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At least 163 records · Page 9Linked to original sources
PubMed2005

RNAi and RNA-based regulation of immune system function.

Gene regulation by short RNAs is a ubiquitous and important mode of control. MicroRNAs are short, single-strand RNAs that bind with partial complementarity to the 3' untranslated region of several genes to silence their expression. This expanding class of endogenous short RNAs are evolutionarily conserved and participate in control of development and cell-specific gene function. Several of these microRNAs have been cloned uniquely from mammalian lymphocytes suggesting specialized roles in lymphocyte development and function. In addition, several genes linked to RNAi in lower eukaryotes have mammalian homologs with specialized roles in adaptive immunity. For example, in worms, the nonsense-mediated decay (NMD) and RNAi pathways appear to be intricately linked. NMD plays a key role in regulating antigen-receptor expression in lymphocytes and there are mammalian homologs for factors identified in worms that appear to be common in both RNAi and NMD pathways. On the other hand, RNA editing and RNAi have an inverse relationship and RNA editing has an important role in viral immunity. These observations indicate unique roles for dsRNAs in the mammalian immune system.

APOBEC-1 Deaminase↗
PubMed2000

RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals.

Double-stranded RNA (dsRNA) directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). Using a recently developed Drosophila in vitro system, we examined the molecular mechanism underlying RNAi. We find that RNAi is ATP dependent yet uncoupled from mRNA translation. During the RNAi reaction, both strands of the dsRNA are processed to RNA segments 21-23 nucleotides in length. Processing of the dsRNA to the small RNA fragments does not require the targeted mRNA. The mRNA is cleaved only within the region of identity with the dsRNA. Cleavage occurs at sites 21-23 nucleotides apart, the same interval observed for the dsRNA itself, suggesting that the 21-23 nucleotide fragments from the dsRNA are guiding mRNA cleavage.

Adenosine Triphosphate↗
PubMed2002

The dsRNA binding protein RDE-4 interacts with RDE-1, DCR-1, and a DExH-box helicase to direct RNAi in C. elegans.

Double-stranded (ds) RNA induces potent gene silencing, termed RNA interference (RNAi). At an early step in RNAi, an RNaseIII-related enzyme, Dicer (DCR-1), processes long-trigger dsRNA into small interfering RNAs (siRNAs). DCR-1 is also required for processing endogenous regulatory RNAs called miRNAs, but how DCR-1 recognizes its endogenous and foreign substrates is not yet understood. Here we show that the C. elegans RNAi pathway gene, rde-4, encodes a dsRNA binding protein that interacts during RNAi with RNA identical to the trigger dsRNA. RDE-4 protein also interacts in vivo with DCR-1, RDE-1, and a conserved DExH-box helicase. Our findings suggest a model in which RDE-4 and RDE-1 function together to detect and retain foreign dsRNA and to present this dsRNA to DCR-1 for processing.

Amino Acid Motifs↗
PubMedDate not supplied

RNAi analysis of genes expressed in the ovary of Caenorhabditis elegans.

As a step towards comprehensive functional analysis of genomes, systematic gene knockout projects have been initiated in several organisms [1]. In metazoans like C. elegans, however, maternal contribution can mask the effects of gene knockouts on embryogenesis. RNA interference (RNAi) provides an alternative rapid approach to obtain loss-of-function information that can also reveal embryonic roles for the genes targeted [2,3]. We have used RNAi to analyze a random set of ovarian transcripts and have identified 81 genes with essential roles in embryogenesis. Surprisingly, none of them maps on the X chromosome. Of these 81 genes, 68 showed defects before the eight-cell stage and could be grouped into ten phenotypic classes. To archive and distribute these data we have developed a database system directly linked to the C. elegans database (Wormbase). We conclude that screening cDNA libraries by RNAi is an efficient way of obtaining in vivo function for a large group of genes. Furthermore, this approach is directly applicable to other organisms sensitive to RNAi and whose genomes have not yet been sequenced.

Animals↗
PubMed2002

PPW-1, a PAZ/PIWI protein required for efficient germline RNAi, is defective in a natural isolate of C. elegans.

One of the remarkable aspects about RNA interference (RNAi) in Caenorhabditis elegans is that the trigger molecules, dsRNA, can be administered via the animal's food. We assayed whether this feature is a universal property of the species by testing numerous strains that have been isolated from different parts of the globe. We found that one isolate from Hawaii had a defect in RNAi that was specific to the germline and was a result of multiple mutations in a PAZ/PIWI domain-containing protein, which we named PPW-1. Deleting ppw-1 in the canonical C. elegans strain Bristol N2 makes it resistant to feeding of dsRNA directed against germline-expressed genes. PPW-1 belongs to the Argonaute family of proteins, which act in posttranscriptional gene silencing and development, and is homologous to the RNAi gene rde-1. Our data indicate that at least two members of this family are required for complete and effective RNAi in C. elegans.

Amino Acid Sequence↗
PubMed2002

The role of RNA editing by ADARs in RNAi.

Adenosine deaminases that act on RNA (ADARs) are RNA-editing enzymes that deaminate adenosines to create inosines in double-stranded RNA (dsRNA). Here we demonstrate that ADARs are not required for RNA interference (RNAi) and that they do not antagonize the pathway to a detectable level when RNAi is initiated by injecting dsRNA. We find, however, that transgenes expressed in the somatic tissues of wild-type animals are silenced in strains with deletions in the two genes encoding ADARs, adr-1 and adr-2. Transgene-induced gene silencing in adr-1;adr-2 mutants depends on genes required for RNAi, suggesting that a dsRNA intermediate is involved. In wild-type animals we detect edited dsRNA corresponding to transgenes, and we propose that editing of this dsRNA prevents somatic transgenes from initiating RNAi in wild-type animals.

Adenosine Deaminase↗
PubMed2002

Evidence that siRNAs function as guides, not primers, in the Drosophila and human RNAi pathways.

In Drosophila, two features of small interfering RNA (siRNA) structure--5' phosphates and 3' hydroxyls--are reported to be essential for RNA interference (RNAi). Here, we show that as in Drosophila, a 5' phosphate is required for siRNA function in human HeLa cells. In contrast, we find no evidence in flies or humans for a role in RNAi for the siRNA 3' hydroxyl group. Our in vitro data suggest that in both flies and mammals, each siRNA guides endonucleolytic cleavage of the target RNA at a single site. We conclude that the underlying mechanism of RNAi is conserved between flies and mammals and that RNA-dependent RNA polymerases are not required for RNAi in these organisms.

Animals↗
PubMed2002

RNAi in human cells: basic structural and functional features of small interfering RNA.

We investigated the mechanism of RNA interference (RNAi) in human cells. Here we demonstrate that the status of the 5' hydroxyl terminus of the antisense strand of a siRNA determines RNAi activity, while a 3' terminus block is tolerated in vivo. 5' hydroxyl termini of antisense strands isolated from human cells were phosphorylated, and 3' end biotin groups were not efficiently removed. We found no requirement for a perfect A-form helix in siRNA for interference effects, but an A-form structure was required for antisense-target RNA duplexes. Strikingly, crosslinking of the siRNA duplex by psoralen did not completely block RNA interference, indicating that complete unwinding of the siRNA helix is not necessary for RNAi activity in vivo. These results suggest that RNA amplification by RNA-dependent RNA polymerase is not essential for RNAi in human cells.

Animals↗
PubMed2001

Genome-wide RNAi.

In many species, double-stranded RNA can specifically and effectively silence genes. This newly discovered biological phenomenon, called RNA interference (RNAi), has practical implications for functional genomics. As shown by two recent reports, RNAi provides a rapid method to test the function of genes in the nematode Caenorhabditis elegans; most of the genes on C. elegans chromosome I and III have now been tested for RNAi phenotypes. The results validate RNAi as a powerful functional genomics tool for C. elegans, and point the way for similar large-scale studies in other species.

Animals↗
PubMed2003

Transposon silencing in the Caenorhabditis elegans germ line by natural RNAi.

Transposable elements are stretches of DNA that can move and multiply within the genome of an organism. The Caenorhabditis elegans genome contains multiple Tc1 transposons that jump in somatic cells, but are silenced in the germ line. Many mutants that have lost this silencing have also lost the ability to execute RNA interference (RNAi), a process whereby genes are suppressed by exposure to homologous double-stranded RNA (dsRNA). Here we show how RNAi causes transposon silencing in the nematode germ line. We find evidence for transposon-derived dsRNAs, in particular to the terminal inverted repeats, and show that these RNAs may derive from read-through transcription of entire transposable elements. Small interfering RNAs of Tc1 were detected. When a germline-expressed reporter gene is fused to a stretch of Tc1 sequence, this transgene is silenced in a manner dependent on functional mutator genes (mut-7, mut-16 and pk732). These results indicate that RNAi surveillance is triggered by fortuitous read-through transcription of dispersed Tc1 copies, which can form dsRNA as a result of 'snap-back' of the terminal inverted repeats. RNAi mediated by this dsRNA silences transposase gene expression.

Alleles↗
PubMed2003

An epi-allelic series of p53 hypomorphs created by stable RNAi produces distinct tumor phenotypes in vivo.

The application of RNA interference (RNAi) to mammalian systems has the potential to revolutionize genetics and produce novel therapies. Here we investigate whether RNAi applied to a well-characterized gene can stably suppress gene expression in hematopoietic stem cells and produce detectable phenotypes in mice. Deletion of the Trp53 tumor suppressor gene greatly accelerates Myc-induced lymphomagenesis, resulting in highly disseminated disease. To determine whether RNAi suppression of Trp53 could produce a similar phenotype, we introduced several Trp53 short hairpin RNAs (shRNAs) into hematopoietic stem cells derived from E(mu)-Myc transgenic mice, and monitored tumor onset and overall pathology in lethally irradiated recipients. Different Trp53 shRNAs produced distinct phenotypes in vivo, ranging from benign lymphoid hyperplasias to highly disseminated lymphomas that paralleled Trp53-/- lymphomagenesis in the E(mu)-Myc mouse. In all cases, the severity and type of disease correlated with the extent to which specific shRNAs inhibited p53 activity. Therefore, RNAi can stably suppress gene expression in stem cells and reconstituted organs derived from those cells. In addition, intrinsic differences between individual shRNA expression vectors targeting the same gene can be used to create an 'epi-allelic series' for dissecting gene function in vivo.

Alleles↗
PubMed2006

Genome-scale loss-of-function screening with a lentiviral RNAi library.

The discovery that RNA interference (RNAi) is functional in mammalian cells led us to form The RNAi Consortium (TRC) with the goal of enabling large-scale loss-of-function screens through the development of genome-scale RNAi libraries and methodologies for their use. These resources form the basis of a loss-of-function screening platform created at the Broad Institute. Our human and mouse libraries currently contain >135,000 lentiviral clones targeting 27,000 genes. Initial screening efforts have demonstrated that these libraries and methods are practical and powerful tools for high-throughput lentivirus RNAi screens.

Animals↗
PubMed2005

RNAi-mediated pathways in the nucleus.

RNA interference (RNAi) is an evolutionarily conserved mechanism that uses short antisense RNAs that are generated by 'dicing' dsRNA precursors to target corresponding mRNAs for cleavage. However, recent developments have revealed that there is also extensive involvement of RNAi-related processes in regulation at the genome level. dsRNA and proteins of the RNAi machinery can direct epigenetic alterations to homologous DNA sequences to induce transcriptional gene silencing or, in extreme cases, DNA elimination. Furthermore, in some organisms RNAi silences unpaired DNA regions during meiosis. These mechanisms facilitate the directed silencing of specific genomic regions.

Animals↗
PubMed2006

Building mammalian signalling pathways with RNAi screens.

Technological advances in mammalian systems are providing new tools to identify the molecular components of signalling pathways. Foremost among these tools is the ability to knock down gene function through the use of RNA interference (RNAi). The fact that RNAi can be scaled up for use in high-throughput techniques has motivated the creation of genome-wide RNAi reagents. We are now at the brink of being able to harness the power of RNAi for large-scale functional discovery in mammalian cells.

Animals↗
PubMed2005

Specific and potent RNAi in the nucleus of human cells.

RNA interference (RNAi) has become a research tool to control gene expression in various organisms and holds potential as a new therapeutic strategy. The mechanism of small interfering RNA (siRNA)-mediated RNAi involves target mRNA cleavage and destruction in the cytoplasm. We investigated siRNA-mediated induction of RNAi in the nucleus of human cells. Notably, we observed highly efficient knockdown of small nuclear RNA 7SK by siRNA. siRNA- and microRNA-programmed RNA-induced silencing complexes (RISCs) were present in both cytoplasmic and nuclear compartments and specifically cleaved their perfectly matched target RNA with markedly high efficiencies. Our results provide the first evidence that human RISCs programmed with siRNA are present in the nucleus and can knock down target RNA levels. These studies reveal new roles for the RNAi machinery in modulating post-transcriptional gene expression in the nucleus.

Cell Nucleus↗
PubMed2002

Killing of leukemic cells with a BCR/ABL fusion gene by RNA interference (RNAi).

Short 21-mer double-stranded RNA (dsRNA) molecules have recently been employed for the sequence-specific silencing of endogenous human genes. This mechanism, called RNA interference (RNAi), is extremely potent and requires only a few dsRNA molecules per cell to silence homologous gene mRNA expression. We used dsRNA targeting the M-BCR/ABL fusion site to kill leukemic cells with such a rearrangement. Transfection of dsRNA specific for the M-BCR/ABL fusion mRNA into K562 cells depleted the corresponding mRNA and the M-BCR/ABL oncoprotein. This was demonstrated by real-time quantitative PCR and Western blots. The BCR/ABL knockdown was accompanied by strong induction of apoptotic cell death. Leukemic cells without BCR/ABL rearrangement were not killed by M-BCR/ABL-dsRNA. In addition, to corroborate the extraordinary sequence specificity of RNAi, we designed another RNA oligo matching the M-BCR/ABL fusion site but having two point mutations within its central region. We show that these two point mutations abolished both p210 reduction and induction of apoptosis in K562 cells. Finally, we compared leukemic cell killing by RNAi to that caused by the ABL kinase tyrosine inhibitor, STI 571, Imatinib. For full induction of apoptosis, dsRNA targeting M-BCR/ABL required 24 h more than Imatinib. This may be caused by the relatively long half-life of the BCR/ABL oncoprotein, which is not targeted by the RNAi mechanism, but is affected by STI 571. When we applied ds M-BCR/ABL RNA and STI 571 in combination, we did not observe a further increase in the induction of apoptosis. Nevertheless, these data may open a field for further studies towards gene-therapeutic approaches using RNA interference to kill tumor cells with specific genetic abnormalities.

Apoptosis↗
PubMed2003

A chemical-regulated inducible RNAi system in plants.

Constitutive expression of an intron-containing self-complementary 'hairpin' RNA (ihpRNA) has recently been shown to efficiently silence target genes in transgenic plants. However, this technique cannot be applied to genes whose silencing may block plant regeneration or result in embryo lethality. To obviate these potential problems, we have used a chemical-inducible Cre/loxP (CLX) recombination system to trigger the expression of an intron-containing inverted-repeat RNA (RNAi) in plants. A detailed characterization of the inducible RNAi system in transgenic Arabidopsis thaliana and Nicotiana benthamiana plants demonstrated that this system is stringently controlled. Moreover, it can be used to induce silencing of both transgenes and endogenous genes at different developmental stages and at high efficiency and without any detectable secondary affects. In addition to inducing complete silencing, the RNAi can be produced at various times after germination to initiate and obtain different degrees of gene silencing. Upon induction, transgenic plants with genetic chimera were obtained as demonstrated by PCR analysis. Such chimeric plants may provide a useful system to study signaling mechanisms of gene silencing in Arabidopsis as well as other cases of long-distance signaling without grafting. The merits of using the inducible CLX system for RNAi expression are discussed.

Arabidopsis↗
PubMed2003

Selective silencing by RNAi of a dominant allele that causes amyotrophic lateral sclerosis.

RNA interference (RNAi) can achieve sequence-selective inactivation of gene expression in a wide variety of eukaryotes by introducing double-stranded RNA corresponding to the target gene. Here we explore the potential of RNAi as a therapy for amyotrophic lateral sclerosis (ALS) caused by mutations in the Cu, Zn superoxide dismutase (SOD1) gene. Although the mutant SOD1 is toxic, the wild-type SOD1 performs important functions. Therefore, the ideal therapeutic strategy should be to selectively inhibit the mutant, but not the wild-type SOD1 expression. Because most SOD1 mutations are single nucleotide changes, to selectively silence the mutant requires single-nucleotide specificity. By coupling rational design of small interfering RNAs (siRNAs) with their validation in RNAi reactions in vitro and in vivo, we have identified siRNA sequences with this specificity. A similarly designed sequence, when expressed as small hairpin RNA (shRNA) under the control of an RNA polymerase III (pol III) promoter, retains the single-nucleotide specificity. Thus, RNAi is a promising therapy for ALS and other disorders caused by dominant, gain-of-function gene mutations.

Alleles↗