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PubMed2004

RNA interference against retroviruses.

Bang and Ellerman, and later Peyton Rous, reported the first identification of transmissible cancer-causing agents, which later turned out to be avian retroviruses. Today avian retroviruses are important models for study of retrovirus replication and pathogenesis, and also important pathogens of domestic fowl. Here we describe the use of RNA interference (RNAi) in live chick embryos to block replication of an avian retrovirus. We also describe inhibition of ASLV and HIV replication in cell culture with RNAi.

Animals↗
PubMed2001

Size constraints for targeting post-transcriptional gene silencing and for RNA-directed methylation in Nicotiana benthamiana using a potato virus X vector.

Using a recombinant potato virus X (PVX) vector, we investigated the relationship between the length of RNA sequence identity with a transgene and the ability to promote post-transcriptional gene silencing (PTGS) and transgene methylation. The lower size limit required for targeting reporter transgene mRNA de novo using PTGS was 23 nucleotides (nt) of complete identity, a size corresponding to that of small RNAs associated with PTGS in plants and RNA interference (RNAi) in animals. The size and sequence specificity were also explored for PTGS-associated transgene methylation and for the targeting of the vector RNA. The PTGS-competent short sequences resulted in similar patterns of methylation. In all cases, including specific sequences of 33 nt with or without symmetrical cytosine residues, the methylation was distributed throughout the transcribed region of the transgene. In contrast, short sequences lacking symmetrical cytosines were less efficient at promoting PTGS of the transgene mRNA. Short gfp sequences in the PVX vector provided as effective a target for the degradation of viral RNA as was found for PVX carrying the complete gfp cDNA. Short sequences were able to initiate PTGS of an endogenous gene, phyotene desaturase, although this occurred in the absence of DNA methylation. This experimental approach provides important insights into the relationship between short RNA sequences and PTGS.

DNA Methylation↗
PubMed2001

RNA interference in Trypanosoma brucei: cloning of small interfering RNAs provides evidence for retroposon-derived 24-26-nucleotide RNAs.

In animals and protozoa, gene-specific double-stranded RNA (dsRNA) triggers degradation of homologous cellular RNAs, a phenomenon known as RNA interference (RNAi). In vitro and in vivo dsRNA is processed by a nuclease to produce 21-25-nt small interfering RNAs (siRNAs) that guide target RNA degradation. Here we show that activation of RNAi in Trypanosoma bruceiby expression or electroporation of actin dsRNA results in production of actin siRNAs and that 10% of these RNAs sediment as high-molecular-weight complexes at 100,000 x g. To characterize actin siRNAs, we established a cloning and enrichment strategy starting from 20-30 nt RNAs isolated from high-speed pellet and supernatant fractions. Sequence analysis revealed that actin siRNAs are 24-26 nt long and their distribution relative to actin dsRNA was similar in the two fractions. By sequencing over 1,300 fragments derived from the high-speed pellet fraction RNA, we found abundant 24-26-nt-long fragments homologous to the ubiquitous retroposon INGI and the site-specific retroposon SLACS. Northern hybridization with strand-specific probes confirmed that retroposon-derived 24-26-nt RNAs are present in both supernatant and high-speed pellet fractions and that they are constitutively expressed. We speculate that RNAi in trypanosomes serves a housekeeping function and is likely to be involved in silencing retroposon transcripts.

Actins↗
PubMed2006

Contributions of 3'-overhang to the dissociation of small interfering RNAs from the PAZ domain: molecular dynamics simulation study.

RNA interference (RNAi) is a 'knock-down' reaction to reduce expression of a specific gene through highly regulated, enzyme-mediated processes. Small interfering RNAs (siRNAs) are RNA molecules that play an effector role in RNAi and can bind the PAZ domains present in Dicer and RISC. We investigated the interaction between the PAZ domain and the siRNA-like duplexes through dissociation molecular dynamics (DMD) simulations. Specifically, we focused on the response of the PAZ domain to various 3'-overhang structures of the siRNA-like duplexes. We found that the siRNA-like duplex with the 3' UU-overhang made relatively more stable complex with the PAZ domain compared to those with 3' CC-, AA-, and GG-overhangs. The siRNA-like duplex with UU-overhang was easily dissociated from the PAZ domain once the structural stability of the complex is impaired. Interestingly, the 3' UU-overhang spent the least time at the periphery region of the binding pocket during the dissociation process, which can be mainly attributable to UU-overhang's smallest number of hydrogen bonds.

Binding Sites↗
PubMed2005

The prospect of silencing disease using RNA interference.

The discovery of RNA interference (RNAi), an endogenous cellular gene-silencing mechanism, has already provided a powerful tool for basic science researchers to study gene function. The subsequent finding that RNAi also operates in mammalian cells has generated excitement regarding potential therapeutic applications. In this article we discuss the basic mechanism of RNAi and the therapeutic opportunities and obstacles for harnessing RNAi for therapy of human disease.

Animals↗
PubMed2007

Exploring in vitro roles of siRNA in cardiovascular disease.

RNA interference (RNAi) is an adaptive defense mechanism through which double stranded RNAs silence cognate genes in a sequence-specific manner. It has been employed widely as a powerful tool in functional genomics studies, target validation and therapeutic product development. Similarly, the application of small interfering RNA (siRNA) to the silencing of the disease-causing genes involved in cardiovascular diseases has made great progress. In this overview, we attempt to provide a brief outline of the current understanding of the mechanism of RNAi and its potential application to the cardiovascular system, with particular emphasis on its ability to identify the pathophysiological function of genes related to several important cardiovascular disorders. The prospects of RNAi-based therapeutics, as well as the advantages and potential problems, are also discussed.

Animals↗
PubMed2007

Guidelines for the selection of effective short-interfering RNA sequences for functional genomics.

To avoid long-double-stranded-RNA-dependent interferon response, short-interfering RNAs (siRNAs) are widely used for RNA interference (RNAi) in mammalian cells. siRNA-based RNAi, however, may not be readily available for the large-scale gene silencing essential for systematic functional genomics, because only a limited fraction of siRNAs is capable of inducing effective mammalian RNAi. siRNAs correctly designed for the knockdown of a particular gene may also destroy the functions of unrelated genes. Here, we describe algorithms by which these serious setbacks can be eliminated in mammalian functional genomics using RNAi and a Web-based online software system for computing highly functional siRNA sequences with maximal target-specificity in mammalian RNAi.

Algorithms↗
PubMed2004

SKAR is a specific target of S6 kinase 1 in cell growth control.

BACKGROUND: The mammalian target of rapamycin (mTOR) and phosphatidylinositol 3-kinase (PI3K) signaling pathways promote cell growth and cell cycle progression in response to nutritional, energy, and mitogenic cues. In mammalian cells, the ribosomal protein S6 kinases, S6K1 and S6K2, lie downstream of mTOR and PI3K, suggesting that translational control through the phosphorylation of S6 regulates cell growth. Interestingly, genetic experiments predict that a substrate that is specific to S6K1 but not S6K2 regulates cell growth. RESULTS: Here we identify SKAR as a novel and specific binding partner and substrate of S6K1 but not S6K2. We find that serines 383 and 385 of human SKAR are insulin-stimulated and rapamycin-sensitive S6K1 phosphorylation sites. Quantitative mass spectrometry reveals that serine 383/385 phosphorylation is sensitive to RNA interference (RNAi)-mediated S6K1 reduction, but not S6K2 reduction. Furthermore, RNAi-mediated reduction of SKAR decreases cell size. SKAR is nuclear protein with homology to the Aly/REF family of RNA binding proteins, which has been proposed to couple transcription with pre-mRNA splicing and mRNA export. CONCLUSIONS: We have identified a novel and specific target of S6K1, SKAR, which regulates cell growth. The homology of SKAR to the Aly/REF family links S6K1 with mRNA biogenesis in the control of cell growth.

Amino Acid Sequence↗
PubMed2005

Chromatin and RNAi factors protect the C. elegans germline against repetitive sequences.

Protection of genomes against invasion by repetitive sequences, such as transposons, viruses, and repetitive transgenes, involves strong and selective silencing of these sequences. During silencing of repetitive transgenes, a trans effect ("cosuppression") occurs that results in silencing of cognate endogenous genes. Here we report RNA interference (RNAi) screens performed to catalog genes required for cosuppression in the Caenorhabditis elegans germline. We find factors with a putative role in chromatin remodeling and factors involved in RNAi. Together with molecular data also presented in this study, these results suggest that in C. elegans repetitive sequences trigger transcriptional gene silencing using RNAi and chromatin factors.

Animals↗
PubMed2004

RNAi of COL1A1 in mesenchymal progenitor cells.

Given that mutant COL1A1 is known to cause Osteogenesis Imperfecta (OI), tools to modulate COL1A1 expression are likely to be of significant therapeutic value. In this context, we have evaluated RNA interference (RNAi) as a means to downregulate COL1A1 expression in Cos-7 cells and in human mesenchymal progenitor stem cells (MPCs), the latter cells giving rise to bone and therefore representing a target cell type for collagen-related disorders. In addition, allele-specificity, a key factor to the success of RNAi-based suppression, was explored with a view to developing a mutation-independent RNAi-based therapeutic for OI by targeting an intragenic SNP within transcripts derived from the COL1A1 gene. Preferential suppression of individual polymorphic alleles that differed by a single nucleotide was observed.

Alleles↗
PubMed2006

Pol II-expressed shRNA knocks down Sod2 gene expression and causes phenotypes of the gene knockout in mice.

RNA interference (RNAi) has been used increasingly for reverse genetics in invertebrates and mammalian cells, and has the potential to become an alternative to gene knockout technology in mammals. Thus far, only RNA polymerase III (Pol III)-expressed short hairpin RNA (shRNA) has been used to make shRNA-expressing transgenic mice. However, widespread knockdown and induction of phenotypes of gene knockout in postnatal mice have not been demonstrated. Previous studies have shown that Pol II synthesizes micro RNAs (miRNAs)-the endogenous shRNAs that carry out gene silencing function. To achieve efficient gene knockdown in mammals and to generate phenotypes of gene knockout, we designed a construct in which a Pol II (ubiquitin C) promoter drove the expression of an shRNA with a structure that mimics human miRNA miR-30a. Two transgenic lines showed widespread and sustained shRNA expression, and efficient knockdown of the target gene Sod2. These mice were viable but with phenotypes of SOD2 deficiency. Bigenic heterozygous mice generated by crossing these two lines showed nearly undetectable target gene expression and phenotypes consistent with the target gene knockout, including slow growth, fatty liver, dilated cardiomyopathy, and premature death. This approach opens the door of RNAi to a wide array of well-established Pol II transgenic strategies and offers a technically simpler, cheaper, and quicker alternative to gene knockout by homologous recombination for reverse genetics in mice and other mammalian species.

3' Untranslated Regions↗
PubMed2001

Specific inhibition of HIV-1 gene expression by double-stranded RNA.

RNA interference (RNAi) is phenomenon in which introduced double-stranded RNAs (ds-RNAs) silence gene expression through specific degradation of their cognate mRNAs. RNAi has been observed in a wide variety of organisms and is considered to be a feature of nearly all eukaryotes. The mediators of sequence specific messenger RNA degradation are 21-23 nucleotide small interfering RNAs generated by ribonucleaseIII cleavage from longer dsRNAs. To investigate the potential of dsRNA to interfere with the function of HIV-1 genes, we have designed six longer dsRNAs containing the HIV-1 gag and env genes. Double-stranded RNAs were tested for inhibitory effects using monkey COS cells. In these anti-HIV-1 activity tests, the dsRNAs targeted to the HIV-1 env sequence showed more than 90% anti-HIV-1 efficiency. Our results show that dsRNA interference can be used as a powerful method for the inhibition of HIV-1 gene expression.

Animals↗
PubMed2005

Delivering RNA interference to the mammalian brain.

RNA interference (RNAi) is a new modality in gene therapy which can elicit down-regulation of gene expression and has enormous potential in the treatment of neurological diseases. RNAi is a conserved system through which double stranded RNA (dsRNA) guides sequence specific mRNA degradation. The RNAi apparatus may be artificially triggered by delivery of naked siRNA molecules or by plasmid-based expression of dsRNA. Before these techniques can be used as effective treatments in the brain, efficient methods of in vivo delivery must be devised. This review first describes the mechanism of RNAi, and then critically examines both viral and non-viral methods for delivery of RNAi to the mammalian brain. There have been a number of important recent publications in this field and the progress towards effective in vivo delivery of RNAi to the central nervous system is discussed. Finally, potential problems that must be considered before applying this technology to the human brain are outlined.

Animals↗
PubMed2004

RNA interference for the identification of disease-associated genes.

RNA interference (RNAi) has emerged as a novel cellular mechanism regulating gene expression at the post-transcriptional level and as a powerful tool to control gene function experimentally. Recent advances in the biology and application of RNAi include the definition of improved criteria for selecting effective small interfering RNA (siRNA) sequences, and the generation of vectors for the delivery of siRNAs and stable silencing of genes in mammalian cells, tissues and animals. High-throughput screening projects based on RNAi have been initiated to search for genes involved in basic biological processes and in complex pathological conditions such as cancer, autoimmunity and degenerative disorders. This research is helping to identify novel therapeutic targets for a range of diseases and may translate into novel clinical applications for RNAi.

Animals↗
PubMed2005

Use of RNA interference to dissect the roles of trans-acting factors in alternative pre-mRNA splicing.

RNA interference (RNAi) is becoming a popular method for analyzing gene function in a variety of biological processes. We have used RNAi in cultured Drosophila cells to identify trans-acting factors that regulate the alternative splicing of endogenously transcribed pre-mRNAs. We have generated a dsRNA library comprising approximately 70% of the Drosophila genes encoding RNA binding proteins and assessed the function of each protein in the regulation of alternative splicing. This approach not only identifies trans-acting factors regulating specific alternative splicing events, but also can provide insight into the alternative splicing regulatory networks of Drosophila. Here, we describe this RNAi approach to identify alternative splicing regulatory proteins in detail.

Alternative Splicing↗
PubMed2007

Gene silencing by RNA interference in the koji mold Aspergillus oryzae.

We found the orthologous genes required for RNA interference (RNAi) in the Aspergillus oryzae genome database, and constructed a set of tools for gene silencing using RNAi in A. oryzae. This system utilizes compatible restriction enzyme sites so that only a single target gene fragment is required to create the hairpin RNA cassette. For ease of handling, we also separated the construction of the hairpin RNA cassette for the target gene from its subsequent introduction into the expression vector. Using the brlA gene as a target for RNAi, we detected decreased mRNA levels and a delayed conidiation phenotype in the transformants. Furthermore, even though A. oryzae possesses three copies of the alpha-amylase gene, a single copy of an alpha-amylase RNAi construct was sufficient to downregulate the mRNA levels and decrease the enzymatic activity to 10% of control levels. Gene silencing by RNAi should provide a powerful genetic tool for post-genomic studies of the industrially important fungus A. oryzae.

Aspergillus oryzae↗
PubMed2006

A structural basis for discriminating between self and nonself double-stranded RNAs in mammalian cells.

Nonspecific effects triggered by small interfering RNAs (siRNAs) complicate the use of RNA interference (RNAi) to specifically downregulate gene expression. To uncover the basis of these nonspecific activities, we analyzed the effect of chemically synthesized siRNAs on mammalian double-stranded RNA (dsRNA)-activated signaling pathways. siRNAs ranging from 21 to 27 nucleotides (nt) in length activated the interferon system when they lacked 2-nt 3' overhangs, a characteristic of Dicer products. We show that the recognition of siRNAs is mediated by the RNA helicase RIG-I and that the presence of 3' overhangs impairs its ability to unwind the dsRNA substrate and activate downstream signaling to the transcription factor IRF-3. These results suggest a structural basis for discrimination between microRNAs that are endogenous Dicer products, and nonself dsRNAs such as by-products of viral replication. These findings will enable the rational design of siRNAs that avoid nonspecific effects or, alternatively, that induce bystander effects to potentially increase the efficacy of siRNA-based treatments of viral infections or cancer.

Animals↗
PubMed2005

Inhibition of hepatitis B virus replication by recombinant small interfering RNAs.

RNA interference (RNAi) is a biological phenomenon in which introduction of a small, double-stranded interfering RNAs (siRNAs) into a cell causes a specific degradation of homologous single-stranded RNA. siRNA can be delivered into the cell by different approaches including synthetic RNA, in vitro transcribed RNA and RNA transcribed from polymerase III-based recombinant vectors. As hepatitis B (HB) represents a worldwide health problem, we attempted to develop a fast and easy approach to generation and screening of specific siRNA-targeted HB virus (HBV) genes. Using PCR amplification, specific siRNA expression cassettes (SECs) were developed and used to generate effective siRNAs against HB virus (HBV) replication and gene expression in mammalian cells. After screening, we identified two SECs that expressed siRNAs which efficiently decreased the level of HBV pre-c/c gene expression in transfected Bel-7402 cells by 81.9% and 87.3%, respectively. In addition, the level of HBV DNA was decreased by 83.5% and 85.2% in HepG2 2.2.15 cells, respectively. This study provides (i) a new effective application of RNA interference to study viral gene function and viral replication and (ii) a new tool for the prevention and treatment of human HBV infection.

DNA, Viral↗