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PubMed2004

Function of the Trypanosome Argonaute 1 protein in RNA interference requires the N-terminal RGG domain and arginine 735 in the Piwi domain.

Argonaute proteins are central components of RNA interference (RNAi) and related phenomena in a wide variety of eukaryotes, including the early diverging protozoan Trypanosoma brucei. The single T. brucei Argonaute protein (TbAGO1) is in a complex with small interfering RNAs (siRNAs), and a fraction of this ribonucleoprotein particle is associated with polyribosomes. In this study, we generated a panel of insertion, deletion, and single point mutants of TbAGO1 and assayed them in vivo for their function in RNAi. In addition to the signature domains of Argonaute proteins, PAZ and Piwi, TbAGO1 has an N-terminal domain with a high abundance of RGG repeats. Deletion of the N-terminal domain blocked association of AGO1 with polyribosomes and severely affected mRNA cleavage. Nevertheless, the mutant protein was in a complex with siRNAs. In contrast, deletion of the Piwi domain led to a loss of siRNAs but did not abolish polyribosome association. Site-directed mutagenesis of conserved amino acids in the Piwi domain identified arginine 735 as essential for RNAi. Although the R735A mutant bound siRNAs and associated with polyribosomes, it displayed a severe defect in the cleavage of target mRNA.

Animals↗
PubMedDate not supplied

RNA interference and human disease.

The completion of the human genome project has left researchers searching for an efficient method to study gene function in mammalian cells. RNA interference (RNAi) is an evolutionarily conserved post-transcriptional gene silencing (PTGS) mechanism mediated by double-stranded RNA (dsRNA). The dsRNA is processed into small duplex RNA molecules of approximately 21-22 nucleotides (nts) termed small interfering RNAs (siRNAs) by a RNase III enzyme called Dicer. Interaction of siRNAs with a multi-protein complex, termed the RNA-induced silencing complex (RISC), results in sequence specific association of the activated RISC complex with the cognate RNA transcript. This interaction leads to sequence-specific cleavage of the target transcript. Originally discovered in Caenorhabditis elegans, the study of RNAi in mammalian cells has blossomed in the last couple of years with the discovery that introduction of siRNA molecules directly into somatic mammalian cells circumvents the non-specific response vertebrate cells have against larger dsRNA molecules. Emerging as a powerful tool for reverse genetic analysis, RNAi is rapidly being applied to study the function of many genes associated with human disease, in particular those associated with oncogenesis and infectious disease. This review summarizes the mechanism of RNAi and provides an overview of its current applications in medicine.

Animals↗
PubMed2005

Inhibition of multiple gene expression and virus replication of HBV by stable RNA interference in 2.2.15 cells.

BACKGROUND/AIMS: Hepatitis B virus (HBV) infection is a world-wide health problem. Recent studies have demonstrated the efficacy of RNA interference (RNAi) against HBV replication at cell culture and animal levels using transient transfection. The present study was to determine whether the stable transfection of short hairpin RNA (shRNA)-producing vector could achieve potent and sustained inhibition of the HBV replication in 2.2.15 cells. METHODS: shRNA-producing vector against HBV and the empty vector were stably transfected into the 2.2.15 cells respectively. A series of experiments were performed in the producing stable lines to determine the changes of viral protein expression and replication. RESULTS: The HBV protein expression and viral replication were suppressed dramatically and stably by the integrated shRNA-producing vectors. Most importantly, this suppression effect persists after 30 passages. CONCLUSIONS: Our data provided the possibility of continuous and stable inhibition of HBV protein expression and replication in patients using RNAi, suggesting a potential clinical application of this novel approach. Furthermore, the established stable transfected cell lines provided a good platform for understanding the mechanism of anti-HBV by RNAi.

Blotting, Northern↗
PubMed2005

Silencing the major apple allergen Mal d 1 by using the RNA interference approach.

BACKGROUND: Apple allergy is dominated by IgE antibodies against Mal d 1 in areas where birch pollen is endemic. Apples with significantly decreased levels of Mal d 1 would allow most patients in these areas to eat apples without allergic reactions. OBJECTIVE: The aim of this study was to inhibit the expression of Mal d 1 in apple plants by RNA interference. METHODS: In vitro -grown apple plantlets were transformed with a construct coding for an intron-spliced hairpin RNA containing a Mal d 1-specific inverted repeat sequence separated by a Mal d 1-specific intron sequence. The presence of the construct in transformants was checked by PCR. Expression of Mal d 1 in leaves was monitored by prick-to-prick skin testing in 3 patients allergic to apples and by immunoblotting with a Mal d 1-reactive mAb and with IgE antibodies against Mal d 1. RESULTS: After transformation, plantlets were selected on the basis of having a normal phenotype and growth rate. With PCR, in 6 of 9 selected plantlets, the presence of the gene-silencing construct was demonstrated. By skin prick test it was shown that a wild-type plantlet had significantly ( P < .05) higher allergenicity than 5 of the transformants. Reduction of expression of Mal d 1 was confirmed by immunoblotting. In wild-type and unsuccessful transformants, a strong band was detected with Mal d 1-reactive mAb 5H8 at the expected apparent M r of 17 kDa. This band was virtually absent in the transformants that carried the gene-silencing construct. With human IgE antibodies, the same observations were made. CONCLUSIONS: Mal d 1 expression was successfully reduced by RNA interference. This translated into significantly reduced in vivo allergenicity. These observations support the feasibility of the production by gene silencing of apples hypoallergenic for Mal d 1.

Adult↗
PubMed2004

Inhibition of epidermal growth factor receptor expression by RNA interference in A549 cells.

AIM: To investigate the biological features of A549 cells in which epidermal growth factor (EGF) receptors expression were suppressed by RNA interference (RNAi). METHODS: A549 cells were transfected using short small interfering RNAs (siRNAs) formulated with Lipofectamine 2000. The EGF receptor numbers were determined by Western blotting and flowcytometry. The antiproliferative effects of sequence specific double stranded RNA (dsRNA) were assessed using cell count, colony assay and scratch assay. The chemosensitivity of transfected cells to cisplatin was measured by MTT. RESULTS: Sequence specific dsRNA-EGFR down-regulated EGF receptor expression dramatically. Compared with the control group, dsRNA-EGFR reduced the cell number by 85.0 %, decreased the colonies by 63.3 %, inhibited the migration by 87.2 %, and increased the sensitivity of A549 to cisplatin by four-fold. CONCLUSION: Sequence specific dsRNA-EGFR were capable of suppressing EGF receptor expression, hence significantly inhibiting cellular proliferation and motility, and enhancing chemosensitivity of A549 cells to cisplatin. The successful application of dsRNA-EGFR for inhibition of proliferation in EGF receptor overexpressing cells can help extend the list of available therapeutic modalities in the treatment of non-small-cell lung carcinoma (NSCLC).

Adenocarcinoma↗
PubMed2005

Potential applications of RNA interference technology in the treatment of cancer.

Inhibition of growth and progression of cancer cells is a challenge with major potential impact. RNA interference (RNAi) technology has been rapidly developed as a laboratory tool for the downregulation of the expression of a gene of interest. Moreover, RNAi offers a new potential for gene therapy of particular neoplasms by the specific inhibition of a cancer-associated target. This article will briefly describe the mechanism and application possibilities of RNAi, and illustrate the therapeutic potential in cancer gene therapy. The utilization of RNAi technology as a potential therapeutic tool for the treatment of cancer will be discussed in detail for two specific targets; the Bcr-Abl tyrosine kinase and the multidrug transporter MDR1/P-glycoprotein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2002

RNA interference directed against viral and cellular targets inhibits human immunodeficiency Virus Type 1 replication.

Human immunodeficiency virus type 1 (HIV-1) gene expression is regulated by both cellular transcription factors and Tat. The ability of Tat to stimulate transcriptional elongation is dependent on its binding to TAR RNA in conjunction with cyclin T1 and CDK9. A variety of other cellular factors that bind to the HIV-1 long terminal repeat, including NF-kappaB, SP1, LBP, and LEF, are also important in the control of HIV-1 gene expression. Although these factors have been demonstrated to regulate HIV-1 gene expression by both genetic and biochemical analysis, in most cases a direct in vivo demonstration of their role on HIV-1 replication has not been established. Recently, the efficacy of RNA interference in mammalian cells has been shown utilizing small interfering RNAs (siRNAs) to result in the specific degradation of host mRNAs and decreases the levels of their corresponding proteins. In this study, we addressed whether siRNAs directed against either HIV-1 tat or reverse transcriptase or the NF-kappaB p65 subunit could specifically decrease the levels of these proteins and thus alter HIV-1 replication. Our results demonstrate the specificity of siRNAs for decreasing the expression of these viral and cellular proteins and inhibiting HIV-1 replication. These studies suggest that RNA interference is useful in exploring the biological role of cellular and viral regulatory factors involved in the control of HIV-1 gene expression.

Gene Products, tat↗
PubMed2004

Control of HIV-1 replication by RNA interference.

Small interfering RNAs (siRNAs) have been shown to direct sequence-specific inhibition of gene expression in mammalian cells. siRNAs are RNA duplexes of 21-23 nucleotides (nts) with approximately 2nt 3' overhangs that can induce degradation of their homologous target mRNAs without interferon responses in mammalian cells. The degradation of the target occurs at the post-transcriptional level, meaning a post-transcriptional gene silencing (PTGS) mechanism called as RNA interference (RNAi). RNAi has emerged as an efficient method to inhibit gene expression in mammalian cells with increasingly successful cases of knockdown of many specific genes. Recent works have shown that the use of RNAi could inhibit HIV-1 replication by targeting viral or cellular genes. RNAi can be considered as a gene-specific therapeutic option for controlling HIV-1 replication. However, the control of HIV-1 replication has become complex because of the limited effectiveness of existing anti-HIV-1 agents and the high speed mutation rate of the HIV-1 genome. Careful assessments are required for the potential of RNAi as a gene therapy approach for controlling HIV-1 replication. This review will discuss the status of the science using RNAi for controlling HIV-1 replication and will describe possible problems for therapeutic applications of RNAi-mediated technologies for HIV-1 behind this novel mechanism.

Gene Expression Regulation, Viral↗
PubMed2003

Applications of RNA interference for studies in DNA damage processing, genome stability, mutagenesis, and cancer.

RNA interference, discovered only five years ago, is an important system for the control of gene expression. It is also quickly becoming a valuable experimental tool as it allows investigators to knock down the level of expression of specific genes. In this paper, we review some applications of this technology for studies in DNA damage processing, genome stability, mutagenesis, and cancer.

Animals↗
PubMed2005

Alu-linked hairpins efficiently mediate RNA interference with less toxicity than do H1-expressed short hairpin RNAs.

RNA interference has become a powerful tool for specific inhibition of gene expression in mammalian cells. Expression constructs allow for the long-term delivery of short interfering RNAs, usually through the expression of Pol III-transcribed hairpins. In some instances, these expression systems have been shown to have side effects, including induction of the interferon response and cytotoxicity. Here we demonstrate that H1-expressed hairpins, as well as the cloning vector, reduce the plating efficiency of HeLa cells. This toxicity is abrogated by coexpression of the hairpin in the same transcript as a human Alu repetitive element. These Alu-linked hairpins retain the ability to knock down expression of target mRNAs. This modification, which we term SINE (short interspersed repetitive element)-enhanced short hairpin RNA, provides an alternative expression system for hairpins with reduced side effects.

Alu Elements↗
PubMed2004

RNA interference in Agrobacterium rhizogenes-transformed roots of Arabidopsis and Medicago truncatula.

RNA interference (RNAi) is a powerful reverse genetic tool to study gene function. The data presented here show that Agrobacterium rhizogenes-mediated RNAi is a fast and effective tool to study genes involved in root biology. The Arabidopsis gene KOJAK, involved in root hair development, was efficiently knocked down. A. rhizogenes-mediated root transformation is a fast method to generate adventitious, genetically transformed roots. In order to select for co-transformed roots a binary vector was developed that enables selection based on DsRED1 expression, with the additional benefit that chimaeric roots can be discriminated. The identification of chimaeric roots provided the opportunity to examine the extent of systemic spread of the silencing signal in the composite plants of both Arabidopsis and Medicago truncatula. It is shown that RNA silencing does not spread systemically to non-co-transformed (lateral) roots and only inefficiently to the non-transgenic shoot. Furthermore, evidence is presented which shows that RNAi is cell autonomous in the root epidermis.

Arabidopsis↗
PubMed2006

A systematic RNA interference screen reveals a cell migration gene network in C. elegans.

Cell migration is essential during embryonic development and tissue morphogenesis. During gonadogenesis in the nematode Caenorhabditis elegans, migration of the distal tip cells forms two U-shaped gonad arms. Malformation results if the distal tip cells stop prematurely or follow an aberrant path, and abnormalities are easily visualized in living nematodes. Here we describe the first comprehensive in vivo RNA interference screen for genes required for cell migration. In this non-biased screen, we systematically analyzed 16,758 RNA-interference depletion experiments by light microscopy and identified 99 genes required for distal tip cell migration. Genetic and physical interaction data connect 59 of these genes to form a cell migration gene network that defines distal tip cell migration in vivo.

Animals↗
PubMed2004

Approach for functional analysis of glycan using RNA interference.

The elucidation of the biological role of glycan is one of the most important issues to be resolved following the genome project. RNA interference is becoming an efficient reverse genetic tool for studying gene function in model organisms, including C.elegans and Drosophila melanogaster. Our molecular evolutionary study has shown that a prototype of glycosyltransferases, which synthesize a variety of glycan structures in the Golgi apparatus, was conserved between mammals and Drosophila. For analyses of the basic physiological functions of glycans, we established the Drosophila inducible RNAi knockdown system and applied it to one glycosyltransferase and one transporter, proteoglycan UDP-galactose: beta-xylose beta1,4galactosyltransferase I and the PAPS-transporter, respectively. If on the silencing of each gene induced ubiquitously under the control of a cytoplasmic actin promoter, the RNAi knockdown fly died, then the protein was indispensable for life. The expression of the target gene was disrupted specifically and the degree of interference was well correlated with the phenotype. The inducible RNAi knockdown fly obtained using the GAL4-UAS system will pave the way for the functional analysis of glycans.

Actins↗
PubMed2004

Analysis of gene function in Trypanosoma brucei using RNA interference.

Trypanosoma brucei, a flagellate protozoa of the family Trypanosomatidae, has become one of the model systems for unicellular pathogens to study fundamentally important biological phenomena. The method of choice today to examine gene function in these organisms is RNA interference (RNAi). Messenger RNA (mRNA) degradation is triggered by double-stranded RNA (dsRNA) produced in vivo from transgenes transcribed from opposing tetracycline (tet)-inducible T7 RNA polymerase promoters, or hairpin RNA transcribed from the tet-inducible procyclic acidic repetitive protein promoter. This chapter describes some of the methods we employ for ablation of gene expression by RNAi in T. brucei with particular emphasis on transfection and cloning of procyclic cells, induction of dsRNA expression, isolation of RNA, and analysis of dsRNA and target mRNA.

Animals↗
PubMed2005

Simple, robust strategies for generating DNA-directed RNA interference constructs.

We describe two complementary strategies for preparing DNA-directed RNA interference (ddRNAi) constructs designed to express hpRNA. The first, oligonucleotide assembly (OA), uses a very simple annealing protocol to combine up to 20 short nucleotides. These are then cloned into appropriately designed restriction sites in expression vectors. OA can be used to prepare simple hairpin (hp)-expressing constructs, but we prefer to use the approach to generate longer constructs. The second strategy, long-range cloning (LRC), uses a novel adaptation of long-range PCR protocols. For LRC, entire vectors are amplified with primers that serve to introduce short sequences into plasmids at defined anchor sites during PCR. The LCR strategy has proven highly reliable in our hands for generating simple ddRNAi constructs. Moreover, LCR is likely to prove useful in many situations in which conventional cloning strategies might prove problematic. In combination, OA and LRC can greatly simplify the design and generation of many expression constructs, including constructs for ddRNAi.

Base Sequence↗
PubMed2003

RNA interference in biology and medicine.

First discovered in plants the nematode Caenorhabditis elegans, the production of small interfering RNAs (siRNAs) that bind to and induce the degradation of specific endogenous mRNAs is now recognized as a mechanism that is widely employed by eukaryotic cells to inhibit protein production at a post-transcriptional level. The endogenous siRNAs are typically 19- to 23-base double-stranded RNA oligonucleotides, produced from much larger RNAs that upon binding to target mRNAs recruit RNases to a protein complex that degrades the targeted mRNA. Methods for expressing siRNAs in cells in culture and in vivo using viral vectors, and for transfecting cells with synthetic siRNAs, have been developed and are being used to establish the functions of specific proteins in various cell types and organisms. RNA interference methods provide several major advantages over prior methods (antisense DNA or antibody-based techniques) for suppressing gene expression. Recent preclinical studies suggest that RNA interference technology holds promise for the treatment of various diseases. Pharmacologists have long dreamed of the ability to selectively antagonize or eliminate the function of individual proteins--RNAi technology may eventually make that dream a reality.

Animals↗
PubMed2004

Inhibition of gene expression with double strand RNA interference in Entamoeba histolytica.

In order to inhibit gene expression in Entamoeba histolytica, we have developed a method based on expressing double strand RNA interference constructs in stable transformants. The 5' end of Eh Dia was cloned head to head with an intervening non-specific stuffer fragment in the E. histolytica expression vector pJST4. This construct was transformed in E. histolytica HM1:IMSS trophozoites and stable transformants were selected with 20microg/ml G418. Our results show that expression of Eh Dia was completely inhibited in these transformants. These stable transformants could be maintained indefinitely without expression of Eh Dia. This method therefore provides an effective tool to study the phenotypic changes, which occur due to inhibition of gene expression in the absence of mutants and other microbiological manipulations in this protozoan parasite.

Animals↗