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Results for “RNA interference (RNAi)”

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PubMed2004

Gene silencing in alveolar type II cells using cell-specific promoter in vitro and in vivo.

RNA interference (RNAi) is a sequence-specific post-transcriptional gene silencing process. Although it is widely used in the loss-of-function studies, none of the current RNAi technologies can achieve cell-specific gene silencing. The lack of cell specificity limits its usage in vivo. Here, we report a cell-specific RNAi system using an alveolar epithelial type II cell-specific promoter--the surfactant protein C (SP-C) promoter. We show that the SP-C-driven small hairpin RNAs specifically depress the expression of the exogenous reporter (enhanced green fluorescent protein) and endogenous genes (lamin A/C and annexin A2) in alveolar type II cells, but not other lung cells, using cell and organ culture in vitro as well as in vivo. The present study provides an efficient strategy in silencing a gene in one type of cell without interfering with other cell systems, and may have a significant impact on RNAi therapy.

Adenoviridae↗
PubMed2003

Members of the Arabidopsis-SKP1-like gene family exhibit a variety of expression patterns and may play diverse roles in Arabidopsis.

Ubiquitin-mediated proteolysis by the proteasome is a critical regulatory mechanism controlling many biological processes. In particular, SKP1, cullin/CDC53, F-box protein (SCF) complexes play important roles in selecting substrates for proteolysis by facilitating the ligation of ubiquitin to specific proteins. In plants, SCF complexes have been found to regulate auxin responses and jasmonate signaling and may be involved in several other processes, such as flower development, circadian clock, and gibberellin signaling. Although 21 Skp1-related genes, called Arabidopsis-SKP1-like (ASK), have been uncovered in the Arabidopsis genome, ASK1 is the only gene that has been analyzed genetically. As a first step toward understanding their functions, we tested for expression of 20 ASK genes using reverse transcription-polymerase chain reaction experiments. Also, we examined the expression patterns of 11 ASK genes by in situ hybridizations. The ASK genes exhibit a spectrum of expression levels and patterns, with a large subset showing expression in the flower and/or fruit. In addition, the ASK genes that have similar sequences tend to have similar expression patterns. On the basis of the expression results, we selectively suppressed the expression of a few ASK genes using RNA interference. Compared with the ask1 mutant, the strong ASK1 RNA interference (RNAi) line exhibited similar or enhanced phenotypes in both vegetative and floral development, whereas ASK11 RNAi plants had normal vegetative growth but mild defects in flower development. The diverse expression patterns and distinct defects observed in RNAi plants suggest that the ASK gene family may collectively perform a range of functions and may regulate different developmental and physiological processes.

Arabidopsis↗
PubMed2006

Fission yeast Cid12 has dual functions in chromosome segregation and checkpoint control.

Fission yeast Cid12 is a member of the Cid1 family of specialized poly(A) polymerases. Like cells lacking cid1, cid12Delta mutants were shown to have checkpoint defects when DNA replication was inhibited. Here, we show that Cid12 is also required for faithful chromosome segregation and that mutation of amino acid residues predicted to be essential for poly(A) polymerase activity resulted in loss of Cid12 function in vivo. Cells lacking Cid12 had an increased chromosome segregation failure rate due to precocious loss of sister chromatid cohesion at the centromere but not along the chromosome arms. In keeping with a recently described function for Cid12 in RNA interference (RNAi)-mediated heterochromatin assembly, this was accompanied by an accumulation of polyadenylated transcripts corresponding to naturally silenced repeat elements within heterochromatic domains, with consequent defects in centromeric gene silencing. These cells also suffered increased meiotic defects, and their viability was dependent on the spindle checkpoint protein Bub1. To account for the effects of Cid12 on various aspects of DNA metabolism, including chromosome segregation and the checkpoint control, we suggest that Cid12 has dual functions in RNAi silencing and regulating mRNA stability.

Amino Acid Sequence↗
PubMed2006

L1 retrotransposition is suppressed by endogenously encoded small interfering RNAs in human cultured cells.

LINE-1s, or L1s, are highly abundant retrotransposons comprising 17% of the human genome. Most L1s are retrotransposition defective; nonetheless, there are approximately 100 full-length L1s potentially capable of retrotransposition in the diploid genome. L1 retrotransposition may be detrimental to the host and thus needs to be controlled. Previous studies have identified sense and antisense promoters in the 5' UTR of full-length human L1. Here we show that the resulting bidirectional transcripts can be processed to small interfering RNAs (siRNAs) that suppress retrotransposition by an RNA interference (RNAi) mechanism. We thus provide evidence that RNAi triggered by antisense transcripts may modulate human L1 retrotransposition efficiently and economically. L1-specific siRNAs are among the first natural siRNAs reported in mammalian systems. This work may contribute to understanding the regulatory role of abundant antisense transcripts in eukaryotic genomes.

5' Untranslated Regions↗
PubMed2006

Modeling CTLA4-linked autoimmunity with RNA interference in mice.

The CTLA4 gene is important for T lymphocyte-mediated immunoregulation and has been associated with several autoimmune diseases, in particular, type 1 diabetes. To model the impact of natural genetic variants of CTLA4, we constructed RNA interference (RNAi) "knockdown" mice through lentiviral transgenesis. Variegation of expression was observed in founders but proved surmountable because it reflected parental imprinting, with derepression by transmission from male lentigenics. Unlike the indiscriminate multiorgan autoimmune phenotype of the corresponding knockout mice, Ctla4 knockdown animals had a disease primarily focused on the pancreas, with rapid progression to diabetes. As with the human disease, the knockdown phenotype was tempered by genetic-modifier loci. RNAi should be more pertinent than gene ablation in modeling disease pathogenesis linked to a gene-dosage variation.

Animals↗
PubMed2006

Isolation and identification of gene-specific microRNAs.

Prediction of microRNA (miRNA) candidates using computer programming has identified hundreds and hundreds of genomic hairpin sequences, of which, the functions remain to be determined. Because direct transfection of hairpin-like miRNA precursors (pre)-miRNAs in mammalian cells is not always sufficient to trigger effective RNA-induced gene-silencing complex (RISC) assembly, a key step for RNA interference (RNAi)-related gene silencing, we developed an intronic miRNA-expressing system to overcome this problem, and successfully increased the efficiency and effectiveness of miRNA-associated RNAi induction in vitro and in vivo. By insertion of a hairpin-like pre-miRNA structure into the intron region of a gene, this intronic miRNA biogenesis system has been found to depend on a coupled interaction of nascent precursor messenger RNA transcription and intron excision within a specific nuclear region proximal to genomic perichromatin fibrils. The intronic miRNA was transcribed by RNA type II polymerases, coexpressed with a primary gene transcript, and excised out of its encoding gene transcript by intracellular RNA splicing and processing mechanisms. Currently, some ribonuclease III endonucleases have been found to be involved in the processing of spliced introns and probably facilitating the intronic miRNA maturation. Using this miRNA-expressing system, we have shown for the first time that the intron-derived miRNAs were able to induce strong RNAi effects in not only human and mouse cells but also zebrafish, chicken embryos, and adult mice. Based on the strand complementarity between the designed miRNA and its target gene sequence, we have also developed a miRNA isolation protocol to purify and identify the mature miRNAs generated by the intronic miRNA-expressing system. Several intronic miRNA identities and structures are currently confirmed to be active in vitro and in vivo. According to this proof- of-principle method, we now have the knowledge to design pre-miRNA inserts that are more efficient and effective for the intronic miRNA-expressing system.

Animals↗
PubMed2005

[Suppression of enhanced green fluorescent protein expression in cells by RNA interference].

OBJECTIVE: To evaluate the role of RNA interference (RNAi) in silencing the enhanced green fluorescent protein (eGFP) expression in 293T and Mel cells. METHODS: Nested-PCR was used to amplify H1 promoter from human 293T cells for driving RNAi synthesis. RNAi vectors (TR1) for silencing the eGFP expression was constructed. The eGFP vector and RNAi vector (TR1) were then co-transfected into the 293T and Mel cells, in which the silencing effect on eGFP expression was investigated by fluorescence microscopy, reverse transcription-PCR(RT-PCR), fluorescence-assited cell sorting(FACS) analysis and real-time RT-PCR. RESULTS: RNAi could effectively reduce more than 50 percent of eGFP expression in 293T cells as well as in Mel cells. CONCLUSION: The RNAi vector constructed in this way paper can effectively inhibit eGFP expression in cells.

Cell Line↗
PubMed2005

Silencing the epidermal growth factor receptor gene with RNAi may be developed as a potential therapy for non small cell lung cancer.

Lung cancer has emerged as a leading cause of cancer death in the world. Non-small cell lung cancer (NSCLC) accounts for 75-80% of all lung cancers. Current therapies are ineffective, thus new approaches are needed to improve the therapeutic ratio. Double stranded RNA (dsRNA)-mediated RNA interference (RNAi) has shown promise in gene silencing, the potential of which in developing new methods for the therapy of NSCLC needs to be tested. We report here RNAi induced effective silencing of the epidermal growth factor receptor (EGFR) gene, which is over expressed in NSCLC. NSCLC cell lines A549 and SPC-A1 were transfected with sequence- specific dsRNA as well as various controls. Immune fluorescent labeling and flow cytometry were used to monitor the reduction in the production of EGFR protein. Quantitative reverse-transcriptase PCR was used to detect the level of EGFR mRNA. Cell count, colony assay, scratch assay, MTT assay in vitro and tumor growth assay in athymic nude mice in vivo were used to assess the functional effects of EGFR silencing on tumor cell growth and proliferation. Our data showed transfection of NSCLC cells with dsRNA resulted in sequence specific silencing of EGFR with 71.31% and 71.78 % decreases in EGFR protein production and 37.04% and 54.92% in mRNA transcription in A549 and SPC-A1 cells respectively. The decrease in EGFR protein production caused significant growth inhibition, i.e.: reducing the total cell numbers by 85.0% and 78.3%, and colony forming numbers by 63.3% and 66.8%. These effects greatly retarded the migration of NSCLC cells by more than 80% both at 24 h and at 48 h, and enhanced chemo-sensitivity to cisplatin by four-fold in A549 cells and seven-fold in SPC-A1. Furthermore, dsRNA specific for EGFR inhibited tumor growth in vivo both in size by 75.06% and in weight by 73.08%. Our data demonstrate a new therapeutic effect of sequence specific suppression of EGFR gene expression by RNAi, enabling inhibition of tumor proliferation and growth. However, in vivo use of dsRNA for gene transfer to tumor cells would be limited because dsRNA would be quickly degraded once delivered in vivo. We thus tested a new bovine lentiviral vector and showed lentivector-mediated RNAi effects were efficient and specific. Combining RNAi with this gene delivery system may enable us to develop RNAi for silencing EGFR into an effective therapy for NSCLC.

Journal Article↗
PubMed2002

An efficient protein complex purification method for functional proteomics in higher eukaryotes.

The ensemble of expressed proteins in a given cell is organized in multiprotein complexes. The identification of the individual components of these complexes is essential for their functional characterization. The introduction of the 'tandem affinity purification' (TAP) methodology substantially improved the purification and systematic genome-wide characterization of protein complexes in yeast. The use of this approach in higher eukaryotic cells has lagged behind its use in yeast because the tagged proteins are normally expressed in the presence of the untagged endogenous version, which may compete for incorporation into multiprotein complexes. Here we describe a strategy in which the TAP approach is combined with double-stranded RNA interference (RNAi) to avoid competition from corresponding endogenous proteins while isolating and characterizing protein complexes from higher eukaryotic cells. This strategy allows the determination of the functionality of the tagged protein and increases the specificity and the efficiency of the purification.

Animals↗
PubMed2005

Overexpression of fasciculation and elongation protein zeta-1 (FEZ1) induces a post-entry block to retroviruses in cultured cells.

Two mutant Rat2 fibroblast cell lines, R3-2 and R4-7, have been previously isolated by a selection for retrovirus resistance. We have now further analyzed the basis of the block to retroviral infection in the R3-2 line. Using Affymetrix GeneChip analysis, several genes were identified as differentially expressed in the mutant R3-2 line compared with the wild-type cells. One of the candidate gene products, FEZ1 (fasciculation and elongation protein zeta-1), a protein kinase C (PKC)zeta-interacting protein homologous to the Caenorhabditis elegans synaptic transport protein UNC-76, was found to be up-regulated >30-fold in the resistant R3-2 line. FEZ1 overexpression in Rat2 cells conferred a potent resistance to infection by genetically marked retroviruses, and the degree of retroviral resistance in both Rat2 fibroblasts and 293T cells tightly correlated with the expression level of FEZ1 transcripts. FEZ1-overexpressing Rat2 cells showed a similar phenotype to that of the mutant R3-2 line: Infection resulted in normal viral DNA synthesis but a reduction in the formation of circular DNA, indicating a block after reverse transcription but before nuclear entry. Partial knockdown of FEZ1 expression in R3-2 by RNA interference (RNAi) significantly reduced the resistance of this line to infection. Thus, our data suggest that FEZ1 overexpression is sufficient to explain the resistant phenotype of R3-2 cells and identify FEZ1 as a new gene capable of causing retrovirus resistance.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Selective degradation of maternal and embryonic transcripts in in vitro produced bovine oocytes and embryos using sequence specific double-stranded RNA.

RNA interference (RNAi) has been used for selective degradation of an mRNA transcript or inhibiting its translation to a functional protein in various species. Here, we applied the RNAi approach to suppress the expression of the maternal transcript C-mos and embryonic transcripts Oct-4 in bovine oocytes and embryos respectively, using microinjection of sequence-specific double-stranded RNA (dsRNA). For this, 435 bp C-mos and 341 bp Oct-4 dsRNA were synthesized and microinjected into the cytoplasm of immature oocytes and zygotes respectively. In experiment 1, immature oocytes were categorized into three groups: those injected with C-mos dsRNA, RNase-free water and uninjected controls. In experiment 2, in vitro produced zygotes were categorized into three groups: those injected with Oct-4 dsRNA, RNase-free water and uninjected controls. The developmental phenotypes, the level of mRNA and protein expression were investigated after treatment in both experiments. Microinjection of C-mos dsRNA has resulted in 70% reduction of C-mos transcript after maturation compared to the water-injected and uninjected controls (P<0.01). Microinjection of zygotes with Oct-4 dsRNA has resulted in 72% reduction in transcript abundance at the blastocyst stage compared to the uninjected control zygotes (P<0.01). Moreover, a significant reduction in the number of inner cell mass (ICM) cells was observed in Oct-4 dsRNA-injected embryos compared to the other groups. From oocytes injected with C-mos dsRNA, 60% showed the extrusion of the first polar body compared to 50% in water-injected and 44% in uninjected controls. Moreover, only oocytes injected with C-mos dsRNA showed spontaneous activation. In conclusion, our results demonstrated that sequence-specific dsRNA can be used to knockdown maternal or embryonic transcripts in bovine embryogenesis.

Animals↗
PubMed2000

Genetic interference in Trypanosoma brucei by heritable and inducible double-stranded RNA.

The use of double-stranded RNA (dsRNA) to disrupt gene expression has become a powerful method of achieving RNA interference (RNAi) in a wide variety of organisms. However, in Trypanosoma brucei this tool is restricted to transient interference, because the dsRNA is not stably maintained and its effects are diminished and eventually lost during cellular division. Here, we show that genetic interference by dsRNA can be achieved in a heritable and inducible fashion. To show this, we established stable cell lines expressing dsRNA in the form of stem-loop structures under the control of a tetracycline-inducible promoter. Targeting a-tubulin and actin mRNA resulted in potent and specific mRNA degradation as previously observed in transient interference. Surprisingly, 10-fold down regulation of actin mRNA was not fatal to trypanosomes. This type of approach could be applied to study RNAi in other organisms that are difficult to microinject or electroporate. Furthermore, to quickly probe the consequences of RNAi for a given gene we established a highly efficient in vivo T7 RNA polymerase system for expression of dsRNA. Using the alpha-tubulin test system we obtained greater than 98% transfection efficiency and the RNAi response lasted at least two to three cell generations. These new developments make it possible to initiate the molecular dissection of RNAi both biochemically and genetically.

Actins↗
PubMed2005

RNA interference-mediated virus clearance from cells both acutely and chronically infected with the prototypic arenavirus lymphocytic choriomeningitis virus.

Several arenaviruses, including Lassa fever virus, cause severe, often lethal hemorrhagic fever in humans. No licensed vaccines are available in the United States, and currently there is no efficacious therapy to treat this viral infection. Therefore the importance of developing effective antiviral approaches to combat pathogenic arenaviruses is clear. Moreover, the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is an important model for the study of viral persistence and associated diseases, as well as for exploring therapies to treat viral chronic infections. The use of small interfering RNAs (siRNAs) to downregulate gene expression via RNA interference (RNAi) has emerged as a powerful genetic tool for the study of gene function. In addition, the successful use of siRNAs to target a variety of animal viruses has led us to consider RNAi as a potential novel antiviral strategy. We have investigated the use of RNAi therapy against LCMV. Here, we show that siRNAs targeting sequences within the viral L polymerase and Z mRNAs inhibit LCMV multiplication in cultured cells. Unexpectedly, the antiviral efficacy of RNAi-based therapy against LCMV was highly dependent on the method used to deliver effector siRNA molecules. Thus, transfection of chemically synthesized siRNA pools to L and Z was ineffective in preventing virus multiplication. In contrast, targeting of the same viral L and Z gene products with siRNAs produced inside cells using a replication-deficient recombinant adenovirus expression system inhibited LCMV multiplication very efficiently. Notably, transduction with the replication-deficient recombinant adenovirus expression system to Z and L effectively cured persistently LCMV-infected cells, suggesting the feasibility of using RNAi therapy to combat viral chronic infections by riboviruses.

Adenoviridae↗
PubMed2006

Progress towards in vivo use of siRNAs.

RNA interference (RNAi) has become the method of choice to suppress gene expression in vitro. It is also emerging as a powerful tool for in vivo research with over 90 studies published using synthetic small interfering RNAs in mammals. These reports demonstrate the potential for use of synthetic small interfering RNAs (siRNAs) as therapeutic agents, especially in the areas of cancer and viral infection. The number of reports using siRNAs for functional genomics applications, for validation of targets for small-molecule drug development programs, and to address questions of basic biology will rapidly grow as methods and protocols for use in animals become more established. This review will first discuss aspects of RNAi biochemistry and biology that impact in vivo use, especially as relates to experimental design, and will then provide an overview of published work with a focus on methodology.

Animals↗
PubMed2004

Application of RNAi to confirm theobromine as the major intermediate for caffeine biosynthesis in coffee plants with potential for construction of decaffeinated varieties.

The caffeine biosynthetic pathway in coffee plants has been proposed to involve three distinct N -methyltransferases, xanthosine methyltransferase (XMT), 7- N -methylxanthine methyltransferase (MXMT; theobromine synthase), and 3,7-dimethylxanthine methyltransferase (DXMT; caffeine synthase). We previously isolated all corresponding cDNAs designated as CaXMT1 , CaMXMT1 , CaMXMT2 and CaDXMT1 , respectively, and showed that caffeine was indeed synthesized in vitro by the combination of their gene products. In order to regulate caffeine biosynthesis in planta , we suppressed expression of CaMXMT1 by the double stranded RNA interference (RNAi) method. For this purpose, we first established a protocol for efficient somatic embryogenesis of Coffea arabica and C. canephora , and then Agrobacterium -mediated transformation techniques. The RNAi transgenic lines of embryogenic tissues derived from C. arabica and transgenic plantlets of C. canephora demonstrated a clear reduction in transcripts for CaMXMT1 in comparison with the control plants. Transcripts for CaXMT1 and CaDXMT1 were also reduced in the most cases. Both embryonic tissues and plantlets exhibited a concomitant reduction of theobromine and caffeine contents to a range between 30% and 50% of that of the control. These results suggest that the CaMXMT1 -RNAi sequence affected expression of not only CaMXMT1 itself, but also CaXMT1 and CaDXMT1 , and that, since the reduction in theobromine content was proportional to that for caffeine, it is involved in the major synthetic pathway in coffee plants. The results also indicate that the method can be practically applied to produce decaffeinated coffee plants.

Caffeine↗
PubMed2006

More complete gene silencing by fewer siRNAs: transparent optimized design and biophysical signature.

Highly accurate knockdown functional analyses based on RNA interference (RNAi) require the possible most complete hydrolysis of the targeted mRNA while avoiding the degradation of untargeted genes (off-target effects). This in turn requires significant improvements to target selection for two reasons. First, the average silencing activity of randomly selected siRNAs is as low as 62%. Second, applying more than five different siRNAs may lead to saturation of the RNA-induced silencing complex (RISC) and to the degradation of untargeted genes. Therefore, selecting a small number of highly active siRNAs is critical for maximizing knockdown and minimizing off-target effects. To satisfy these needs, a publicly available and transparent machine learning tool is presented that ranks all possible siRNAs for each targeted gene. Support vector machines (SVMs) with polynomial kernels and constrained optimization models select and utilize the most predictive effective combinations from 572 sequence, thermodynamic, accessibility and self-hairpin features over 2200 published siRNAs. This tool reaches an accuracy of 92.3% in cross-validation experiments. We fully present the underlying biophysical signature that involves free energy, accessibility and dinucleotide characteristics. We show that while complete silencing is possible at certain structured target sites, accessibility information improves the prediction of the 90% active siRNA target sites. Fast siRNA activity predictions can be performed on our web server at http://optirna.unl.edu/.

Artificial Intelligence↗
PubMed2004

Global expression changes resulting from loss of telomeric DNA in fission yeast.

BACKGROUND: Schizosaccharomyces pombe cells lacking the catalytic subunit of telomerase (encoded by trt1+) lose telomeric DNA and enter crisis, but rare survivors arise with either circular or linear chromosomes. Survivors with linear chromosomes have normal growth rates and morphology, but those with circular chromosomes have growth defects and are enlarged. We report the global gene-expression response of S. pombe to loss of trt1+. RESULTS: Survivors with linear chromosomes had expression profiles similar to cells with native telomeres, whereas survivors with circular chromosomes showed continued upregulation of core environmental stress response (CESR) genes. In addition, survivors with circular chromosomes had altered expression of 51 genes compared to survivors with linear chromosomes, providing an expression signature. S. pombe progressing through crisis displayed two waves of altered gene expression. One coincided with crisis and consisted of around 110 genes, 44% of which overlapped with the CESR. The second was synchronized with the emergence of survivors and consisted of a single class of open reading frames (ORFs) with homology both to RecQ helicases and to dh repeats at centromeres targeted for heterochromatin formation via an RNA interference (RNAi) mechanism. Accumulation of transcript from the ORF was found not only in trt1- cells, but also in dcr1- and ago1- RNAi mutants, suggesting that RNAi may control its expression. CONCLUSIONS: These results demonstrate a correlation between a state of cellular stress, short telomeres and growth defects in cells with circular chromosomes. A putative new RecQ helicase was expressed as survivors emerged and appears to be transcriptionally regulated by RNAi, suggesting that this mechanism operates at telomeres.

Adenosine Triphosphatases↗
PubMed2006

Reproducible and inducible knockdown of gene expression in mice.

RNA interference (RNAi) has emerged as an efficient approach for rapid analysis of gene function. In mammalian cells, vector-based expression of small hairpin RNAs (shRNA) produces potent and stable gene knockdown effects. An inducible RNAi system with reproducible levels of siRNA expression will extend the usefulness of this methodology to the identification of gene functions within the developing or adult mouse. We present evidence that an RNA polymerase III-driven U6 promoter with stuffer sequences flanked by loxP sites inserted at three different sites within the promoter drives shRNA expression in a Cre recombinase-dependent manner. We utilized this approach to develop a generic strategy for the reproducible knockdown of gene expression in mice. By placing the inducible shRNA cassette into the ROSA26 locus of the mouse, we were able to generate reproducible levels of controlled expression of shRNA to produce discernable phenotypes in vitro and in vivo. This approach circumvents the prescreening of random integration in embryonic stem cell clones and further enables conditional gene knockdown with temporal and/or tissue specificity. This methodology should expedite large-scale functional studies.

Animals↗