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PubMed2003

Farnesyl pyrophosphate synthase is an essential enzyme in Trypanosoma brucei. In vitro RNA interference and in vivo inhibition studies.

We report the cloning and sequencing of a gene encoding the farnesyl pyrophosphate synthase (FPPS) of Trypanosoma brucei. The protein (TbFPPS) is an attractive target for drug development because the growth of T. brucei has been shown to be inhibited by analogs of its substrates, the nitrogen containing bisphosphonates currently in use in bone resorption therapy. The protein predicted from the nucleotide sequence of the gene has 367 amino acids and a molecular mass of 42 kDa. Several sequence motifs found in other FPPSs are present in TbFPPS, including an 11-mer peptide insertion present also in the Trypanosoma cruzi FPPS. Heterologous expression of TbFPPS in Escherichia coli produced a functional enzyme that was inhibited by several nitrogen-containing bisphosphonates, such as pamidronate and risedronate. Risedronate was active in vivo against T. brucei infection in mice (giving a 60% survival rate), but pamidronate was not effective. The essential nature of TbFPPS was studied using RNA interference (RNAi) to inhibit the expression of the gene. Expression of TbFPPS double-stranded RNA in procyclic trypomastigotes caused specific degradation of mRNA. After 4 days of RNAi, the parasite growth rate declined and the cells subsequently died. Similar results were obtained with bloodstream form trypomastigotes, except that the RNAi system in this case was leaky and mRNA levels and parasites recovered with time. Molecular modeling and structure-activity investigations of enzyme and in vitro growth inhibition data resulted in similar pharmacophores, further validating TbFPPS as the target for bisphosphonates. These results establish that FPPS is essential for parasite viability and validate this enzyme as a target for drug development.

Alkyl and Aryl Transferases↗
PubMed2001

RNA interference is mediated by 21- and 22-nucleotide RNAs.

Double-stranded RNA (dsRNA) induces sequence-specific posttranscriptional gene silencing in many organisms by a process known as RNA interference (RNAi). Using a Drosophila in vitro system, we demonstrate that 21- and 22-nt RNA fragments are the sequence-specific mediators of RNAi. The short interfering RNAs (siRNAs) are generated by an RNase III-like processing reaction from long dsRNA. Chemically synthesized siRNA duplexes with overhanging 3' ends mediate efficient target RNA cleavage in the lysate, and the cleavage site is located near the center of the region spanned by the guiding siRNA. Furthermore, we provide evidence that the direction of dsRNA processing determines whether sense or antisense target RNA can be cleaved by the siRNA-protein complex.

Animals↗
PubMed2006

Development and validation of vectors containing multiple siRNA expression cassettes for maximizing the efficiency of gene silencing.

BACKGROUND: RNA interference (RNAi) was originally identified as a biological process in which short double-stranded RNA (dsRNA) suppress the expression of genes complimentary to the dsRNA. This cellular intrinsic gene silencing mechanism has subsequently been developed as a useful tool for studies of gene function. A major strategy for producing small interfering RNA (siRNA) in cultured cells involves the use of siRNA expression vectors in which a RNA polymerase III (Pol III) promoter and transcription stop signal are designed to constitute a functional siRNA expression cassette for production of siRNA. However, most of the available vectors contain only one siRNA expression cassette. RESULTS: In order to maximize the efficiency and versatility of the vector-based siRNA approach, we have developed vectors containing multiple (up to six) tandem siRNA expression cassettes. Moreover, we demonstrated that these vectors can be used not only to produce different siRNA to simultaneously suppress the expression of multiple genes but also to maximize the silencing of a single gene. CONCLUSION: The vectors containing multiple siRNA expression cassettes can serve as useful tools for maximizing the efficiency of gene silencing.

Base Sequence↗
PubMed2005

The diagnosis and therapy of brain tumours.

Neoplasms arising from glial cells make up the most common group of primary brain tumors. The clinical outcome, especially the survival rates of the patients with brain tumours depend on tumour grade expressing its malignancy. A prognosis for glioblastomas (WHO IV) is very poor, but for astrocytomas (WHO I and II) it is relatively favourable. For oligodendrogliomas a longer survival time than for glioblastomas is observed. There is evidence that oxidative stress and reactive oxygen species (ROS) are crucial in the etiology and progression of a number of human diseases, including neoplasms. An oxidative damage of DNA, lipids and proteins is caused mainly with hydroxyl radical (*OH), the most reactive ROS species and may be seriously deleterious. In addition to all four basic nucleotides: adenosine (A), guanosine (G), tymidine (T) and cytosine (C), 5-methylcytosine (m5C) is a rare but normal component of cellular DNA and occurs mainly within a sequence of a structural gene or in regulatory regions. In the reaction with hydroxyl radical all DNA components can be modified, but m5C is relatively easily deaminated to thymine, which, in turn, pairs with adenine and after a round of replication, CG to TA transition occurs. Because thymine is a normal DNA base, therefore the product of spontaneous deamination of m5C is not so easily detected by cell's DNA repair system. Thus, 5-methylcytosine residue constitutes a mutational hotspot and DNA methylation pattern in patients might be useful as a primary diagnostic tool or as a marker for early detection of relapse of the disease. In recent years a new mechanism of posttranscriptional gene silencing has been discovered and named RNA interference (RNAi). This phenomenon is based on mRNA degradation mediated by small double-stranded RNA molecules, approximately 19-28 nucleotides in length, called short interfering RNAs (siRNAs). These molecules are produced from long dsRNAs by a dsRNA-specific endonuclease (DICER) and form 300 kD multi-enzyme complex (RISC) which by Watson-Crick base-pairing of noncoding strand with their mRNA-targets induce specific degradation. The high sequence-specificity of RNAi makes it a new, promising tool in a gene-function analysis as well as in potential therapeutics development.

Animals↗
PubMed2004

RNA interference: from biology to drugs and therapeutics.

RNA interference (RNAi) is a newly discovered and popular technology platform among researchers not only in the fields of RNA biology and molecular cell biology. It has created excitement in clinical sciences such as oncology, neurology, endocrinology, infectious diseases and drug discovery. There is an urgent need to educate and connect academic and industry researchers for the purpose of knowledge transfer. Thus, GeneExpression Systems of Waltham organized its Second International Conference in Waltham City (May 2-4, 2004, MA, USA) on the theme of 'RNA interference: From Biology to Drugs & Therapeutics.' About 200 participants and 32 speakers attended this two and half-day event which was arranged in six scientific and three technology sessions and ended with a panel discussion. This report covers a few representative talks from academia, biotech and the drug industry.

Animals↗
PubMed2001

Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate.

Duplexes of 21-23 nucleotide (nt) RNAs are the sequence-specific mediators of RNA interference (RNAi) and post-transcriptional gene silencing (PTGS). Synthetic, short interfering RNAs (siRNAs) were examined in Drosophila melanogaster embryo lysate for their requirements regarding length, structure, chemical composition and sequence in order to mediate efficient RNAi. Duplexes of 21 nt siRNAs with 2 nt 3' overhangs were the most efficient triggers of sequence-specific mRNA degradation. Substitution of one or both siRNA strands by 2'-deoxy or 2'-O-methyl oligonucleotides abolished RNAi, although multiple 2'-deoxynucleotide substitutions at the 3' end of siRNAs were tolerated. The target recognition process is highly sequence specific, but not all positions of a siRNA contribute equally to target recognition; mismatches in the centre of the siRNA duplex prevent target RNA cleavage. The position of the cleavage site in the target RNA is defined by the 5' end of the guide siRNA rather than its 3' end. These results provide a rational basis for the design of siRNAs in future gene targeting experiments.

Animals↗
PubMed2004

Those interfering little RNAs! Silencing and eliminating chromatin.

RNA interference (RNAi) is widely used for knocking down expression of genes of interest and in systematic screens for desired phenotypes. In post-transcriptional gene silencing, double-stranded RNA triggers are processed to small interfering RNAs, which act to seek out and destroy homologous transcripts. A variety of organisms utilise the RNAi pathway to silence expression of potentially harmful endogenous mobile elements and to eliminate unnecessary sequences. In plants and fission yeast, RNAi can also mediate chromatin-based silencing resulting in transcriptional shutdown of homologous transcription units (transcriptional gene silencing) and the formation of centromeric heterochromatin. In metazoans, the expression of non-coding RNAs is often associated with the formation of silent chromatin domains but it remains to be determined if RNAi is involved.

Animals↗
PubMed2006

ATP-binding cassette transporters are required for efficient RNA interference in Caenorhabditis elegans.

RNA interference (RNAi) is a conserved gene-silencing phenomenon that can be triggered by delivery of double-stranded RNA (dsRNA) to cells and is a widely exploited technology in analyses of gene function. Although a number of proteins that facilitate RNAi have been identified, current descriptions of RNAi and interrelated mechanisms are far from complete. Here, we report that the Caenorhabditis elegans gene haf-6 is required for efficient RNAi. HAF-6 is a member of the ATP-binding cassette (ABC) transporter gene superfamily. ABC transporters use ATP to translocate small molecule substrates across the membranes in which they reside, often against a steep concentration gradient. Collectively, ABC transporters are involved in a variety of activities, including protective or barrier mechanisms that export drugs or toxins from cells, organellar biogenesis, and mechanisms that protect against viral infection. HAF-6 is expressed predominantly in the intestine and germline and is localized to intracellular reticular organelles. We further demonstrate that eight additional ABC genes from diverse subfamilies are each required for efficient RNAi in C. elegans. Thus, the ability to mount a robust RNAi response to dsRNA depends upon the deployment of two ancient systems that respond to environmental assaults: RNAi mechanisms and membrane transport systems that use ABC proteins.

ATP-Binding Cassette Transporters↗
PubMed2006

RNAi technology: a revolutionary tool for the colorectal cancer therapeutics.

With the many changes that have taken place in people's diet and lifestyle, colorectal cancer (CRC) has become a global concern. There were approximately 950000 new cases diagnosed and 500000 deaths recorded worldwide in 2000. It is the second most common type of cancer in the Western world, and it is the third most common type of digestive tumor in China. It is reported that the morbidity of CRC is 4.08/100000 for men and 3.30/100000 for women in China. Despite the rate of improvements in surgery, radiotherapy and chemotherapy, the overall five-year survival is around 50%. Therefore, novel treatment need to be developed in order to add to the therapeutic armamentarium. RNA interference (RNAi) is a sequence-specific post-transcriptional gene silencing mechanism, which is triggered by double-stranded RNA (dsRNA) and causes degradation of mRNA homologous in sequence to the dsRNA. This new approach has been successfully adopted to inhibit virus replication and tumorigenicity. Recent reports have described DNA vector-based strategies for delivery of small interfering RNA (siRNA) into mammalian cells, further expanding the utility of RNAi. With the development of the RNAi technology and deeper understanding of this field, a promising new modality of treatment appeared, which can be used in combination with the existing therapies. We reviewed the proceedings on the actualities and advancement of RNAi technology for colorectal cancer therapeutics.

Animals↗
PubMed2006

Robust statistical methods for hit selection in RNA interference high-throughput screening experiments.

RNA interference (RNAi) high-throughput screening (HTS) experiments carried out using large (>5000 short interfering [si]RNA) libraries generate a huge amount of data. In order to use these data to identify the most effective siRNAs tested, it is critical to adopt and develop appropriate statistical methods. To address the questions in hit selection of RNAi HTS, we proposed a quartile-based method which is robust to outliers, true hits and nonsymmetrical data. We compared it with the more traditional tests, mean +/- k standard deviation (SD) and median +/- 3 median of absolute deviation (MAD). The results suggested that the quartile-based method selected more hits than mean +/- k SD under the same preset error rate. The number of hits selected by median +/- k MAD was close to that by the quartile-based method. Further analysis suggested that the quartile-based method had the greatest power in detecting true hits, especially weak or moderate true hits. Our investigation also suggested that platewise analysis (determining effective siRNAs on a plate-by-plate basis) can adjust for systematic errors in different plates, while an experimentwise analysis, in which effective siRNAs are identified in an analysis of the entire experiment, cannot. However, experimentwise analysis may detect a cluster of true positive hits placed together in one or several plates, while platewise analysis may not. To display hit selection results, we designed a specific figure called a plate-well series plot. We thus suggest the following strategy for hit selection in RNAi HTS experiments. First, choose the quartile-based method, or median +/- k MAD, for identifying effective siRNAs. Second, perform the chosen method experimentwise on transformed/normalized data, such as percentage inhibition, to check the possibility of hit clusters. If a cluster of selected hits are observed, repeat the analysis based on untransformed data to determine whether the cluster is due to an artifact in the data. If no clusters of hits are observed, select hits by performing platewise analysis on transformed data. Third, adopt the plate-well series plot to visualize both the data and the hit selection results, as well as to check for artifacts.

Cluster Analysis↗
PubMed2006

Tissue-specific RNAi reveals that WT1 expression in nurse cells controls germ cell survival and spermatogenesis.

Using a novel tissue-specific RNA interference (RNAi) approach that mimics the principle by which naturally occurring microRNAs (miRNA) are made, we demonstrate that the Wilms' tumor 1 (WT1) transcription factor has an essential role in spermatogenesis. Mice depleted of WT1 in Sertoli nurse cells suffered from increased germ cell apoptosis, loss of adherens junctions, disregulation of adherence junction-associated genes, and impaired fertility. These effects were recapitulated in transgenic mice expressing a dominant-negative form of WT1 in Sertoli cells, demonstrating the validity of our RNAi approach. Our results indicate that the tumor suppressor WT1 promotes Sertoli cell-germ cell signaling events driving spermatogenesis.

Adherens Junctions↗
PubMed2005

RNAi-induced silencing of embryonic tryptophan oxygenase in the Pyralid moth, Plodia interpunctella.

Gene silencing through the introduction of double-stranded RNA (RNA interference, RNAi) provides a powerful tool for the elucidation of gene function in many systems, including those where genomics and proteomics are incomplete. The use of RNAi technology for gene silencing in Lepidoptera has lacked significant attention compared to other systems. To demonstrate that RNAi can be utilized in the lepidopteran, Plodia interpunctella, we cloned a cDNA for tryptophan oxygenase, and showed that silencing of tryptophan oxygenase through RNAi during embryonic development resulted in loss of eye-color pigmentation. The complete amino acid sequence of Plodia tryptophan oxygenase can be accessed through NCBI Protein Database.

Amino Acid Sequence↗
PubMed2000

The efficacy of RNAi in the study of the plant cytoskeleton.

Recent studies on a variety of organisms point to the ubiquity of RNA interference (RNAi) as a means to induce a gene-specific block to translation. RNAi has gained popularity in the last few years in the study of a number of problems in development. In this review, we highlight recent findings with RNAi using several different kinds of animals and fungi, and we show how these responses parallel cosuppression effects described in plants nearly a decade earlier. We then point to the efficacy of RNAi in studying minor and regulatory components of the plant cytoskeleton, and we highlight some recent studies using this approach with the water fern, Marsilea vestita.

Actins↗
PubMed2004

The RNA interference proteins and vasa locus are involved in the silencing of retrotransposons in the female germline of Drosophila melanogaster.

RNA interference (RNAi) is considered as a defense against expansion of transposable elements. The proteins related to RNA helicase and Argonaute families are involved in RNAi process in different organisms. It was shown that Argonaute AUBERGINE and putative RNA helicase SPINDLE-E proteins were essential for RNAi in Drosophila. Here, we describe the role of aubergine (aub) and spindle-E (spn-E) genes in the control of LTR retrotransposon copia and nonLTR telomeric Het-A and I retrotransposons in ovaries. spn-E mutation causes a drastically increased lacZ expression driven by copia LTR. For the first time we show the involvement of AUBERGINE protein and VASA RNA helicase, essential for oocyte patterning, in the retrotransposon silencing. spn-E, vasa and aub mutations cause similar accumulation of both I element and Het-A transcripts in the developing oocyte. VASA and AUBERGINE proteins are known as components of perinuclear ribonucleoprotein particles in germ cells, and spn-E mutation disturbs protein content of the particles. We suggest participation of these proteins in the same silencing pathway.

Animals↗
PubMed2006

Interference of porcine reproductive and respiratory syndrome virus replication on MARC-145 cells using DNA-based short interfering RNAs.

Porcine reproductive and respiratory syndrome (PRRS) is an economically important disease in swine-producing areas of the world. Many vaccine strategies developed to control the disease are not yet completely successful. The objective of this study was to determine if RNA interference (RNAi) could be utilized to inhibit PRRSV replication on MARC-145 cells. Four short interfering RNA (siRNA) sequences (N95, N179, N218 and N294) directed against a well-conserved region of PRRSV genome ORF7 gene were selected. Sense and antisense siRNA encode sequences separated by a hairpin loop sequence were designed as short hairpin RNA (shRNA) expression cassettes driven by mouse U6 promoter. Using a polymerase chain reaction (PCR)-based approach, shRNAs were generated from shRNA expression cassettes. The PCR products were cloned into pEGFP-N1 vector and shRNA expression vectors were constructed. When MARC-145 cells were transfected with shRNA expression vectors and then infected with PRRSV, N179 was found to be the most effective inhibition site in decreasing cytopathic effect (CPE) induced by PRRSV. Western blot, indirect immunofluorescence and fluorescence quantitative PCR (FQ-PCR) confirmed that the expression of ORF7 was reduced both at protein and RNA levels comparing to controls. The results presented here indicated that DNA-based siRNA could effectively inhibit the replication of PRRS virus (approximately 681-fold reduction of viral titers) on MARC-145 cells.

Animals↗
PubMed2005

RNA interference spreading in C. elegans.

The phenomenon of RNA interference (RNAi) occurs in eukaryotic organisms from across the boundaries of taxonomic kingdoms. In all cases, the basic mechanism of RNAi appears to be conserved--an initial trigger [double-stranded RNA (dsRNA) containing perfect homology over at least 19-21/bp with an endogenous gene] is processed into short interfering RNA (siRNA) molecules and these siRNAs stimulate degradation of the homologous mRNA. In the vast majority of species, RNAi can only be initiated following the deliberate introduction of dsRNA into a cell by microinjection, electroporation, or transfection. However, in the nematode worm Caenorhabditis elegans, RNAi can be simply initiated by supplying dsRNA in the surrounding medium or in the diet. Following uptake, this dsRNA triggers a systemic effect, initiating RNAi against the corresponding target gene in tissues that are not in direct contact with the external milieu. This phenomenon of systemic RNAi, or RNAi spreading, is notably absent from mammalian species, a fact that is likely to prove a substantial barrier to the wider use of RNAi as a clinical therapy. An understanding of the mechanism of systemic RNAi is therefore of considerable importance, and several advances of the last few years have begun to shed light on this process. Here we review our current understanding of systemic RNAi in C. elegans and draw comparisons with systemic RNAi pathways in other organisms.

Animals↗
PubMed2004

A protein sensor for siRNA asymmetry.

To act as guides in the RNA interference (RNAi) pathway, small interfering RNAs (siRNAs) must be unwound into their component strands, then assembled with proteins to form the RNA-induced silencing complex (RISC), which catalyzes target messenger RNA cleavage. Thermodynamic differences in the base-pairing stabilities of the 5' ends of the two approximately 21-nucleotide siRNA strands determine which siRNA strand is assembled into the RISC. We show that in Drosophila, the orientation of the Dicer-2/R2D2 protein heterodimer on the siRNA duplex determines which siRNA strand associates with the core RISC protein Argonaute 2. R2D2 binds the siRNA end with the greatest double-stranded character, thereby orienting the heterodimer on the siRNA duplex. Strong R2D2 binding requires a 5'-phosphate on the siRNA strand that is excluded from the RISC. Thus, R2D2 is both a protein sensor for siRNA thermodynamic asymmetry and a licensing factor for entry of authentic siRNAs into the RNAi pathway.

Animals↗
PubMed2004

RNA interference as an antiviral approach: targeting HIV-1.

RNA interference (RNAi) is an evolutionary conserved gene-silencing mechanism in which 21- to 23-mer double-stranded short interfering RNA (siRNA) mediates the sequence-specific degradation of mRNA. The recent discovery that exogenously delivered siRNA can trigger RNAi in mammalian cells raises the possibility of using this technology as a therapeutic tool against pathogenic viruses. This review describes the antiviral RNAi field, which is barely two years-old, with an emphasis on recent studies aimed at suppression of HIV-1.

Animals↗