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PubMed2006

Distinct populations of primary and secondary effectors during RNAi in C. elegans.

RNA interference (RNAi) is a phylogenetically widespread gene-silencing process triggered by double-stranded RNA. In plants and Caenorhabditis elegans, two distinct populations of small RNAs have been proposed to participate in RNAi: "Primary siRNAs" (derived from DICER nuclease-mediated cleavage of the original trigger) and "secondary siRNAs" [additional small RNAs whose synthesis requires an RNA-directed RNA polymerase (RdRP)]. Analyzing small RNAs associated with ongoing RNAi in C. elegans, we found that secondary siRNAs constitute the vast majority. The bulk of secondary siRNAs exhibited structure and sequence indicative of a biosynthetic mode whereby each molecule derives from an independent de novo initiation by RdRP. Analysis of endogenous small RNAs indicated that a fraction derive from a biosynthetic mechanism that is similar to that of secondary siRNAs formed during RNAi, suggesting that small antisense transcripts derived from cellular messenger RNAs by RdRP activity may have key roles in cellular regulation.

Animals↗

PubMed2006

Drosophila Paf1 modulates chromatin structure at actively transcribed genes.

The Paf1 complex in yeast has been reported to influence a multitude of steps in gene expression through interactions with RNA polymerase II (Pol II) and chromatin-modifying complexes; however, it is unclear which of these many activities are primary functions of Paf1 and are conserved in metazoans. We have identified and characterized the Drosophila homologs of three subunits of the yeast Paf1 complex and found striking differences between the yeast and Drosophila Paf1 complexes. We demonstrate that although Drosophila Paf1, Rtf1, and Cdc73 colocalize broadly with actively transcribing, phosphorylated Pol II, and all are recruited to activated heat shock genes with similar kinetics; Rtf1 does not appear to be a stable part of the Drosophila Paf1 complex. RNA interference (RNAi)-mediated depletion of Paf1 or Rtf1 leads to defects in induction of Hsp70 RNA, but tandem RNAi-chromatin immunoprecipitation assays show that loss of neither Paf1 nor Rtf1 alters the density or distribution of phosphorylated Pol II on the active Hsp70 gene. However, depletion of Paf1 reduces trimethylation of histone H3 at lysine 4 in the Hsp70 promoter region and significantly decreases the recruitment of chromatin-associated factors Spt6 and FACT, suggesting that Paf1 may manifest its effects on transcription through modulating chromatin structure.

Animals↗

PubMed2006

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.

Alleles↗

PubMed2005

Targeting Id1 and Id3 inhibits peritoneal metastasis of gastric cancer.

Inhibitor of DNA binding (Id) proteins are essential for cell differentiation, proliferation, migration, invasion and angiogenesis. Recently, they have been shown to correlate with less differentiated phenotypes, high malignant potential and poor clinical outcome in various kinds of tumors. In an attempt to develop new strategies for the treatment of peritoneal metastasis of gastric cancer, we prepared an Id1, 3 double-knockdown gastric cancer cell line, MKN45, by RNA interference and investigated its effects on the development of metastatic nodules in the peritoneal cavity. Both cell proliferation and migration capabilities were decreased in Id1, 3 double-knockdown cells, as was their ability to bind to laminin, which could be explained by the decreased expression of integrin alpha6. These are important steps in the metastatic process. In a mouse model, the number of peritoneal metastatic nodules formed by Id1, 3 double-knockdown cells was reduced compared to mock-transfected control cells, as was the size of individual tumors. In this study, we clearly demonstrated that Id1, 3 double-knockdown significantly impaired the ability of gastric cancer cells to form peritoneal metastasis. Id should be considered an ideal target for the treatment and prevention of gastric cancer, and RNA interference is an attractive and promising strategy to achieve it.

Animals↗

PubMed2006

Functional genomics identifies TOR-regulated genes that control growth and division.

BACKGROUND: The TOR (target of rapamycin) ser/thr protein kinase is the central component of a eukaryotic signaling pathway that regulates growth and is the direct target of the clinically useful drug rapamycin. Recent efforts have identified at least two multiprotein complexes that contain TOR, but little is known in higher eukaryotes about the genes downstream of TOR that control growth. RESULTS: By combining the use of a small molecule inhibitor (rapamycin), transcriptional profiling, and RNA interference in Drosophila tissue culture cells, we identified genes whose expression responds to Drosophila TOR (dTOR) inhibition and that regulate cell size. Several of the dTOR-regulated genes that function in cell size control have additional roles in cell division. Most of these genes are conserved in mammals and several are linked to human disease. This set of genes is highly enriched for regulators of ribosome biogenesis, which emphasizes the importance of TOR-dependent transcription in building the protein synthesis machinery in higher eukaryotes. In addition, we identify two dTOR-regulated genes, CG3071 and CG6677, whose human orthologs, SAW and ASH2L, are also under TOR-dependent transcriptional control and encode proteins with conserved functional roles in growth. CONCLUSIONS: We conclude that combining RNA interference with genomic analysis approaches, such as transcriptional profiling, is an effective way to identify genes functioning in a particular biological process. Moreover, this strategy, if applied in model systems with simpler genomes, can identify genes with conserved functions in mammals.

Animals↗

PubMed2003

Functional studies of the PI(3)-kinase signalling pathway employing synthetic and expressed siRNA.

RNA interference (RNAi) is a RNA-mediated sequence-specific gene silencing mechanism. Recently, this mechanism has been used to down-regulate protein expression in mammalian cells by applying synthetic- or vector-generated small interfering RNAs (siRNAs). However, for the evaluation of this new knockdown technology, it is crucial to demonstrate biological consequences beyond protein level reduction. Here, we demonstrate that this new siRNA-based technology is suitable to analyse protein functions using the phosphatidylinositol (PI) 3-kinase signal transduction pathway as a model system. We demonstrate stable and transient siRNA-mediated knockdown of one of the PI 3-kinase catalytic subunits, p110beta, which leads to inhibition of invasive cell growth in vitro as well as in a tumour model system. Importantly, this result is consistent with loss-of-function phenotypes induced by conventional RNase H-dependent antisense molecules or treatment with the PI 3-kinase inhibitor LY294002. RNAi knockdown of the downstream kinases Akt1 and Akt2 does not reduce cell growth on extracellular matrix. Our data show that synthetic siRNAs, as well as vector-based expression of siRNAs, are a powerful new tool to interfere with signal transduction processes for the elucidation of gene function in mammalian cells.

Animals↗

PubMed2002

The PML gene is not involved in the regulation of MHC class I expression in human cell lines.

The promyelocytic leukemia gene, PML, is a growth and transformation suppressor. An additional role for PML as a regulator of major histocompatibility complex (MHC) class I antigen presentation has been proposed in a murine model, which would account for evasion from host immunity of tumors bearing malfunctioning PML, such as acute promyelocytic leukemia. Here we investigated a possible role of PML for the control MHC class I expression in human cells. PML function was perturbed in human cell lines either by PML/RAR alpha transfection or by PML- specific RNA interference. Impairment of wild-type PML function was proved by a microspeckled disassembly of nuclear bodies (NBs), where the protein is normally localized, or by their complete disappearance. However, no MHC class I down-regulation was observed in both instances. We next constructed a PML mutant, PML mut ex3, that is a human homolog of the murine PML mutant, truncated in exon 3, that was shown to down-regulate murine MHC class I. PML mut ex3 transfected in human cell lines exerted a dominant-negative effect since no PML molecules were detected in NBs but, instead, in perinuclear and cytoplasmic larger dot-like structures. Nevertheless, no down-regulation of MHC class I expression was evident. Moreover, neither transfection with PML mut ex3 nor PML-specific RNA interference affected the ability of gamma-interferon to up-regulate MHC class I expression. We conclude that, in human cell lines, PML is not involved directly in the regulation of MHC class I expression.

Amino Acid Sequence↗

PubMed2004

Short hairpin RNA system to inhibit human p16 in squamous cell carcinoma.

OBJECTIVE: To develop short hairpin RNA (shRNA) vectors and virions to trigger RNA interference against human p16. DESIGN AND INTERVENTIONS: A modular vector-based shRNA system was used to construct multiple distinct shRNA vectors against the unique exon 1alphaof p16. Target sequences were designed using rigid criteria for length and GC content, along with basic local alignment search tool (BLAST) evaluation to ensure targeting specificity. Individual shRNA-p16 cassettes were then cotransfected with a p16 expression vector and evaluated by Western blot. Cassettes showing a high level of p16 repression were used to construct shRNA-p16 expressing adenoviral and retroviral vectors and tested in a human head and neck squamous cell carcinoma line expressing endogenous and exogenous p16. RESULTS: Adenoviral and retroviral transfer of shRNA-p16 significantly reduced p16 protein levels, while control constructs left p16 expression unchanged. CONCLUSIONS: Although p16 is a common target of inactivation in head and neck cancer, its biological role remains ill defined. RNA interference is rapidly becoming the standard to target selected genes of interest for inactivation. We have successfully inhibited p16 expression using shRNA cassettes with strong activity against human p16 and integrated these constructs into adenoviral and retroviral vectors for transient and integrated expression in human cells. Application of this novel modular system to primary human cells will allow the biological consequences of p16 loss to be examined both in vitro and in vivo.

Adenoviridae↗

PubMed2006

[The role of protein tyrosine phosphatases Shp-2 involved in the formation of the neuromuscular junction].

OBJECTIVE: To investigate the involvement of protein tyrosine phosphatases Shp-2 in regulating postsynaptic signaling at the NMJ. METHODS: Cultured C2 mouse myotubes were used to mimic NMJ formation; immunoprecipitation, immuno-blot, RNA interference and immunofluorescent labeling were used in this study. RESULTS: We first showed that the general tyrosine phosphatase inhibitor pervanadate functionally activated MuSK and enhanced both agrin-independent and agrin-dependent AChR clustering in muscle cells: the MuSK band at 115 kD showed increased tyrosine phosphorylation after pervanadate treatment; 10 micromol/L pervanadate increased AChR clustering (mean = 6.43/cell) more than six-fold compared with control (mean = 0.96/cell), P < 0.0001, t-test; inclusion of pervanadate with agrin increased the size of agrin-induced clusters (difference = 1.53-fold, P < 0.0001, t-test) without significantly increasing the number of clusters (P = 0.08, t-test). Next, by immuno-screening we identified the SH2 domain-containing phosphatase Shp2 as a major tyrosine phosphatase in C2 myotubes and demonstrated that its selective down-regulation by RNA interference increased MuSK activation and AChR clustering: MuSK phosphorylation observed in the presence of pervanadate alone was increased in Shp2-depleted cells relative to control cells; AChR clusters in untreated myotubes were counted and data pooled from four experiments showed a doubling of their number (2.21-fold) in cells transfected with the Shp2 siRNA (n = 198) compared to those transfected with the control siRNA (n = 220); the number of AChR clusters was also increased in Shp2 siRNA-transfected cells compared to controls following treatment with pervanadate (1.5-fold; n = 149 cells) and agrin (1.41-fold; n = 125), P < 0.001, t-test. We also asked how Shp2 affect AChR clustering after increasing Shp2 activity: C2C12 cells were transfected with Shp-2 active form (E76A) and wild type Shp2 (used as control) respectively, average length of AChR clusters in active form (E76A) was decreased about 20% compared with wild type Shp2, P < 0.01, t-test. CONCLUSION: These results suggest that Shp2 functions in a feedback loop to regulate agrin/MuSK signaling and these findings highlight the importance of protein tyrosine phosphatases in postsynaptic signaling at the NMJ and provide insights into how balancing actions of protein kinases and phosphatases may determine the formation of synaptic specializations in the developing nervous system.

Agrin↗

PubMed2004

Short hairpin RNA and retroviral vector-mediated silencing of p53 in mammalian cells.

RNA interference (RNAi) is the process of gene silencing, in which the recognition of double-stranded RNA ultimately leads to post-transcriptional suppression of gene expression. RNAi induced by small interfering RNA (siRNA) or short hairpin RNA (shRNA) is an important research approach for the analysis of gene function in mammalian cells. Here we established a shRNA expression retroviral vector in which the sense and antisense sequences targeting wild type human p53 were linked together with a 9-nucleotide loop. Further, we found that shRNA expressed from a RNA polymerase III vector based on the human H1 RNA promoter could effectively inhibit gene expression in a sequence-specific manner. H1-driven shRNA dramatically reduced the expression of wild type human p53 in H1299 cells transfected with wtp53. We also found that the retroviral vector could circumvent the difficulty in transfection for the delivery of siRNA or shRNA and the generation of long-term gene silencing. The use of shRNA expression vector for RNAi might provide a rapid and versatile method for assessing gene function in mammalian cells with possible applications in the treatment of diseases.

Base Sequence↗

PubMed2005

Post-transcriptional gene silencing by siRNAs and miRNAs.

Recent years have seen a rapid increase in our understanding of how double-stranded RNA (dsRNA) and 21- to 25-nucleotide small RNAs, microRNAs (miRNAs) and small interfering RNAs (siRNAs), control gene expression in eukaryotes. This RNA-mediated regulation generally results in sequence-specific inhibition of gene expression; this can occur at levels as different as chromatin modification and silencing, translational repression and mRNA degradation. Many details of the biogenesis and function of miRNAs and siRNAs, and of the effector complexes with which they associate have been elucidated. The first structural information on protein components of the RNA interference (RNAi) and miRNA machineries is emerging, and provides some insight into the mechanism of RNA-silencing reactions.

Animals↗

PubMed2006

The silent treatment: RNAi as a defense against virus infection in mammals.

RNA interference (RNAi) is a mechanism for sequence-specific gene silencing guided by double-stranded RNA. In plants and insects it is well established that RNAi is instrumental in the response to viral infections; whether RNAi has a similar function in mammals is under intense investigation. Recent studies to address this question have identified some unanticipated interactions between the RNAi machinery and mammalian viruses. Furthermore, introduction of virus-specific small interfering RNAs (siRNAs) into cells, thus programming the RNAi machinery to target viruses, is an effective therapeutic approach to inhibit virus replication in vitro and in animal models. Although several issues remain to be addressed, such as delivery and viral escape, these findings hold tremendous potential for the development of RNAi-based antiviral therapeutics.

Animals↗

PubMed2006

RNAi-based knockdown of HBx mRNA in HBx-transformed and HBV-producing human liver cells.

RNA interference (RNAi) is the process of sequence-specific gene silencing induced by 21-23-nt RNA of small interfering RNA (siRNA). The HBx of hepatitis B virus (HBV) causing human liver diseases has been known as a multifunctional protein which affects transcription, cell growth, and apoptotic cell death. Here, we demonstrate that the HBx-specific siRNA (siRNAx) and short hairpin RNA (shRNAx) effectively induce the degradation of HBx mRNA in HBx-transformed and HBV-producing human liver cells by up to 80-90%. Also, the HBx expression in HBx-transformed cells was continuously silenced by retransformation with the shRNAx expression vector. These results imply that HBx-driven RNAi, either delivery of siRNAx or expression of shRNAx, provides a promising anti-HBV approach to suppress the HBx expression in human hepatoma cells.

Base Sequence↗

PubMed2007

Suppression of apoptosis in the liver by systemic and local delivery of small-interfering RNAs.

RNA interference (RNAi) is a sequence-specific gene-silencing mechanism triggered by double-stranded RNA. RNAi was shown to allow transient or stable knockdown of gene expression in a broad range of species and has been used successfully for functional genomic screens in mammalian cells and Caenorhabditis elegans. Standard therapeutic use of RNAi in clinical settings in humans has been hampered by the lack of effective methods to deliver the small-interfering RNAs (siRNAs) or short-hairpin RNA expression vectors into the diseased organs. In mice, systemic delivery of siRNAs by hydrodynamic intravascular injection leads to highly efficient uptake of siRNAs into the liver. Several groups demonstrated therapeutic use of RNAi in mouse models of acute liver failure or hepatitis B virus replication. This chapter will focus on the technical background of hydrodynamic and portal vein delivery techniques in mice and will give practical guidance for using these techniques for siRNA delivery into the liver.

Animals↗

PubMed2006

Inhibition of hypoxia-inducible factor-1alpha and endothelial progenitor cell differentiation by adenoviral transfer of small interfering RNA in vitro.

RNA interference is applied to study gene function in different organisms and in various cell types. Little is known about the effect of RNA interference on human endothelial progenitor cells (EPCs) in vitro. To address this issue, short hairpin RNA targeting the human hypoxia inducible factor-1alpha (HIF-1alpha) was transferred into human EPCs by an adenoviral vector. HIF-1alpha mRNA and protein expression was dramatically and specifically downregulated after adeno-small interfering RNA (siRNA)-HIF-1alpha infection in cells under hypoxia, a condition in which HIF-1alpha would have been induced. This effect persisted for at least 72 h and was accompanied by suppression of vascular endothelial growth factor (VEGF) mRNA and protein expression. The expression of endothelial cell markers CD31, VEGF receptor 2 (Flk-1) and eNOS as well as NO production were also markedly decreased. Functional studies showed HIF-1alpha knockdown via adenoviral siRNA transfer inhibited EPC colony formation, differentiation, proliferation and migration. These data indicate that specific gene knockdown via adenoviral transfer of siRNA is feasible in EPCs, and the effect is long-lasting. Our findings raise the possibility that such long-term modified human EPCs may be used to treat hypoxic tumor metastases that are known to be resistant to conventional therapeutic regimes.

Adenoviridae↗

PubMed2004

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.

Cell Line, Tumor↗

PubMed2003

A biochemical framework for RNA silencing in plants.

RNA silencing phenomena were first discovered in plants, yet only the RNA interference pathway in animals has been subject to biochemical analysis. Here, we extend biochemical analysis to plant RNA silencing. We find that standard wheat germ extract contains Dicer-like enzymes that convert double-stranded RNA (dsRNA) into two classes of small interfering RNAs, as well as an RNA-dependent RNA polymerase activity that can convert exogenous single-stranded RNA into dsRNA. In this plant embryo extract, an endogenous microRNA (miRNA) that lacks perfect complementarity to its RNA targets nonetheless acts as a small interfering RNA. The miRNA guides an endonuclease to cleave efficiently wild-type Arabidopsis PHAVOLUTA mRNA, but not a dominant mutant previously shown to perturb leaf development. This finding supports the view that plant miRNAs direct RNAi and that miRNA-specified mRNA destruction is important for proper plant development. Thus, endonuclease complexes guided by small RNAs are a common feature of RNA silencing in both animals and plants.

Arabidopsis↗

PubMed2003

High-throughput selection of effective RNAi probes for gene silencing.

RNA interference (RNAi) is a process of sequence-specific posttranscriptional gene silencing mediated by double-stranded RNA. RNAi has recently emerged as a powerful genetic tool to analyze gene function in mammalian cells. The power of this method is limited however, by the uncertainty in predicting the efficacy of small interfering RNAs (siRNAs) in silencing a gene. This has imposed serious limitations not only for small-scale but also for high-throughput RNAi screening initiatives in mammalian systems. We have developed a reliable and quantitative approach for the rapid and efficient identification of the most effective siRNA against any gene. The efficacy of siRNA sequences is monitored by their ability to reduce the expression of cognate target-reporter fusions with easily quantified readouts. Finally, using micro array-based cell transfections, we demonstrate an unlimited potential of this approach in high-throughput screens for identifying effective siRNA probes for silencing genes in mammalian systems. This approach is likely to have implications in the use of RNAi as a reverse genetic tool for analyzing mammalian gene function on a genome-wide scale.

Animals↗