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PubMed2005

The RISC subunit Tudor-SN binds to hyper-edited double-stranded RNA and promotes its cleavage.

Long perfect double-stranded RNA (dsRNA) molecules play a role in various cellular pathways. dsRNA may undergo extensive covalent modification (hyper-editing) by adenosine deaminases that act on RNA (ADARs), resulting in conversion of up to 50% of adenosine residues to inosine (I). Alternatively, dsRNA may trigger RNA interference (RNAi), resulting in silencing of the cognate mRNA. These two pathways have previously been shown to be antagonistic. We show a novel interaction between components of the ADAR and RNAi pathways. Tudor staphylococcal nuclease (Tudor-SN) is a subunit of the RNA-induced silencing complex, which is central to the mechanism of RNAi. Here we show that Tudor-SN specifically interacts with and promotes cleavage of model hyper-edited dsRNA substrates containing multiple I.U and U.I pairs. This interaction suggests a novel unsuspected interplay between the two pathways that is more complex than mutual antagonism.

Adenosine Deaminase↗
PubMed2003

Small RNA: can RNA interference be exploited for therapy?

CONTEXT: RNA interference (RNAi) is the sequence-specific gene-silencing induced by double-stranded RNA (dsRNA), and gives information about gene function quickly, easily, and inexpensively. The use of RNAi for genetic-based therapies is widely studied, especially in viral infections, cancers, and inherited genetic disorders. RNAi has been used to make tissue-specific knockdown mice for studying gene function in a whole animal. Combined with genomics data, RNAi-directed gene-silencing could allow functional determination of any gene expressed in a cell or pathway. The term RNAi came from the discovery that the injection of dsRNAs into Caenorhabditis elegans interferes with the expression of specific genes containing a complementary region to the delivered dsRNA. Although stalled for a time by the non-gene-specific interferon response elicited by dsRNA molecules longer than about 30 nucleotides in mammalian cells, Tom Tuschl's group found that transfection of synthetic 21-nucleotide small-interfering RNA (siRNA) duplexes were highly selective and sequence-specific inhibitors of endogenous genes. STARTING POINT: siRNA expression has been studied with siRNA from plasmid and viral vectors that efficiently deliver siRNAs into both dividing and non-dividing cells, stem cells, zygotes, and their differentiated progeny. A collection of RNA interference vectors that suppress 50 human de-ubiquitinating enzymes allowed Thijn Brummelkamp and colleagues to study this gene family and to identify de-ubiquitinating enzymes in cancer-relevant pathways (Nature 2003; 424: 797-801). These researchers found that the familial cylindromatosis tumour suppressor gene (CYLD), previously of unknown function, could enhance the activation of the transcription factor NF-kappaB, leading to increased resistance to apoptosis. They have now started to investigate the use of CYLD inhibitors in clinical trials. WHERE NEXT: The ability to efficiently and stably produce and deliver sufficient amounts of siRNA to the proper target tissues require refinement before this new technology can be tried clinically. Initial in-vivo studies reported effective transgene suppression in adult mice by chemically synthesised siRNAs. More recently many researchers have used plasmid and viral vectors for transcription of short-hairpin RNAs, both in vitro and in vivo. With these expression systems, gene expression was more stably inhibited than with the transient knockdown recorded with chemically synthesised siRNA. Human trials exploiting these latest findings are likely to soon follow.

Animals↗
PubMed2004

RNA interference in neuroscience.

One of the most exciting findings in recent years has been the discovery of RNA interference (RNAi). RNAi is an important system that allows sequence-specific gene silencing through targeted degradation of mRNA by cognate double-stranded RNA (dsRNA). RNAi plays a role in endogenous cellular processes, such as developmental control and heterochromatin formation, and serves as an antiviral defense mechanism. Recent findings suggest that RNAi and related pathways are involved in protecting the genome against instabilities caused by transposons and repetitive sequences. Several rapidly developing RNAi methodologies provide a new approach for elucidation of gene functions. RNAi technology is also currently being evaluated as a potentially useful method to develop highly specific dsRNA-based gene-silencing therapeutics. In this paper, we review the use of RNAi in neuroscience research and as a possible therapeutic tool for treatment of neurological diseases.

Animals↗
PubMed2006

An unusual Dicer-like1 protein fuels the RNA interference pathway in Trypanosoma brucei.

RNA interference (RNAi) is an evolutionarily conserved gene-silencing pathway that is triggered by double-stranded RNA (dsRNA). Central to this pathway are two ribonucleases: Dicer, a multidomain RNase III family enzyme that initiates RNAi by generating small interfering RNAs (siRNAs), and Argonaute or Slicer, an RNase H signature enzyme that affects cleavage of mRNA. Previous studies in the early diverging protozoan Trypanosoma brucei have established a key role for Argonaute 1 in RNAi. However, the identity of Dicer has not been resolved. Here, we report the identification and functional characterization of a T. brucei Dicer-like enzyme (TbDcl1). Using genetic and biochemical approaches, we provide evidence that TbDcl1 is required for the generation of siRNA-size molecules and for RNAi. Whereas Dicer and Dicer-like proteins are endowed with two adjacent RNase III domains at the carboxyl terminus (RNase IIIa and RNase IIIb), the arrangement of these two domains is unusual in TbDcl1. RNase IIIa is close to the amino terminus, and RNase IIIb is located approximately in the center of the molecule. This domain organization is specific to trypanosomatids and further illustrates the variable structures of protozoan Dicer-like proteins as compared to fungal and metazoan Dicer.

Amino Acid Sequence↗
PubMed2001

RNA interference of gene expression (RNAi) in cultured Drosophila cells.

RNA interference (RNAi) can be used to silence genes in a number of taxa, including plants, nematodes, protozoans, flies, and mammals represented by mouse embryos and cultured mammalian cells. To investigate signal transduction pathways, we used RNAi on Drosophila-cultured cells, which affords the opportunity to study protein function in a simple, well-defined cell culture system. Furthermore, the results obtained from experiments performed on cultured cells can be confirmed and extended in the whole organism, which, in the case of Drosophila, is also RNAi responsive. RNAi takes advantage of the unique ability of double-stranded RNA (dsRNA) molecules to induce posttranscriptional gene silencing in a highly specific manner. This silencing is efficacious and long-lived, as it is passed to subsequent generations in insect cell culture. To date, all Drosophila cell lines tested (S2, KC, BG2-C6, and Shi) respond to dsRNAs by ablating expression of the target protein. Furthermore, all dsRNAs tested (more than 15) have been effective at silencing the target gene. Drosophila cell cultures are simple, easily manipulated model systems that will facilitate loss-of-function studies applicable to a wide variety of questions.

Animals↗
PubMed2003

Signaling from Akt to FRAP/TOR targets both 4E-BP and S6K in Drosophila melanogaster.

The eIF4E-binding proteins (4E-BPs) interact with translation initiation factor 4E to inhibit translation. Their binding to eIF4E is reversed by phosphorylation of several key Ser/Thr residues. In Drosophila, S6 kinase (dS6K) and a single 4E-BP (d4E-BP) are phosphorylated via the insulin and target of rapamycin (TOR) signaling pathways. Although S6K phosphorylation is independent of phosphoinositide 3-OH kinase (PI3K) and serine/threonine protein kinase Akt, that of 4E-BP is dependent on PI3K and Akt. This difference prompted us to examine the regulation of d4E-BP in greater detail. Analysis of d4E-BP phosphorylation using site-directed mutagenesis and isoelectric focusing-sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the regulatory interplay between Thr37 and Thr46 of d4E-BP is conserved in flies and that phosphorylation of Thr46 is the major phosphorylation event that regulates d4E-BP activity. We used RNA interference (RNAi) to target components of the PI3K, Akt, and TOR pathways. RNAi experiments directed at components of the insulin and TOR signaling cascades show that d4E-BP is phosphorylated in a PI3K- and Akt-dependent manner. Surprisingly, RNAi of dAkt also affected insulin-stimulated phosphorylation of dS6K, indicating that dAkt may also play a role in dS6K phosphorylation.

3-Phosphoinositide-Dependent Protein Kinases↗
PubMed2006

The endocytic pathway mediates cell entry of dsRNA to induce RNAi silencing.

Many metazoan cells can take up exogenous double-stranded (ds) RNA and use it to initiate an RNA silencing response, however, the mechanism for this uptake is ill-defined. Here, we identify the pathway for dsRNA uptake in Drosophila melanogaster S2 cells. Biochemical and cell biological analyses, and a genome-wide screen for components of the dsRNA-uptake machinery, indicated that dsRNA is taken up by an active process involving receptor-mediated endocytosis. Pharmacological inhibition of endocytic pathways disrupted exogenous dsRNA entry and the induction of gene silencing. This dsRNA uptake mechanism seems to be evolutionarily conserved, as knockdown of orthologues in Caenorhabditis elegans inactivated the RNA interference response in worms. Thus, this entry pathway is required for systemic RNA silencing in whole organisms. In Drosophila cells, pharmacological evidence suggests that dsRNA entry is mediated by pattern-recognition receptors. The possible role of these receptors in dsRNA entry may link RNA interference (RNAi) silencing to other innate immune responses.

Animals↗
PubMed2005

La Crosse virus nonstructural protein NSs counteracts the effects of short interfering RNA.

Through a process known as RNA interference (RNAi), double-stranded short interfering RNAs (siRNAs) silence gene expression in a sequence-specific manner. Recently, several viral proteins, including the nonstructural protein NSs of tomato spotted wilt virus (a plant-infecting bunyavirus), the interferon antagonist protein NS1 of influenza virus, and the E3L protein of vaccinia virus, have been shown to function as suppressors of RNAi, presumably as a counterdefense against cellular mechanisms that decrease viral production. La Crosse virus (LACV), a member of the California serogroup of orthobunyaviruses, has a trisegmented negative-stranded genome comprised of large (L), medium (M), and small (S) segments. To develop a strategy for segment-specific inhibition of transcription, we designed 13 synthetic siRNAs targeting specific RNA segments of the LACV genome that decreased LACV replication and antigen expression in mammalian (293T) and insect (C6/36) cells. Furthermore, NSs, a LACV nonstructural protein, markedly inhibited RNAi directed both against an LACV M segment construct and against a host gene (glyeraldehyde-3-phosphate dehydrogenase), suggesting a possible role for this viral protein in the suppression of RNA silencing. Segment-specific siRNAs will be useful as a tool to analyze LACV transcription and replication and to obtain recombinant viruses. Additionally, NSs will help us to identify molecular pathways involved in RNAi and further define its role in the innate immune system.

Animals↗
PubMed2005

Molecular mechanisms of protein kinase C-induced apoptosis in prostate cancer cells.

Protein kinase C (PKC) isozymes, a family of serine-threonine kinases, are important regulators of cell proliferation and malignant transformation. Phorbol esters, the prototype PKC activators, cause PKC translocation to the plasma membrane in prostate cancer cells, and trigger an apoptotic response. Studies in recent years have determined that each member of the PKC family exerts different effects on apoptotic or survival pathways. PKCdelta, one of the novel PKCs, is a key player of the apoptotic response via the activation of the p38 MAPK pathway. Studies using RNAi revealed that depletion of PKCdelta totally abolishes the apoptotic effect of the phorbol ester PMA. Activation of the classical PKCalpha promotes the dephosphorylation and inactivation of the survival kinase Akt. Studies have assigned a pro-survival role to PKCepsilon, but the function of this PKC isozyme remains controversial. Recently, it has been determined that the PKC apoptotic effect in androgen-dependent prostate cancer cells is mediated by the autocrine secretion of death factors. PKCdelta stimulates the release of TNFalpha from the plasma membrane, and blockade of TNFalpha secretion or TNFalpha receptors abrogates the apoptotic response of PMA. Molecular analysis indicates the requirement of the extrinsic apoptotic cascade via the activation of death receptors and caspase-8. Dissecting the pathways downstream of PKC isozymes represents a major challenge to understanding the molecular basis of phorbol ester-induced apoptosis.

Androgens↗
PubMed2005

Two novel proteins, dos1 and dos2, interact with rik1 to regulate heterochromatic RNA interference and histone modification.

BACKGROUND: Chromosomal behavior during mitosis and meiosis depends in part on heterochromatic modifications such as histone H3 lysine-9 methylation (H3K9me). In fission yeast, the Heterochromatin Protein 1 homolog Swi6 recognizes H3K9me, silences transcription, and retains cohesin at pericentromeric repeats. Heterochromatin formation also depends on processing of transcripts derived from centromeric repeats by the RNAi machinery. The DDB1 homolog, Rik1, and histone methyltransferase, Clr4, act in a complex to promote H3K9me. However, the mechanism underlying this interaction is poorly understood. RESULTS: Using a cytological screen, we have identified two novel genes, dos1(+) and dos2(+), which are required for localization of Swi6. Deletion of either of these genes results in mitotic and meiotic chromosome missegregation, defects in mitotic centromeric cohesion and meiotic telomere clustering, and loss of heterochromatic silencing. Dos1 is predominantly located in the nucleus in a Dos2-dependent manner and directly interacts with Rik1. Each of these genes is required for the association of H3K9me with centromeric repeats, as well as for the production of small interfering RNAs. CONCLUSIONS: Dos1 and Dos2 are required for the formation of heterochromatin in fission yeast. We hypothesize that the physical interaction between Dos1 and Rik1 represents a role in regulating activity of the Rik1/Clr4 complex. Dos2 contributes to this role by regulating Dos1 localization. Our findings suggest a mechanism for recruitment of Clr4 in the RNAi-dependent heterochromatin pathway, in which Dos1 and Dos2 are essential.

Carrier Proteins↗
PubMed2006

The T-box factor TBX-2 and the SUMO conjugating enzyme UBC-9 are required for ABa-derived pharyngeal muscle in C. elegans.

The C. elegans pharynx is produced from the embryonic blastomeres ABa and MS. Pharyngeal fate in the ABa lineage is specified by the combined activities of GLP-1/Notch-mediated signals and the TBX-37 and TBX-38 T-box transcription factors. Here, we show another T-box factor TBX-2 also functions in ABa-derived pharyngeal development. tbx-2 mutants arrest as L1 larvae lacking most or all ABa-derived pharyngeal muscles. In comparison, tbx-2 mutants retain ABa-derived marginal cells and pharyngeal muscles derived from MS. A tbx-2Colon, two colonsgfp translational fusion is expressed in a dynamic pattern in C. elegans embryos beginning near the 100-cell stage. Early expression is limited to a small number of cells, which likely include the ABa-derived pharyngeal precursors, while later expression is observed in body wall muscles and a subset of pharyngeal neurons. TBX-2 contains 2 consensus sumoylation sites, and it interacts in a yeast two-hybrid assay with the UBC-9 and GEI-17 components of the C. elegans SUMO-conjugating pathway. ubc-9(RNAi) has been previously shown to cause variable embryonic and larval arrest, and we find that, like tbx-2 mutants, ubc-9(RNAi) animals lack ABa-derived pharyngeal muscles. ubc-9(RNAi) also alters the subnuclear distribution of TBX-2::GFP fusion protein, suggesting that UBC-9 and TBX-2 interact in C. elegans. Together, these results indicate that TBX-2 and SUMO-conjugating enzymes are necessary for ABa-derived pharyngeal muscle, and we hypothesize that TBX-2 function requires sumoylation. Sumoylation is increasingly recognized as an important mechanism controlling activity of many nuclear factors, and these results provide the first evidence that T-box factor activity may require sumoylation.

Animals↗
PubMed2003

RNAi: an ever-growing puzzle.

In recent years, sequence-specific gene silencing has been an area of increasing focus, both because of its interesting biology and because of its power as an experimental tool. A growing understanding of one such phenomenon, RNA interference (RNAi), has provided clues that many homology-dependent gene-silencing mechanisms share a common trigger, double-stranded RNA. Recent findings that RNAi and related pathways are involved not only in the response to exogenous pathogenic and endogenous parasitic nucleic acids but also in basic cellular processes, such as gene regulation and heterochromatin formation, have further fueled interest in this rapidly expanding field.

Animals↗
PubMed2006

Angiopoietin-2 functions as an autocrine protective factor in stressed endothelial cells.

Angiopoietin (Ang)-2, a context-dependent agonist/antagonist for the vascular-specific Tie2 receptor, is highly expressed by endothelial cells at sites of normal and pathologic angiogenesis. One prevailing model suggests that in these settings, Ang-2 acts as an autocrine Tie2 blocker, inhibiting the stabilizing influence of the Tie2 activator Ang-1, thereby promoting vascular remodeling. However, the effects of endogenous Ang-2 on cells that are actively producing it have not been studied in detail. Here, we demonstrate that Ang-2 expression is rapidly induced in endothelial cells by the transcription factor FOXO1 after inhibition of the phosphatidylinositol 3-kinase/Akt pathway. We employ RNAi and blocking antibodies to show that in this setting, Ang-2 unexpectedly functions as a Tie2 agonist, bolstering Akt activity so as to provide negative feedback on FOXO1-regulated transcription and apoptosis. In addition, we show that Ang-2, like Ang-1, activates Tie2/Akt signaling in vivo, thereby inhibiting the expression of FOXO1 target genes. Consistent with a role for Ang-2 as a Tie2 activator, we demonstrate that Ang-2 inhibits vascular leak. Our data suggests a model in which Ang-2 expression is induced in stressed endothelial cells, where it acts as an autocrine Tie2 agonist and protective factor.

Androstadienes↗
PubMed2006

RNA interference and antisense-mediated gene silencing in Dictyostelium.

Knockouts by homologous recombination are frequently used to investigate the function of genes in Dictyostelium and other organisms. Antisense-mediated gene silencing and RNA interference (RNAi) are convenient alternatives to reduce gene expression to different levels and to silence multigene families. We describe here the methods for efficient RNA interference in Dictyostelium and some useful mutant strains that enhance the success rate or may serve as convenient controls. We believe that it is helpful to also discuss failed attempts to optimize and expand the system because these are rarely discussed in the literature. In addition, a list of in silico and experimentally identified components in the RNAi and antisense pathway is presented.

Animals↗
PubMed2005

Single small-interfering RNA expression vector for silencing multiple transforming growth factor-beta pathway components.

Although RNA interference (RNAi) is a popular technique, no method for simultaneous silencing of multiple targets by small-hairpin RNA (shRNA)-expressing RNAi vectors has yet been established. Although gene silencing can be achieved by synthetic small-interfering RNA (siRNA) duplexes, the approach is transient and largely dependent on the transfection efficiency of the host cell. We offer a solution: a simple, restriction enzyme-generated stable RNAi technique that can efficiently silence multiple targets with a single RNAi vector and a single selection marker. In this study, we succeeded in simultaneous stable knockdown of transforming growth factor beta (TGF-beta) pathway-related Smads--Smad2, Smad3 and Smad4--at the cellular level. We observed distinct phenotypic changes in TGF-beta-dependent cellular functions such as invasion, wound healing and apoptosis. This method is best suited for an analysis of complex signal transduction pathways in which silencing of a single gene cannot account for the whole process.

Cell Line↗
PubMed2005

Somatic misexpression of germline P granules and enhanced RNA interference in retinoblastoma pathway mutants.

Caenorhabditis elegans homologues of the retinoblastoma (Rb) tumour suppressor complex specify cell lineage during development. Here we show that mutations in Rb pathway components enhance RNA interference (RNAi) and cause somatic cells to express genes and elaborate perinuclear structures normally limited to germline-specific P granules. Furthermore, particular gene inactivations that disrupt RNAi reverse the cell lineage transformations of Rb pathway mutants. These findings suggest that mutations in Rb pathway components cause cells to revert to patterns of gene expression normally restricted to germ cells. Rb may act by a similar mechanism to transform mammalian cells.

Alleles↗
PubMed2006

Knockdown of berberine bridge enzyme by RNAi accumulates (S)-reticuline and activates a silent pathway in cultured California poppy cells.

Reticuline is a key compound in the biosynthetic pathway for isoquinoline alkaloids in plants, which include morphine, codeine and berberine. We established cultured California poppy (Eschscholzia californica) cells, in which berberine bridge enzyme (BBE) was knocked down by RNA interference, to accumulate the important key intermediate reticuline. Both BBE mRNA accumulation and enzyme activity were effectively suppressed in transgenic cells. In these transgenic cells, end-products of isoquinoline alkaloid biosynthesis, such as sanguinarine, were considerably reduced and reticuline was accumulated at a maximum level of 310 mug/g-fresh weight. In addition, 1 g-fresh weight of these cells secreted significant amounts of reticuline into the medium, with a maximum level of 6 mg/20 mL culture medium. These cells also produced a methylated derivative of reticuline, laudanine, which could scarcely be detected in control cells. We discuss the potential application of RNAi technology in metabolic modification and the flexibility of plant secondary metabolism.

Alkaloids↗
PubMed2006

Transient depletion of Ku70 and Xrcc4 by RNAi as a means to manipulate the non-homologous end-joining pathway.

Non-homologous end joining (NHEJ) is the major DNA double-strand break (DSB) repair pathway in mammalian cells and is likely responsible for the non-homologous integration of transgenes. In higher eukaryotes, this pathway predominates over the homologous recombination (HR) pathway and therefore may account for the low level of HR events that occur in mammalian cells. We evaluated the effects of transient RNAi-induced down-regulation of key components of the NHEJ pathway in human HCT116 cells. Treatment with siRNA targeting Ku70 and Xrcc4 reduced corresponding protein levels by 80-90% 48h after transfection, with a return to normal levels by 96h. Additionally, down-regulation of Ku70 and Xrcc4 resulted in a concomitant depletion of both Ku70 and Ku86 proteins. Biological consequences of transient RNAi-mediated depletion of Ku70 and Xrcc4 included sensitization to gamma radiation and a significant decrease in the expression of a linear GFP reporter gene. The results highlight the possibility of a successful means to manipulate the NHEJ pathway by RNAi.

Antigens, Nuclear↗