Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “RNA interference (RNAi)”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

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

At least 793 records · Page 44Linked to original sources
PubMed2001

ATP requirements and small interfering RNA structure in the RNA interference pathway.

We examined the role of ATP in the RNA interference (RNAi) pathway. Our data reveal two ATP-dependent steps and suggest that the RNAi reaction comprises at least four sequential steps: ATP-dependent processing of double-stranded RNA into small interfering RNAs (siRNAs), incorporation of siRNAs into an inactive approximately 360 kDa protein/RNA complex, ATP-dependent unwinding of the siRNA duplex to generate an active complex, and ATP-independent recognition and cleavage of the RNA target. Furthermore, ATP is used to maintain 5' phosphates on siRNAs. A 5' phosphate on the target-complementary strand of the siRNA duplex is required for siRNA function, suggesting that cells check the authenticity of siRNAs and license only bona fide siRNAs to direct target RNA destruction.

Adenosine Triphosphate↗
PubMed2002

Induction of RNA interference in Caenorhabditis elegans by RNAs derived from plants exhibiting post-transcriptional gene silencing.

The term 'gene silencing' refers to transcriptional and post-transcriptional control of gene expression. Related processes are found across kingdoms in plants and animals. We intended to test whether particular RNA constituents of a silenced plant can induce silencing in an animal. We generated Nicotiana benthamiana lines that expressed green fluorescent protein (GFP) from a transgene. Plants in which GFP expression was spontaneously silenced showed siRNAs characteristic of post-transcriptional gene silencing (PTGS). RNA extracts prepared from silenced plants were injected into a GFP-expressing strain of Caenorhabditis elegans, where they induced RNA interference (RNAi). Extracts from non-silenced plants were inactive. This directly demonstrates a relationship and a mechanistic link between PTGS and RNAi. Controls confirmed that the silencing agent was an RNA. Size fractionation on denaturing gels revealed that an RNA of approximately 85 nt was most active in inducing silencing in the worm. Northern blot analysis of the region in question did not detect a prominent GFP-specific RNA of sense or antisense polarity, indicating that the RNA species which induced silencing was present only in low concentration or did not hybridize due to formation of an intramolecular double strand. In view of its high activity, it is possible that this agent is responsible for the systemic spread of silencing in plants and it might represent the aberrant RNA, a previously postulated inducer of silencing.

Animals↗
PubMed2006

Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways.

RNA interference (RNAi) is a universal and evolutionarily conserved phenomenon of post-transcriptional gene silencing by means of sequence-specific mRNA degradation, triggered by small double-stranded RNAs. Because this mechanism can be efficiently induced in vivo by expressing target-complementary short hairpin RNA (shRNA) from non-viral and viral vectors, RNAi is attractive for functional genomics and human therapeutics. Here we systematically investigate the long-term effects of sustained high-level shRNA expression in livers of adult mice. Robust shRNA expression in all the hepatocytes after intravenous infusion was achieved with an optimized shRNA delivery vector based on duplex-DNA-containing adeno-associated virus type 8 (AAV8). An evaluation of 49 distinct AAV/shRNA vectors, unique in length and sequence and directed against six targets, showed that 36 resulted in dose-dependent liver injury, with 23 ultimately causing death. Morbidity was associated with the downregulation of liver-derived microRNAs (miRNAs), indicating possible competition of the latter with shRNAs for limiting cellular factors required for the processing of various small RNAs. In vitro and in vivo shRNA transfection studies implied that one such factor, shared by the shRNA/miRNA pathways and readily saturated, is the nuclear karyopherin exportin-5. Our findings have fundamental consequences for future RNAi-based strategies in animals and humans, because controlling intracellular shRNA expression levels will be imperative. However, the risk of oversaturating endogenous small RNA pathways can be minimized by optimizing shRNA dose and sequence, as exemplified here by our report of persistent and therapeutic RNAi against human hepatitis B virus in vivo.

Animals↗
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↗
PubMed2006

RNA interference in cancer.

In the recent years, RNA interference (RNAi) has emerged as a major regulatory mechanism in eukaryotic gene expression. The realization that changes in the levels of microRNAs are directly associated with cancer led to the recognition of a new class of tumor suppressors and oncogenes. Moreover, RNAi has been turned into a potent tool for artificially modulating gene expression through the introduction of short interfering RNAs. A plethora of individual inhibitory RNAs as well as several large collections of these reagents have been generated. The systems for stable and regulated expression of these molecules emerged as well. These tools have helped to delineate the roles of various cellular factors in oncogenesis and tumor suppression and laid the foundation for new approaches in gene discovery. Furthermore, successful inhibition of tumor cell growth by RNAi aimed at oncogenes in vitro and in vivo supports the enthusiasm for potential therapeutic applications of this technique. In this article we review the evidence of microRNA involvement in cancer, the use of short interfering RNAs in forward and reverse genetics of this disease, and as well as both the benefits and limitations of experimental RNAi.

Animals↗
PubMed2006

RNA interference-based gene silencing as an efficient tool for functional genomics in hexaploid bread wheat.

Insertional mutagenesis and gene silencing are efficient tools for the determination of gene function. In contrast to gain- or loss-of-function approaches, RNA interference (RNAi)-induced gene silencing can possibly silence multigene families and homoeologous genes in polyploids. This is of great importance for functional studies in hexaploid wheat (Triticum aestivum), where most of the genes are present in at least three homoeologous copies and conventional insertional mutagenesis is not effective. We have introduced into bread wheat double-stranded RNA-expressing constructs containing fragments of genes encoding Phytoene Desaturase (PDS) or the signal transducer of ethylene, Ethylene Insensitive 2 (EIN2). Transformed plants showed phenotypic changes that were stably inherited over at least two generations. These changes were very similar to mutant phenotypes of the two genes in diploid model plants. Quantitative real-time polymerase chain reaction revealed a good correlation between decreasing mRNA levels and increasingly severe phenotypes. RNAi silencing had the same quantitative effect on all three homoeologous genes. The most severe phenotypes were observed in homozygous plants that showed the strongest mRNA reduction and, interestingly, produced around 2-fold the amount of small RNAs compared to heterozygous plants. This suggests that the effect of RNAi in hexaploid wheat is gene-dosage dependent. Wheat seedlings with low mRNA levels for EIN2 were ethylene insensitive. Thus, EIN2 is a positive regulator of the ethylene-signaling pathway in wheat, very similar to its homologs in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa). Our data show that RNAi results in stably inherited phenotypes and therefore represents an efficient tool for functional genomic studies in polyploid wheat.

Carotenoids↗
PubMed2006

An inducible T7 RNA polymerase-dependent plasmid system.

RNA interference (RNAi) has become a powerful tool for the specific silencing of gene transcription. Especially the targeting of genes in mammalian cells has been greatly improved by generating plasmid based and viral vector-based systems. This permits expression of short hairpin RNA (shRNA) on a longterm basis. However, an inducible expression of shRNA is required, if the target is essential for cell survival. We developed a doxycycline-inducible two-plasmid system for the expression of a ribozyme-processed shRNA. In contrast to other existing systems, we use the highly specific T7 phage RNA polymerase, which does not interact with cellular factors; therefore, interference with cellular functions is limited. One plasmid is responsible for doxycycline-dependent expression of T7 RNA polymerase and a second plasmid expresses a ribozyme-processed shRNA under the control of a T7 promoter. Our results showed that doxycycline- dependent expression of T7 RNA polymerase was tightly controlled and expression of an shRNA against firefly luciferase inhibited 86% of luciferase activity. In conclusion, our plasmid system provides a very useful tool for analyzing essential gene functions in vitro.

Base Sequence↗
PubMed2006

[Inhibitory effects of RNA interference on expression of matrix metalloproteinase-9 gene and invasiveness and adhesion in ovarian cancer cells].

OBJECTIVE: To investigate the inhibitory effects of RNA interference (RNAi) on the expression of matrix metalloproteinase-9 (MMP-9) gene and invasiveness and adhesion of ovarian cancer cells. METHODS: Four groups of different specific target sequence in coding region of MMP-9 and one non-specific sequence were chosen, which were Site1, Site2, Site3, Site4 and Site5. Small interference RNA (siRNA) expression cassettes (SEC) were constructed by PCR and transfected into ovarian cancer HO-8910PM cells. RT-PCR and western blot were used to detect mRNA and protein expression of MMP-9 gene; the abilities of invasion and adhesion were detected by Matrigel invasion assay and cell adhesion assay. RESULTS: The expression of MMP-9 was inhibited and the inhibitory effects of different sequence were varied. The mRNA expression was 0.64 +/- 0.06, 0.47 +/- 0.07, 0.55 +/- 0.10 in Site1, Site2, Site3 group, and protein expression was 0.30 +/- 0.09, 0.27 +/- 0.08, 0.37 +/- 0.12, respectively. Site2 group had the most efficient inhibitory effect, followed by Site1 and Site3 groups. Cell growth curve revealed that cell growth was significantly inhibited in Site2 group. Invasiveness and adhesion were significantly reduced, the inhibitory rate on invasion in Site1, Site2, Site3 groups were 50.0%, 50.0% and 37.5%, respectively; the inhibitory rate on adhesion in Site1, Site2, Site3, Site4 groups were 43.8%, 48.8%, 33.9%, 24.2% at 60 min and 41.6%, 40.2%, 35.1%, 16.0% at 90 min, respectively. CONCLUSIONS: RNAi exists in ovarian HO-8910PM cells. MMP-9 siRNA can specifically down-regulate MMP-9 expression and lead to the inhibition of invasiveness and adhesion in ovarian cancer cells.

Blotting, Western↗
PubMed2006

Chemical and structural diversity of siRNA molecules.

Short interfering RNAs (siRNAs) are 21-23 nt long double-stranded oligoribonucleotides which in mammalian cells exhibit a potency for sequence-specific gene silencing via an RNA interference (RNAi) pathway. It has been already proven that exogenous, chemically synthesized siRNA molecules are effective inhibitors of gene expression and are widely applied for analysis of protein function and proteomics-based target identification. Moreover, since their discovery siRNA molecules have been implemented as potential candidates for therapeutic applications. Variously modified siRNA molecules containing sugar modifications (2'-OMe, -F, -O-allyl, -amino, orthoesters and LNA analogues), internucleotide phospodiester bond modifications (phosphorothioates, boranophosphates), base modifications (s(2)U) as well as 3'-terminal cholesterol-conjugated constructs were investigated as potential candidates for effective inhibition of gene expression. This chapter reviews an impact of chemical and structural modifications of siRNA molecules on their serum and thermal stability, cellular and in vivo activity, cellular uptake, biodistribution and cytotoxicity. Functional analysis of chemically modified siRNA molecules allows for better understanding of the mechanism of the RNA interference process as well as demonstrates immense efforts in optimizing in vivo potency of siRNA molecules for RNAi-based drug design.

Animals↗
PubMed2004

Cyclophilin A retrotransposition into TRIM5 explains owl monkey resistance to HIV-1.

In Old World primates, TRIM5-alpha confers a potent block to human immunodeficiency virus type 1 (HIV-1) infection that acts after virus entry into cells. Cyclophilin A (CypA) binding to viral capsid protects HIV-1 from a similar activity in human cells. Among New World primates, only owl monkeys exhibit post-entry restriction of HIV-1 (ref. 1). Paradoxically, the barrier to HIV-1 in owl monkey cells is released by capsid mutants or drugs that disrupt capsid interaction with CypA. Here we show that knockdown of owl monkey CypA by RNA interference (RNAi) correlates with suppression of anti-HIV-1 activity. However, reintroduction of CypA protein to RNAi-treated cells did not restore antiviral activity. A search for additional RNAi targets unearthed TRIMCyp, an RNAi-responsive messenger RNA encoding a TRIM5-CypA fusion protein. TRIMCyp accounts for post-entry restriction of HIV-1 in owl monkeys and blocks HIV-1 infection when transferred to otherwise infectable human or rat cells. It seems that TRIMCyp arose after the divergence of New and Old World primates when a LINE-1 retrotransposon catalysed the insertion of a CypA complementary DNA into the TRIM5 locus. This is the first vertebrate example of a chimaeric gene generated by this mechanism of exon shuffling.

Amino Acid Sequence↗
PubMed2003

Transgene-induced RNA interference: a strategy for overcoming gene redundancy in polyploids to generate loss-of-function mutations.

Gene redundancy in polyploid species complicates genetic analyses by making the generation of recessive, loss-of-function alleles impractical. We show that this problem can be circumvented using RNA interference (RNAi) to achieve dominant loss of function of targeted genes. Arabidopsis suecica is an allotetraploid (amphidiploid) hybrid of A. thaliana and A. arenosa. We demonstrate that A. suecica can be genetically transformed using the floral dip method for Agrobacterium-mediated transformation. Transgenes segregate as in a diploid, indicating that chromosome pairing occurs exclusively (or almost so) among homologs and not among homeologs. Expressing a double-stranded (ds) RNA corresponding to the A. thaliana gene, decrease in DNA methylation 1 (DDM1) caused the elimination of DDM1 mRNAs and the loss of methylation at both A. thaliana- and A. arenosa-derived centromere repeats. These results indicate that a single RNAi-inducing transgene can dominantly repress multiple orthologs.

Arabidopsis↗
PubMed2003

Comparison of the suppressive effects of antisense oligonucleotides and siRNAs directed against the same targets in mammalian cells.

RNA interference appears to be a potentially powerful tool for studies of genes of unknown function. However, differences in efficacy at different target sites remain problematic when small interfering RNA (siRNA) is used as an effector. Similar problems are associated with attempts at gene inactivation using antisense oligonucleotides (ODNs) and ribozymes. We performed a comparative analysis of the suppressive effects of three knockdown methods, namely, methods based on RNA interference (RNAi), antisense ODNs, and ribozymes, using a luciferase reporter system. Dose-response experiments revealed that the IC50 value for the siRNA was about 100-fold lower than that of the antisense ODN. Our results provide useful information about the positional effects in RNAi, which might help to improve the design of effective siRNAs.

Gene Expression Regulation↗
PubMed2003

Transport of dsRNA into cells by the transmembrane protein SID-1.

RNA interference (RNAi) spreads systemically in plants and nematodes to silence gene expression distant from the site of initiation. We previously identified a gene, sid-1, essential for systemic but not cell-autonomous RNAi in Caenorhabditis elegans. Here, we demonstrate that SID-1 is a multispan transmembrane protein that sensitizes Drosophila cells to soaking RNAi with a potency that is dependent on double-stranded RNA (dsRNA) length. Further analyses revealed that SID-1 enables passive cellular uptake of dsRNA. These data indicate that systemic RNAi in C. elegans involves SID-1-mediated intercellular transport of dsRNA.

Adenosine Triphosphate↗
PubMed2006

pWormgatePro enables promoter-driven knockdown by hairpin RNA interference of muscle and neuronal gene products in Caenorhabditis elegans.

Recent advances in genome research and RNA interference (RNAi) technology have accelerated the adoption of genome-wide experimental approaches for determining gene function in the model organism Caenorhabditis elegans. Despite recent successes, the application of RNAi is limited when gene knockdown causes complex phenotypes or embryonic lethality. Recently, the high-throughput pWormgate cloning system has been introduced as a tool to efficiently generate heat-shock-inducible hairpin RNA constructs using the Gateway recombination technology. We have modified pWormgate into a versatile hairpin cloning plasmid, pWormgatePro, which facilitates temporally and spatially inducible hairpin RNAi using constitutively active, tissue-specific promoters. To demonstrate its utility we knocked down unc-22 in body wall muscles as well as the axon guidance gene unc-5 in the nervous system indicating that promoter-driven hairpins can overcome the neuronal resistance to RNAi. Using pWormgatePro we also show that RNAi in the nervous system of C. elegans is non-autonomous and that spreading of the RNAi signal from neurons to muscle is substantially reduced but not abolished in spreading-defective sid-1 mutant animals. Our findings illustrate the effectiveness of pWormgatePro for gene silencing in muscle cells and neurons and bring forward the possibility of applying tissue-specific RNAi on a genome-wide scale.

Animals↗
PubMed2005

Modulation of angiogenesis with siRNA inhibitors for novel therapeutics.

Cancer and many other serious diseases are characterized by the uncontrolled growth of new blood vessels. Recently, RNA interference (RNAi) has reinvigorated the therapeutic prospects for inhibiting gene expression and promises many advantages over binding inhibitors, including high specificity, which is essential for targeted therapeutics. This article describes the latest developments using small-interfering RNA (siRNA) inhibitors to downregulate various angiogenic and tumor-associated factors, both in cell-culture assays and in animal disease models. The majority of research efforts are currently focused on understanding gene function, as well as proof-of-concept for siRNA-mediated anti-angiogenesis. The prospects for siRNA therapeutics, both advantages and looming hurdles, are evaluated.

Cell Adhesion Molecules↗
PubMed2004

Drosophila mitochondrial transcription factor B2 regulates mitochondrial DNA copy number and transcription in schneider cells.

We report the cloning and molecular analysis of Drosophila mitochondrial transcription factor B2 (d-mt-TFB2), a protein that plays a role in mitochondrial transcription and mitochondrial DNA (mtDNA) replication in Drosophila. An RNA interference (RNAi) construct was designed that reduces expression of d-mtTFB2 to 5% of its normal level in Schneider cells. RNAi knock-down of d-mtTFB2 reduces the abundance of specific mitochondrial RNA transcripts 2- to 8-fold and decreases the copy number of mtDNA approximately 3-fold. In a corollary manner, we find that overexpression of d-mtTFB2 increases both the abundance of mitochondrial RNA transcripts and the copy number of mtDNA. In a comparative experiment, we find that overexpression of Drosophila mitochondrial transcription factor A (d-TFAM) increases mtDNA copy number with no significant effect on mitochondrial transcripts. This argues for a direct role for mtTFB2 in mitochondrial transcription and suggests that, if TFAM serves a role in transcription, its endogenous level limits mtDNA copy number but not transcription. Furthermore, we suggest that mtTFB2 increases mtDNA copy number by increasing the frequency of initiation of DNA replication, whereas TFAM serves to stabilize and package mtDNA in mitochondrial nucleoids. Our work represents the first study to document the function of mtTFB2 in vivo, establishing a dual role in regulation of both transcription and replication, and provides a benchmark for comparative biochemical studies in various animal systems.

Amino Acid Sequence↗
PubMed2005

Human mitochondrial tRNAMet is exported to the cytoplasm and associates with the Argonaute 2 protein.

Argonaute (Ago) proteins are the effector proteins of RNA interference (RNAi) and related silencing mechanisms that are mediated by small RNAs. Ago proteins bind directly to microRNAs (miRNAs) and to short interfering RNAs and are the core protein components of RNA induced silencing complexes (RISCs) and microRNPs (miRNPs). Here we report that an ~70-nt RNA associates specifically with immunopurified Ago2 expressed in human 293 cells. By directional cloning we identified this RNA as the mitochondrial tRNA(Met) (mt tRNA(Met)). Various exported (mt) tRNAs were detected in the cytosol of 293 cells, but Ago2 was found selectively bound to (mt) tRNA(Met). The association in the cytosol of exported (mt) tRNA(Met) with Ago2 complements genetic and microscopic data that link mitochondria with RNAi-related components and events.

Active Transport, Cell Nucleus↗
PubMed2006

The silent treatment: siRNAs as small molecule drugs.

As soon as RNA interference (RNAi) was found to work in mammalian cells, research quickly focused on harnessing this powerful endogenous and specific mechanism of gene silencing for human therapy. RNAi uses small RNAs, less than 30 nucleotides in length, to suppress expression of genes with complementary sequences. Two strategies can introduce small RNAs into the cytoplasm of cells, where they are active - a drug approach where double-stranded RNAs are administered in complexes designed for intracellular delivery and a gene therapy approach to express precursor RNAs from viral vectors. Phase I clinical studies have already begun to test the therapeutic potential of small RNA drugs that silence disease-related genes by RNAi. This review will discuss progress in developing and testing small RNAi-based drugs and potential obstacles.

Animals↗