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At least 199 records · Page 11Linked to original sources
PubMed2001

Argonaute2, a link between genetic and biochemical analyses of RNAi.

Double-stranded RNA induces potent and specific gene silencing through a process referred to as RNA interference (RNAi) or posttranscriptional gene silencing (PTGS). RNAi is mediated by RNA-induced silencing complex (RISC), a sequence-specific, multicomponent nuclease that destroys messenger RNAs homologous to the silencing trigger. RISC is known to contain short RNAs ( approximately 22 nucleotides) derived from the double-stranded RNA trigger, but the protein components of this activity are unknown. Here, we report the biochemical purification of the RNAi effector nuclease from cultured Drosophila cells. The active fraction contains a ribonucleoprotein complex of approximately 500 kilodaltons. Protein microsequencing reveals that one constituent of this complex is a member of the Argonaute family of proteins, which are essential for gene silencing in Caenorhabditis elegans, Neurospora, and Arabidopsis. This observation begins the process of forging links between genetic analysis of RNAi from diverse organisms and the biochemical model of RNAi that is emerging from Drosophila in vitro systems.

Amino Acid Sequence↗
PubMed2004

Gene therapy progress and prospects. Downregulating gene expression: the impact of RNA interference.

The control and maintenance of gene expression is critical for cell development and differentiation. Over the last 2 years, our understanding of the role of RNA as a regulator of gene expression has significantly increased. Small RNA molecules are key elements of a machinery that trigger chromosomal modifications, post-transcriptional gene silencing and protein translational blockade depending on the source, the RNA and the nature of the interaction with the target nucleic acid. Currently, the best characterized of this group of RNA-mediated gene regulation pathways is the post-transcriptional gene silencing mechanism known as RNA interference. RNAi is triggered by double-stranded RNA (dsRNA), which induces the formation of a ribonucleoprotein complex that mediates sequence-specific cleavage of the transcript cognate with the input dsRNA. RNAi has been adapted as a functional genomics tool and it has potential as a therapeutic approach. This review will summarize our current understanding of the RNAi mechanism and the various applications of RNAi-based technologies.

Animals↗
PubMed2005

Promises and challenges in developing RNAi as a research tool and therapy for neurodegenerative diseases.

RNA interference (RNAi) is a recently discovered mechanism that is conserved in a wide range of eukaryotic species. Triggered by double-stranded RNA, RNAi identifies and destroys the mRNA that shares homology with the double-stranded RNA. Because of its specificity, RNAi has a high potential for being a powerful investigative and therapeutic tool. Indeed, its use as a reverse genetics tool to determine gene functions in invertebrates and cultured mammalian cells has already been experiencing an explosive growth. Gratifyingly we have also seen its application in dissecting neurodegeneration pathways in vitro. Although early studies suggested that RNAi could be readily adapted for in vivo studies in mammals using the transgenic technology, difficulties including low transgenicity and low RNAi efficacy have emerged, which has prevented the wide use of transgenic RNAi. The potential of RNAi therapy for human diseases has been a great source of excitement. Several new studies have demonstrated this concept in animal models of neurodegenerative disease. In this review, we highlight the recent literature and our own data in applying RNAi in research and therapy in the area of neurodegenerative diseases. We discuss the present and future challenges in the full realization of the potential for RNAi.

Animals↗
PubMed2002

RNAi: gene-silencing in therapeutic intervention.

Several rapidly developing RNA interference (RNAi) methodologies hold the promise to selectively inhibit gene expression in mammals. RNAi is an innate cellular process activated when a double-stranded RNA (dsRNA) molecule of greater than 19 duplex nucleotides enters the cell, causing the degradation of not only the invading dsRNA molecule, but also single-stranded (ssRNAs) RNAs of identical sequences, including endogenous mRNAs. As such, RNAi technology is currently being evaluated not only as an extremely powerful instrument for functional genomic analyses, but also as a potentially useful method to develop highly specific dsRNA based gene-silencing therapeutics.

Animals↗
PubMed2004

Antiapoptotic action of hypoxia-inducible factor-1 alpha in human endothelial cells.

Hypoxia-inducible factor-1 (HIF-1) is the major transcription factor involved in the adaptive response to hypoxia and consists of HIF-1 alpha and HIF-1 beta subunits. Indirect evidence suggests that HIF-1 alpha may exert both proapoptotic and antiapoptotic actions in response to hypoxia. In this study, we evaluated the effects of RNA interference (RNAi) targeting HIF-1 alpha messenger RNA (mRNA) on apoptosis in primary cultured human umbilical vascular endothelial cells (HUVECs) exposed to anoxia and reoxygenation (A/R). HUVECs were transfected with specific 21-nt small interfering RNA (siRNA) duplexes targeting HIF-1 alpha mRNA sequences or scrambled RNA duplexes and subjected either to normoxia for 251/2 h or to anoxia for 11/2 h, and subsequently normoxia for 24 h (A/R). Control samples were subjected to A/R but not transfected. HUVECs apoptosis was evaluated by Tdt-mediated dUTP nick end-labeling (TUNEL) assay and by activated caspase-3 immunostaining and immunoblotting. The efficacy of RNAi was assessed by knockdown of HIF-1 alpha mRNA and protein expression via in situ hybridization, real-time quantitative PCR, immunohistochemistry, and Western blotting. When compared with normoxic cultures, A/R significantly upregulated HIF- 1 alpha mRNA and protein expression in HUVECs, but did not appreciably alter the percentage of apoptotic cells. In contrast, a significantly greater proportion of HUVECs transfected with specific siRNA duplexes and exposed to A/R demonstrated evidence of apoptosis when compared with nontransfected cells. Transfection with specific siRNA duplexes knocked down HIF-1 alpha mRNA and protein expression in A/R-treated cells by approximately 60%, whereas transfection with scrambled siRNA duplexes had no noticeable effect on HIF-1 alpha expression. These findings strongly suggest that HIF-1 alpha exerts an antiapoptotic role in HUVECs stressed by anoxia.

Apoptosis↗
PubMed2006

High potency silencing by single-stranded boranophosphate siRNA.

In RNA interference (RNAi), double-stranded short interfering RNA (ds-siRNA) inhibits expression from complementary mRNAs. Recently, it was demonstrated that short, single-stranded antisense RNA (ss-siRNA) can also induce RNAi. While ss-siRNA may offer several advantages in both clinical and research applications, its overall poor activity compared with ds-siRNA has prevented its widespread use. In contrast to the poor gene silencing activity of native ss-siRNA, we found that the silencing activity of boranophosphate-modified ss-siRNA is comparable with that of unmodified ds-siRNA. Boranophosphate ss-siRNA has excellent maximum silencing activity and is highly effective at low concentrations. The silencing activity of boranophosphate ss-siRNA is also durable, with significant silencing up to 1 week after transfection. Thus, we have demonstrated that boranophosphate-modified ss-siRNA can silence gene expression as well as native ds-siRNA, suggesting that boranophosphate-modified ss-siRNAs should be investigated as a potential new class of therapeutic agents.

Argonaute Proteins↗
PubMed2025

Argonaute 2 targets viral transcripts but not genomes of RNA viruses during antiviral RNA interference in Drosophila.

RNA interference (RNAi) mediated by the small interfering RNA (siRNA) pathway is a major antiviral mechanism in insects. This pathway is triggered when double-stranded RNA (dsRNA) produced during virus replication is recognized by Dicer-2, leading to the formation of virus-derived siRNA duplexes. These siRNAs are loaded onto the programmable nuclease Argonaute-2 (AGO2), with one strand serving as a guide to target and cleave fully complementary sequences of viral RNAs. While siRNAs are generated from viral dsRNA, the specific viral RNA species targeted for silencing during RNA virus replication remains unclear. In this study, we characterized the primary viral RNA targets of the Drosophila siRNA pathway during infections caused by negative and positive RNA viruses, namely Vesicular stomatitis virus (VSV) and Sindbis virus (SINV). Our findings reveal that polyadenylated transcripts of VSV and SINV are the major targets of silencing by the siRNA pathway during infection, likely when they are poised for translation. Consistent with earlier findings, we show that AGO2 is associated with ribosomes in control and virus infected cells. Therefore, we propose that the inhibition of the replication of RNA viruses in Drosophila results from the silencing of incoming viral transcripts, facilitated by the association of AGO2 with ribosomes.

Animals↗
PubMed2006

Therapeutic potential of RNAi in metabolic diseases.

Over the past years RNA interference (RNAi) has exploded as a new approach to manipulate gene expression in mammalian systems. More recently, RNAi has acquired interest as a potential therapeutic strategy. This review focuses on the potential therapeutic use of RNAi for metabolic diseases, the current understanding of RNAi biology, and how RNAi has been utilized to study the role of different genes in the pathogenesis of diabetes and obesity. Also reviewed are the in vivo proof-of-principle experiments that provide the preclinical justification for the development of RNAi-based therapeutics for diabetes and the key challenges that currently limit its application in the clinical setting.

Gene Targeting↗
PubMed2005

New tool for an old problem: can RNAi efficiently resolve the issue of genetic redundancy?

RNA interference (RNAi) has become a generally accepted tool for inhibiting gene expression in many laboratory organisms. Nagel et al.,(1) in a recent paper, give an example of how this tool can also be used to address the question of genetic redundancy. Their focus was on the redundancy in Drosophila melanogaster of the Enhancer of split gene complex [E(spl)-C] which comprises seven highly related genes. Their somewhat conflicting findings are probably the typical scenario for most RNAi experiments: some expected results and some surprises.

Animals↗
PubMed2001

RNAi and brain function: was McConnell on the right track?

RNA interference (RNAi), one of the hottest topics of molecular biology research today, has unique features that are eerily reminiscent of the phenomenon of "RNA-mediated memory transfer," a controversial line of work that was investigated with great enthusiasm in the 1960s. If not a coincidence, then this suggests taking a new look at RNA-mediated modulation of neural function and raises the possibility that RNAi might be one of the physiologic mechanisms that regulate long-term gene expression in the brain.

Animals↗
PubMed2003

Double-stranded RNA-binding proteins could suppress RNA interference-mediated antiviral defences.

RNA interference (RNAi) is a double-stranded (ds)RNA-inducible, sequence-specific RNA-degradation mechanism that operates as a natural antiviral system in plants and animals. Successful virus infection requires evasion or suppression of RNAi. Indeed, RNAi suppressor proteins have been identified in plant and animal viruses, although the molecular mechanism of silencing inhibition is still poorly understood. Because many RNA viruses encode dsRNA-binding proteins (dsRBPs) and as RNAi is triggered by the accumulation of dsRNAs, dsRBPs were examined to see if they inhibit RNAi. Here, it is shown that heterologous dsRBPs suppressed RNAi in plants, indicating that in natural host-virus interactions, pathogen-encoded dsRBPs could inactivate RNAi-mediated host defences.

Plant Proteins↗
PubMed2000

The NED-8 conjugating system in Caenorhabditis elegans is required for embryogenesis and terminal differentiation of the hypodermis.

This work has identified the enzymes involved in the activation and conjugation of the ubiquitin-like protein NED-8 in Caenorhabditis elegans. A C. elegans conjugating enzyme, UBC-12, is highly specific in its ability to utilize NED-8 as a substrate. Immunostaining shows that NED-8 is conjugated in vivo to a major target protein with a conjugate size of 90 kDa. While the amount of this conjugate is developmentally regulated with reduced levels in the larval stages, the mRNA encoding C. elegans UBC-12 is constitutively produced throughout development, as is NED-8 itself. The importance of the NED-8 conjugating system in C. elegans was determined by RNA interference (RNAi) assays using double-stranded RNA encoding NED-8, UBC-12, or the NED-8 activating enzyme component ULA-1. The progeny of both ned-8 and ubc-12 RNAi-treated hermaphrodites either arrested during embryonic development or underwent abnormal postembryonic development. The effect on postembryonic development was pleiotropic, the most frequent gross abnormality being vulval eversion during the L4 stage. Individuals with an everted vulva either burst at the L4 to adult molt or gave rise to adults incapable of egg laying. Additionally, both ned-8 and ubc-12 RNAi induced a striking abnormality in the alae, structures produced by the lateral hypodermal seam cells in the adult nematode. Affected alae were patchy and frequently diverged around a central space. Vulval defects were also produced by RNAi directed at C. elegans ula-1. This is the first demonstration of a requirement for NED-8 conjugation in metazoan development.

Amino Acid Sequence↗
PubMed2006

siRNA-containing liposomes modified with polyarginine effectively silence the targeted gene.

Development of RNA interference (RNAi) technology utilizing the short interfering RNA sequences (siRNA) based 'targeted' therapeutics has focused on creating methods for delivering siRNAs to cells and for enhancing siRNA stability in vitro and in vivo. Here, we describe a novel approach for siRNA cellular delivery using siRNA encapsulated into liposomes additionally bearing arginine octamer (R8) molecules attached to their outer surface (R8-liposomes). The R8-liposomal human double minute gene 2 (HDM2)-siRNA demonstrated a significant stability against degradation in the blood serum (siRNA-loaded R8-liposomes remained intact even after 24-h incubation), and higher transfection efficiency into all three tested lung tumor cell lines. siRNA delivery successfully proceeds in the presence of plasma proteins, and R8-liposomes demonstrate low non-specific toxicity. The mechanism of action of R8-liposome-encapsulated siRNA is associated with the RNAi-mediated degradation of the target mRNA. siRNA in R8-liposomes effectively inhibited the targeted gene and significantly reduced the proliferation of cancer cells. The approach offers the potential for siRNA delivery for various in vitro and in vivo applications.

Cell Line, Tumor↗
PubMed2004

Expression of small hairpin RNA by lentivirus-based vector confers efficient and stable gene-suppression of HIV-1 on human cells including primary non-dividing cells.

RNA interference (RNAi) is a sequence-specific RNA degradation process mediated by short double-stranded RNAs termed small interfering RNAs. Here, we describe the lentivirus-based vector small interfering RNA system expressing. As a pilot study, we generated constructs expressing small hairpin RNA (shRNA) specific for luciferase gene (shLuc) or green fluorescence protein (shGFP) under the control of human H1 RNA polymerase III promoter. The effect of the shRNA was evaluated against HIV-1 infection in a single-round or multiple-round infectious system using an HIV-1 molecular clone carrying the luc or GFP gene. In the single-round infectious system, cells transduced with shLuc by lentiviral vector significantly reduced (approximately 90% reduction) viral gene expression after challenge infection at a multiplicity of infection of 10. These transduced cells continued to resist against at least four sequentially repeated challenge infections. Importantly, this efficient antiviral activity persisted over 35 days in culture. In a multiple-round infectious system using a replication-competent HIV-1 molecular clone carrying the GFP gene, we also observed that a lentiviral vector expressing shGFP could inhibit HIV-1 replication for at least 1 week. The profound effect of lentiviral shRNA was also observed in human primary monocyte-derived macrophages. Thus, shRNA introduced through the lentiviral vector can be useful for efficient and stable gene suppression in human cells including primary non-dividing cells. Moreover, quantitative analysis of viral cDNA synthesis on challenge infection showed that viral genomic RNAs packaged in incoming virus core might not be targeted by shLuc. Instead, the degradation of transcripts from integrated proviral DNAs might be a major cause of the profound reduction in HIV-1 gene expression by shRNA in our system.

Cell Division↗
PubMed2003

The Ref/Aly proteins are dispensable for mRNA export and development in Caenorhabditis elegans.

The mRNA export pathway is highly conserved throughout evolution. We have used RNA interference (RNAi) to functionally characterize bona fide RNA export factors and components of the exon-exon junction complex (EJC) in Caenorhabditis elegans. RNAi of CeNXT1/p15, the binding partner of CeNXF1/TAP, caused early embryonic lethality, demonstrating an essential function of this gene during C. elegans development. Moreover, depletion of this protein resulted in nuclear accumulation of poly(A)(+) RNAs, supporting a direct role of NXT1/p15 in mRNA export in C. elegans. Previously, we have shown that RNAi of CeSRm160, a protein of the EJC complex, resulted in wild-type phenotype; in the present study, we demonstrate that RNAi of CeY14, another component of this complex, results in embryonic lethality. In contrast, depletion of the EJC component CeRNPS1 results in no discernible phenotype. Proteins of the REF/Aly family act as adaptor proteins mediating the recruitment of the mRNA export factor, NXF1/TAP, to mRNAs. The C. elegans genome encodes three members of the REF/Aly family. RNAi of individual Ref genes, or codepletion of two Ref genes in different combinations, resulted in wild-type phenotype. Simultaneous suppression of all three Ref genes did not compromise viability or progression through developmental stages in the affected progeny, and only caused a minor defect in larval mobility. Furthermore, no defects in mRNA export were observed upon simultaneous depletion of all three REF proteins. These results suggest the existence of multiple adaptor proteins that mediate mRNA export in C. elegans.

Amino Acid Sequence↗
PubMed2005

Inhibiting the expression of CD28 costimulatory molecule on human lymphocytes by special siRNA.

BACKGROUND: The B7/CD28 pathway provides critical costimulatory signals for complete T cell activation, and members of this pathway have served as useful targets for immunotherapeutic strategies. In this study, we investigated the RNA interference (RNAi) effect induced by small interfering RNA (siRNA) targeting CD28 mRNA on human lymphocytes and its specificity. METHODS: According to CD28 gene sequence, we designed and synthysized three different siRNAs (siRNA-1, siRNA-2, siRNA-3) containing 21 bases using Silencertrade mark siRNA construction kit. These siRNAs were transfected into freshly isolated human lymphocytes with Lipofectamine 2000 reagent. At 24-hour, 48-hour and 72-hour post transfection, these cells were collected and analyzed. The changes of surface expression of CD28 gene were detected by flow cytometry, and the changes of CD28 mRNA levels were determined by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). The cell viability of transfected lymphocytes was determined by methyl thiazolyl tetrazolium (MTT) assay and trypan blue dye exclusion assay. RESULTS: Three siRNAs (siRNA-1, siRNA-2, siRNA-3) specifically targeting CD28 mRNA were successfully designed and constructed. Flow cytometry analysis showed that a decrease in CD28 expression was detectable at 24-hour post transfection. Different siRNA showed different inhibition effects on CD28 expression. At 48-hour post transfection, the degrees of reduction with siRNA-1, siRNA-2 and siRNA-3 were 22.10% +/- 1.63%, 73.50% +/- 1.02% and 42.90% +/- 0.89% respectively compared with the control (P < 0.001). Neither of the groups transfected only with siRNA or lipo showed marked reduction in CD28 expression (3.15% +/- 0.75% and 4.55% +/- 0.80%) (P > 0.05). Moreover, lymphocytes treated with siRNA-co showed no marked reduction in CD28 expression (5.07% +/- 0.96%) (P > 0.05). The results of semi-quantitative RT-PCR assay indicated CD28 mRNA level was inhibited after transfection of specific siRNAs. At least 4-fold of reduction in siRNA-2 group occurred at 48-hour post transfection compared with the control (P < 0.001). MTT assay and trypan blue dye exclusion assay demonstrated that the viable cell rations of transfected lymphocytes were significantly reduced in siRNA-1, siRNA-2 and siRNA-3 groups at 48-hour post transfection (P < 0.01). The control groups showed no marked reduction in cell viability (P > 0.05). CONCLUSIONS: Three different siRNAs were synthesized and transfected into lymphocytes. They could reduce the expression of CD28 and the CD28 mRNA level. siRNA-2 was the most efficient. The cell viability reduced correspondingly. Therefore, the silencing effect on CD28 mRNA induced by siRNA may contribute to costimulatory blockade. This result show that siRNA may be useful for further study on graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (allo-BMT).

Adolescent↗
PubMed2005

High-throughput screening of effective siRNAs from RNAi libraries delivered via bacterial invasion.

Use of RNA interference (RNAi) as a reverse genetics tool for silencing genes in mammalian cells is achieved by in vitro transfection of small interfering RNAs (siRNAs). For a target gene, several siRNAs must be designed according to the empirical rules. We demonstrated that functional short hairpin RNAs (shRNAs) could be synthesized in Escherichia coli and delivered directly via bacterial invasion to the near entirety of a mammalian cell population to trigger RNAi. Furthermore, using a luciferase-target gene transcript, we identified effective shRNAs and siRNAs from RNAi libraries delivered conveniently through bacterial invasion in 96-well plates without need for preparation, purification and transfection of shRNAs. Notably, several of the most highly effective shRNAs and siRNAs identified do not fit the empirical rules commonly used for siRNA design, suggesting that this approach is a powerful tool for RNAi research, and could be used complementarily to the empirical rules for RNAi applications.

Combinatorial Chemistry Techniques↗
PubMed2003

RNAi: mammalian oocytes do it without RNA-dependent RNA polymerase.

Studies in mutant organisms deficient in RNA interference (RNAi) and related post-transcriptional gene silencing implicated a role for a single class of RNA-dependent RNA polymerases (RdRp). Nevertheless, sequence homologs to these RdRps have not been found in coelomate organisms such as Drosophila or mammals. This lack of homologous sequences does not exclude that an RdRp functions in RNAi in these organisms because an RdRp could be acquired by horizontal transfer from an RNA virus. In fact, such a sequence is found in mice (Aquarius) and we observe that it is expressed in mouse oocytes and early embryos, which exhibit RNAi. We report here that cordycepin, an inhibitor of RNA synthesis, does not prevent Mos double-strand RNA (dsRNA) to target endogenous Mos mRNA in mouse oocytes and that targeting a chimeric Mos-EGFP mRNA with dsRNA to EGFP does not reduce the endogenous Mos mRNA, but does target the chimeric mRNA. These results indicate that an RdRp is not involved in dsRNA-mediated mRNA degradation in mammalian oocytes, and possibly in mammals in general, and therefore that only homologous sequences to the dsRNA are targeted for degradation.

Animals↗