded RNA, indicating that endogenous NtFAD3 mRNA was not a template for an RNA-dependent RNA polymerase. The secondary NtFAD3 siRNAs were produced in the crossbred plants between the NtFAD3 overexpressed plant and the NtFAD3 RNAi plant. Similarly, the secondary siRNAs were generated in the systemically silenced scion. Although these secondary siRNAs originated preferentially from the 3' region downstream of the inducer region, the secondary siRNAs produced in the silenced scion (non-cell autonomous secondary siRNAs) resulted in the strong degradation of the target mRNA, but the secondary siRNAs in the crossbred plants (cell-autonomous secondary siRNAs) showed limited RNA degradation activity. These results showed that this in vivo assay for determination of RNAi efficiency is a useful tool to delineate RNAi mechanisms.

Biolistics↗
PubMed2005

An antiviral role for the RNA interference machinery in Caenorhabditis elegans.

RNA interference (RNAi) is a sequence-specific gene-silencing mechanism triggered by exogenous dsRNA. In plants an RNAi-like mechanism defends against viruses, but the hypothesis that animals possess a similar natural antiviral mechanism related to RNAi remains relatively untested. To test whether genes needed for RNAi defend animal cells against virus infection, we infected wild-type and RNAi-defective cells of the nematode C. elegans with vesicular stomatitis virus engineered to encode a GFP fusion protein. We show that upon infection, cells lacking components of the RNAi apparatus produce more GFP and infective particles than wild-type cells. Furthermore, we show that mutant cells with enhanced RNAi produce less GFP. Our observation that multiple genes required for RNAi are also required for resistance to vesicular stomatitis virus suggests that the RNAi machinery functions in resistance to viruses in nature.

Animals↗
PubMed1997

Importance of structural differences between complementary RNA molecules to control of replication of an IncB plasmid.

Replication of the IncB miniplasmid pMU720 is dependent on the expression of repA, the gene encoding replication initiator protein RepA. Binding of a small antisense RNA (RNAI) to its complementary target (stem-loop I [SLI]) in the RepA mRNA prevents the participation of SLI in the formation of a pseudoknot that is an enhancer of translation of this mRNA. Thus, RNAI regulates the frequency of replication of pMU720 by controlling the efficiency of translation of the RepA mRNA. Mutational analysis of the two seven-base complementary sequences involved in formation of the pseudoknot showed that only the five central bases of each were critical for the formation of the pseudoknot. Physical analysis of SLI showed that despite the complete complementarity of its sequence to that of RNAI, the structures of the two molecules are different. The most prominent difference between the two structures is the presence of a 4-base internal loop immediately below the hairpin loop of SLI but not that of RNAI. Closure of this internal loop in SLI resulted in a 40-fold reduction in repA expression and loss of sensitivity of the residual expression to inhibition by RNAI. By contrast, repA expression was largely unaffected by the closure of a lower internal loop whose presence in SLI and RNAI is essential for effective interaction between these two molecules. These results suggest that the interaction of SLI with the distal pseudoknot bases is fundamentally different from the RNAI-SLI binding interaction and that the differences in structure between RNAI and SLI are necessary to allow SLI to be able to efficiently bind RNAI and to participate in pseudoknot formation.

Bacterial Proteins↗
PubMed2005

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.

Adenoviruses, Human↗
PubMed2001

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.

Animals↗
PubMed2002

[RNA interference for mammalian cells].

Knock-out of mammalian genes is technically troublesome and time-consuming compared to those of lower animals. RNA interference (RNAi) is a strategy of sequence-specific post-translational gene silencing, and it has been successfully applied for disruption of gene transcripts in C. elegans. Gene silencing by RNAi had not been accomplished in mammalian cells until recently, because bystander activation of DNA-dependent protein kinase (PKR) coincides to disturb gene silencing by long double-stranded RNA (dsRNA), resulting in non-specific repression of translation of many proteins. A breakthrough has come recently with a report suggesting that 20- or 21-bp duplex RNA with 2 bp 3' overhang are made by DICER protein that cleaves long dsRNA during RNAi reactions in vivo and the resultant short dsRNAs mediate RNAi. Those short dsRNAs, namely, small interference RNA (siRNA), barely activate PKR. Using synthesized 21 bp siRNA, T. Tuschl's group has challenged to establish an artificial RNAi method suitable for mammalian cells. Their report was favorable in that siRNA specifically suppressed targeted gene translation in mammalian cells during culture without activation of PKR. Recently K. Taira's group developed the vector-based siRNA expression system by which RNAi is feasible in mammalian cells. Almost all genes can be targeted by RNAi. RNAi methods require minimal time and labor; therefore, mammalian gene knockdown by RNAi will become popular in the near future.

Gene Silencing↗