SEARCH · PubMed Health

Results for “RNA interference”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 199 records · Page 11Linked to original sources

PubMed2006

HIV-1 TAR RNA subverts RNA interference in transfected cells through sequestration of TAR RNA-binding protein, TRBP.

TAR RNA-binding protein, TRBP, was recently discovered to be an essential partner for Dicer and a crucial component of the RNA-induced silencing complex (RISC), a critical element of the RNA interference (RNAi) of the cell apparatus. Human TRBP was originally characterized and cloned 15 years ago based on its high affinity for binding the HIV-1 encoded leader RNA, TAR. RNAi is used, in part, by cells to defend against infection by viruses. Here, we report that transfected TAR RNA can attenuate the RNAi machinery in human cells. Our data suggest that TAR RNA sequesters TRBP rendering it unavailable for downstream Dicer-RISC complexes. TAR-induced inhibition of Dicer-RISC activity in transfected cells was partially relieved by exogenous expression of TRBP.

HIV Long Terminal Repeat↗

PubMed2003

The adenosine analog tubercidin inhibits glycolysis in Trypanosoma brucei as revealed by an RNA interference library.

We used an RNA interference (RNAi) library in a forward genetic selection to study the mechanism of toxicity of tubercidin (7-deazaadenosine) to procyclic Trypanosoma brucei. Following transfection of cells with an RNAi-based genomic library, we used 5 microm tubercidin to select a drug-resistant cell line. Surprisingly, we found in these resistant cells that the hexose transporters had been silenced. We subsequently found that silencing of hexokinase, a glycolytic enzyme, also yielded tubercidin-resistant parasites. These observations suggested that glycolysis could be a target of tubercidin action and that RNAi silencing of glycolytic enzymes was gradual enough to allow the parasites to adapt to alternative sources of energy. Indeed, adaptation of procyclic trypanosomes to a glucose-independent metabolism by reduction of glucose in the culture medium caused tubercidin resistance. High pressure liquid chromatography analysis of glycolytic intermediates from parasites treated with tubercidin showed a dose-dependent increase in concentration of 1,3-bisphosphoglycerate, a substrate of phosphoglycerate kinase. Furthermore, tubercidin triphosphate inhibited recombinant T. brucei phosphoglycerate kinase activity in vitro with an IC50 of 7.5 microm. We conclude that 5 microm tubercidin kills trypanosomes by targeting glycolysis, especially by inhibition of phosphoglycerate kinase.

Adaptation, Physiological↗

PubMed2005

Application of RNA interference to study stem cell function: current status and future perspectives.

RNA interference is a mechanism displayed by most eukaryotic cells to rid themselves of foreign double-stranded RNA molecules. In the six years since the initial report, RNA interference has now been demonstrated to function in mammalian cells to alter gene expression, and has been used as a means for genetic discovery as well as a possible strategy for genetic correction. An equally popular topic over the past six years has been the proposal to utilize embryonic stem cells or adult stem cells as cell-based therapies for human diseases. The aim of this review is to provide a general overview of how RNA interference suppresses gene expression and to examine some published RNA interference approaches that have resulted in changes in stem cell function and suggest the possible clinical relevance of this work.

Animals↗

PubMed2005

The structure of the flock house virus B2 protein, a viral suppressor of RNA interference, shows a novel mode of double-stranded RNA recognition.

We report the structure of the flock house virus B2 protein, a potent suppressor of RNA interference (RNAi) in animals and plants. The B2 protein is a homodimer in solution and contains three alpha-helices per monomer. Chemical shift perturbation shows that an antiparallel arrangement of helices (alpha2/alpha2') forms an elongated binding interface with double-stranded RNA (dsRNA). This implies a novel mode of dsRNA recognition and provides insights into the mechanism of RNAi suppression by B2.

Amino Acid Sequence↗

PubMed2000

Functional anatomy of a dsRNA trigger: differential requirement for the two trigger strands in RNA interference.

In RNA-mediated interference (RNAi), externally provided mixtures of sense and antisense RNA trigger concerted degradation of homologous cellular RNAs. We show that RNAi requires duplex formation between the two trigger strands, that the duplex must include a region of identity between trigger and target RNAs, and that duplexes as short as 26 bp can trigger RNAi. Consistent with in vitro observations, a fraction of input dsRNA is converted in vivo to short segments of approximately 25 nt. Interference assays with modified dsRNAs indicate precise chemical requirements for both bases and backbone of the RNA trigger. Strikingly, certain modifications are well tolerated on the sense, but not the antisense, strand, indicating that the two trigger strands have distinct roles in the interference process.

Animals↗

PubMed2006

Editor meets silencer: crosstalk between RNA editing and RNA interference.

The most prevalent type of RNA editing is mediated by ADAR (adenosine deaminase acting on RNA) enzymes, which convert adenosines to inosines (a process known as A-->I RNA editing) in double-stranded (ds)RNA substrates. A-->I RNA editing was long thought to affect only selected transcripts by altering the proteins they encode. However, genome-wide screening has revealed numerous editing sites within inverted Alu repeats in introns and untranslated regions. Also, recent evidence indicates that A-->I RNA editing crosstalks with RNA-interference pathways, which, like A-->I RNA editing, involve dsRNAs. A-->I RNA editing therefore seems to have additional functions, including the regulation of retrotransposons and gene silencing, which adds a new urgency to the challenges of fully understanding ADAR functions.

Adenosine↗

PubMed2006

[Vector-mediated HER-2 RNA interference against HER-2-positive breast cancer].

OBJECTIVE: To study the feasibility of vector-mediated RNA interference for HER-2-positive breast cancer therapy. METHODS: A plasmid vector capable of mediating HER-2 RNA interference was constructed, and HER-2-positive breast cancer cell line SKBR-3 was transfected with this constructed vector. The expression of HER-2 mRNA and protein was analyzed by RT-PCR and Western blotting, and the growth and apoptosis of SKBR-3 cells was analyzed after transfection. RESULTS: The expressions of HER-2 mRNA and HER-2 protein was downregulated in response to vector-mediated HER-2 RNA interference, which also resulted in tumor cell growth inhibition and increased number apoptotic cells. CONCLUSION: HER-2 is a good target for RNA interference and RNA interference targeting HER-2 can lead to HER-2 breast cancer cell apoptosis and growth inhibition.

Apoptosis↗

PubMed2005

A lentiviral microRNA-based system for single-copy polymerase II-regulated RNA interference in mammalian cells.

The advent of RNA interference has led to the ability to interfere with gene expression and greatly expanded our ability to perform genetic screens in mammalian cells. The expression of short hairpin RNA (shRNA) from polymerase III promoters can be encoded in transgenes and used to produce small interfering RNAs that down-regulate specific genes. In this study, we show that polymerase II-transcribed shRNAs display very efficient knockdown of gene expression when the shRNA is embedded in a microRNA context. Importantly, our shRNA expression system [called PRIME (potent RNA interference using microRNA expression) vectors] allows for the multicistronic cotranscription of a reporter gene, thereby facilitating the tracking of shRNA production in individual cells. Based on this system, we developed a series of lentiviral vectors that display tetracycline-responsive knockdown of gene expression at single copy. The high penetrance of these vectors will facilitate genomewide loss-of-function screens and is an important step toward using bar-coding strategies to follow loss of specific sequences in complex populations.

Animals↗

PubMed2005

Escape from the interferon response associated with RNA interference using vectors that encode long modified hairpin-RNA.

In mammalian cells, siRNAs have been used to induce RNA interference (RNAi) in an attempt to prevent nonspecific effects (including the interferon (IFN) response) which are caused by long double-stranded RNAs (dsRNAs) of more than 30 bp. In this report, we describe a novel and simple strategy for avoiding activation of the IFN response by dsRNA. We show that modified hairpin-RNAs (mhRNAs) of more than 100 bp, with multiple specific point-mutations within the sense strand and transcribed from the U6 or tRNA(Val) promoters, can cause RNAi without inducing the IFN pathway genes. Moreover, we demonstrate that the 50-bp mhRNA vector could effectively suppress the replication of multiple hepatitis C viruses (the genomes of which differ slightly, thus the 21-bp siRNA vector failed to suppress one of them). Our findings should enhance the exploitation of RNAi in mammalian cells, especially in the field of RNAi therapy against pathogenic viruses.

Base Sequence↗

PubMed2007

RNA interference technologies and their use in cancer research.

PURPOSE OF REVIEW: Recently, RNA interference has evolved into a powerful research tool to functionally characterize genes. Genome-wide RNA interference reagents can study the loss-of-function phenotypes of candidate genes in the context of various disease model systems. In this review, we discuss the data from the most recent studies using RNA interference reagents with a focus on RNA interference-based genomic screening as a tool to expand our knowledge about the molecular basis of cancer. RECENT FINDINGS: Tumorigenesis is the result of the progressive accumulation of mutations in genes controlling cell proliferation and death. Various genes carrying these alterations are known to be directly linked to tumor growth; however, how to translate this knowledge into effective chemotherapeutics, nontoxic to normal cells, is still a subject of intensive research. SUMMARY: Loss-of-function studies offer a potential for validation of known and unrecognized tumor-associated targets. RNA interference-mediated gene knockdown can be exploited to study the reprogrammed circuitry of genes, discover gene interactions restricted to cancer cells and identify mechanisms of chemoresistance in cancer cells. In addition, the simultaneous use of cancer drugs and RNA interference also provides a paradigm to develop strategies to inactivate essential genes promoting neoplastic growth.

Humans↗

PubMed2001

RNA interference: mechanisms and applications.

RNA interference (RNAi) is a phenomenon induced by double-stranded RNA (dsRNA) in which gene expression is inhibited through specific degradation of mRNA. The mechanism involves conversion of dsRNA into short RNAs that direct ribonucleases to homologous mRNA targets. This process is related to normal defence against viruses and mobilisation of transposons. Treatment with dsRNA has become an important method for analysing gene functions in invertebrate organisms. RNAi has also been demonstrated in several vertebrate species but with lower efficiency. Development of procedures for in vivo production of dsRNA may provide efficient tools for tissue- and stage-specific gene targeting.

Animals↗

PubMed2002

Tissue-specific RNA interference in postimplantation mouse embryos with endoribonuclease-prepared short interfering RNA.

RNA interference (RNAi) using double-stranded RNA has been used for the systematic analysis of gene function in invertebrate organisms. Here we have explored the use of short interfering RNA (siRNA) to knock down gene expression during the development of mammalian postimplantation embryos. The developing CNS system of embryonic day 10 mouse embryos was used as a model tissue. siRNA prepared by endoribonuclease digestion (esiRNA) was injected into the lumen of the neural tube at specific regions and delivered into neuroepithelial cells by directed electroporation. Injected and electroporated embryos were grown for 1 day in whole-embryo culture and the effects of RNAi were examined. esiRNA directed against beta-galactosidase (beta-gal), coelectroporated into neuroepithelial cells together with reporter plasmids expressing GFP and beta-gal, abolished expression of beta-gal but not GFP, showing the specificity of the esiRNA-mediated RNAi. To demonstrate RNAi of endogenous gene expression, we used heterozygous embryos of a knock-in mouse line expressing GFP from the Tis21 locus, a gene turned on in neuroepithelial cells that switch from proliferation to neurogenesis. GFP-directed esiRNA electroporated into neuroepithelial cells of such embryos blocked the GFP expression normally occurring on the onset of neurogenesis. Taken together, our data indicate that esiRNA delivered in a tissue-specific manner by topical injection followed by directed electroporation can efficiently silence endogenous gene expression in mammalian postimplantation embryos.

Animals↗

PubMed2001

Distinct roles for RDE-1 and RDE-4 during RNA interference in Caenorhabditis elegans.

RNA interference (RNAi) is a cellular defense mechanism that uses double-stranded RNA (dsRNA) as a sequence-specific trigger to guide the degradation of homologous single-stranded RNAs. RNAi is a multistep process involving several proteins and at least one type of RNA intermediate, a population of small 21-25 nt RNAs (called siRNAs) that are initially derived from cleavage of the dsRNA trigger. Genetic screens in Caenorhabditis elegans have identified numerous mutations that cause partial or complete loss of RNAi. In this work, we analyzed cleavage of injected dsRNA to produce the initial siRNA population in animals mutant for rde-1 and rde-4, two genes that are essential for RNAi but that are not required for organismal viability or fertility. Our results suggest distinct roles for RDE-1 and RDE-4 in the interference process. Although null mutants lacking rde-1 show no phenotypic response to dsRNA, the amount of siRNAs generated from an injected dsRNA trigger was comparable to that of wild-type. By contrast, mutations in rde-4 substantially reduced the population of siRNAs derived from an injected dsRNA trigger. Injection of chemically synthesized 24- or 25-nt siRNAs could circumvent RNAi resistance in rde-4 mutants, whereas no bypass was observed in rde-1 mutants. These results support a model in which RDE-4 is involved before or during production of siRNAs, whereas RDE-1 acts after the siRNAs have been formed.

Animals↗

PubMed2006

The potential regulation of L1 mobility by RNA interference.

The hypothesis that RNA interference constrains L1 mobility seems inherently reasonable: L1 mobility can be dangerous and L1 RNA, the presumed target of RNAi, serves as a critical retrotransposition intermediate. Despite its plausibility, proof for this hypothesis has been difficult to obtain. Studies attempting to link the L1 retrotransposition frequency to alterations in RNAi activity have been hampered by the long times required to measure retrotransposition frequency, the pleiotropic and toxic effects of altering RNAi over similar time periods, and the possibility that other cellular machinery may contribute to the regulation of L1s. Another problem is that the commonly used L1 reporter cassette may serve as a substrate for RNAi. Here we review the L1-RNAi hypothesis and describe a genetic assay with a modified reporter cassette that detects approximately 4 times more L1 insertions than the conventional retrotransposition assay.

Journal Article↗

PubMed2005

Knocking down transport: applications of RNA interference in the study of drug transport proteins.

RNA interference (RNAi) is a gene silencing process mediated by double-stranded RNA (dsRNA). The silencing process is comprised of an initiation step, in which small interfering RNA (siRNA) is introduced to the cell, and an effector step, which involves degrading mRNA molecules of the target gene. RNA interference has been observed in most organisms from plants to vertebrates. As a gene silencing approach, RNAi has proven to be extremely useful in characterizing gene function and developing new tools in cancer therapy and drug delivery. The development of RNAi-related technologies is an emerging area in biomedical research. In this review, recent progress in the application of RNAi to the study of transport proteins is summarized and evaluated; the advantages, disadvantages and future directions of RNAi technology are discussed.

Animals↗

PubMed2005

Positional effect of chemical modifications on short interference RNA activity in mammalian cells.

A systematic study on the effect of 2'-sugar modifications (2'-F (2'-F-2'-deoxy-nucleoside residues), 2'-O-Me (2'-O-methyl-nucleoside residues), and 2'-O-MOE [2'-O-(2-methoxyethyl)]-nucleoside residues) in the antisense and sense strands of short interference RNA (siRNA) was performed in HeLa cells. The study of the antisense strand of siRNAs demonstrated that activity depends on the position of the modifications in the sequence. The siRNAs with modified ribonucleotides at the 5'-end of the antisense strand were less active relative to the 3'-modified ones. The 2'-F sugar was generally well-tolerated on the antisense strand, whereas the 2'-O-Me showed significant shift in activity depending on the position of modification. The 2'-O-MOE modification in the antisense strand resulted in less active siRNA constructs regardless of placement position in the construct. The incorporation of the modified residues, e.g., 2'-O-Me and 2'-O-MOE, in the sense strand of siRNA did not show a strong positional preference. These results may provide guidelines to design effective and stable siRNAs for RNA interference mediated therapeutic applications.

Animals↗

PubMed2005

Translating RNA interference into therapies for human disease.

RNA interference (RNAi) represents one of the most promising new frontiers in drug discovery. Breakthroughs in understanding RNA's extensive natural role in essential cellular processes have opened up the potential for a whole new class of drugs based on RNAi. Harnessing the natural process of RNAi, short, double-stranded RNA molecules are able to inhibit expression of genes in a sequence-specific manner. By targeting disease-causing genes, RNAi drugs have the potential to be more selective than traditional drugs, and thus more effective as well as less toxic. Over the past few years, important strides have been made in translating the promise of RNAi into therapies for human disease. In contrast to the extensive lead optimization steps typically required in small-molecule and protein drug discovery, RNAi drug candidates can be identified using bioinformatics to select sequences complementary to the target mRNA. The process of selecting an RNAi-based drug candidate may simply involve the synthesis and testing of a relatively small number of short double strands (duplexes) of RNA, incorporating chemical modifications that confer stability and direct these RNA duplexes to the appropriate tissues and cells, and/or formulating these RNA duplexes with appropriate delivery agents to achieve the same goals. As advances in RNAi therapeutics continue, the decades to come should bring a potent new class of drugs based on RNAi.

Animals↗

PubMed2006

Improving RNA interference in mammalian cells by 4'-thio-modified small interfering RNA (siRNA): effect on siRNA activity and nuclease stability when used in combination with 2'-O-alkyl modifications.

A systematic structure-activity relationship study of 4'-thioribose containing small interfering RNAs (siRNAs) has led to the identification of highly potent and stable antisense constructs. To enable this optimization effort for both in vitro and in vivo applications, we have significantly improved the yields of 4'-thioribonucleosides by using a chirally pure (R)-sulfoxide precursor. siRNA duplexes containing strategically placed regions of 4'-thio-RNA were synthesized and evaluated for RNA interference activity and plasma stability. Stretches of 4'-thio-RNA were well tolerated in both the antisense and sense strands. However, optimization of both the number and placement of 4'-thioribonucleosides was necessary for maximal potency. These optimized siRNAs were generally equipotent or superior to native siRNAs and exhibited increased thermal and plasma stability. Furthermore, significant improvements in siRNA activity and plasma stability were achieved by judicious combination of 4'-thioribose with 2'-O-methyl and 2'-O-methoxyethyl modifications. These optimized 4'-thio-siRNAs may be valuable for developing stable siRNAs for therapeutic applications.

Animals↗