RESUMO
RNA interference is a powerful mechanism for sequence-specific inhibition of gene expression. It is widely known that small interfering RNAs (siRNAs) targeting the same region of a target-messenger RNA can have widely different efficacies. In efforts to better understand the siRNA features that influence knockdown efficiency, we analyzed siRNA interactions with a high-molecular weight complex in whole cell extracts prepared from two different cell lines. Using biochemical tools to study the nature of the complex, our results demonstrate that the primary siRNA-binding protein in the whole cell extracts is Dicer. We find that Dicer is capable of discriminating highly functional versus poorly functional siRNAs by recognizing the presence of 2-nt 3' overhangs and the thermodynamic properties of 2-4 bp on both ends of effective siRNAs. Our results suggest a role for Dicer in pre-selection of effective siRNAs for handoff to Ago2. This initial selection is reflective of the overall silencing potential of an siRNA.
Assuntos
Interferência de RNA , RNA Interferente Pequeno/metabolismo , Complexo de Inativação Induzido por RNA/metabolismo , Ribonuclease III/fisiologia , Proteínas Argonautas , Extratos Celulares , Fator de Iniciação 2 em Eucariotos/fisiologia , Células HCT116 , Células HEK293 , Humanos , RNA Interferente Pequeno/química , Proteínas de Ligação a RNA/fisiologiaRESUMO
An efficient RNAi largely depends on optimal design of the siRNA. In recent studies, Dicer substrates were found to be more potent than classical synthetic 21-mer siRNAs, suggesting a coupling of the Dicer-mediated processing step to the efficient assembly of the silencing complex, RISC. We describe the fundamental principles and experimental results leading to optimal Dicer substrates.
Assuntos
Sequência de Bases , Interferência de RNA , RNA Interferente Pequeno/genética , Ribonuclease III , Algoritmos , Animais , Dados de Sequência Molecular , RNA Interferente Pequeno/metabolismo , Ribonuclease III/genética , Ribonuclease III/metabolismoRESUMO
PURPOSE: The coagulation trigger tissue factor has been implicated in tumor growth, angiogenesis, and metastasis. In this study, we explore the effects of ex vivo and in vivo delivery of short interfering RNA (siRNA) targeting tissue factor on B16 melanoma colonization of the lung in a murine model for metastasis. The purposes of this work are to establish a noncytotoxic in vivo model for investigation of tissue factor function and provide preclinical assessment of the therapeutic potential of tissue factor siRNA for prevention of metastasis. EXPERIMENTAL DESIGN AND RESULTS: C57BL/6 mice were evaluated for pulmonary metastases following tail vein injection of B16 cells transfected with either active or inactive siRNA. Mice receiving cells transfected with active siRNA had significantly lower numbers of pulmonary tumors compared with mice injected with control cells (transfected with inactive siRNA). The average time point at which the mice started to exhibit tumor-associated stress was also increased significantly from 22 days for the control group to 27 days for the experimental group (P = 0.01). In a therapeutically more relevant model, where the siRNA was delivered i.p. and the cells (untransfected) by tail vein injection, an inhibitory effect on metastasis was observed when the siRNA treatment was initiated either before or at the time of cell injection. CONCLUSIONS: The results suggest that tissue factor has a crucial function in promoting lung tumor metastasis of blood-borne tumor cells in the early stages of the tumor take process and further suggest that treatment with tissue factor siRNA may become a viable clinical strategy for prevention of tumor metastasis.
Assuntos
Sistemas de Liberação de Medicamentos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/secundário , Melanoma Experimental/tratamento farmacológico , RNA Interferente Pequeno/administração & dosagem , Tromboplastina/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Injeções Intravenosas , Injeções Subcutâneas , Neoplasias Pulmonares/patologia , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , RNA Interferente Pequeno/síntese química , RNA Interferente Pequeno/químicaRESUMO
Synthetic RNA duplexes that are substrates for Dicer are potent triggers of RNA interference (RNAi). Blunt 27mer duplexes can be up to 100-fold more potent than traditional 21mer duplexes. Not all 27mer duplexes show increased potency. Evaluation of the products of in vitro dicing reactions using electrospray ionization mass spectrometry reveals that a variety of products can be produced by Dicer cleavage. Use of asymmetric duplexes having a single 2-base 3'-overhang restricts the heterogeneity that results from dicing. Inclusion of DNA residues at the ends of blunt duplexes also limits heterogeneity. Combination of asymmetric 2-base 3'-overhang with 3'-DNA residues on the blunt end result in a duplex form which directs dicing to predictably yield a single primary cleavage product. It is therefore possible to design a 27mer duplex which is processed by Dicer to yield a specific, desired 21mer species. Using this strategy, two different 27mers can be designed that result in the same 21mer after dicing, one where the 3'-overhang resides on the antisense (AS) strand and dicing proceeds to the 'right' ('R') and one where the 3'-overhang resides on the sense (S) strand and dicing proceeds to the 'left' ('L'). Interestingly, the 'R' version of the asymmetric 27mer is generally more potent in reducing target gene levels than the 'L' version 27mer. Strand targeting experiments show asymmetric strand utilization between the two different 27mer forms, with the 'R' form favoring S strand and the 'L' form favoring AS strand silencing. Thus, Dicer processing confers functional polarity within the RNAi pathway.
Assuntos
Interferência de RNA , RNA de Cadeia Dupla/metabolismo , RNA Interferente Pequeno/metabolismo , Ribonuclease III/metabolismo , Linhagem Celular , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , RNA de Cadeia Dupla/química , RNA Interferente Pequeno/química , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Short interfering RNA (siRNA), the active agent of RNA interference, shows promise of becoming a valuable tool in both basic and clinical research. We explore the tolerance to mutations and chemical modifications in various parts of the two 21-nt strands of a siRNA targeting the blood clotting initiator Tissue Factor. The mutations were G/C transversions. The chemical modifications were 2'-O-methylation, 2'-O-allylation and phosphorothioates. We found that siRNA generally tolerated mutations in the 5' end, while the 3' end exhibited low tolerance. This observation may facilitate the design of siRNA for specific targeting of transcripts containing single nucleotide polymorphisms. We further demonstrate that in our system the single antisense strand of the wild-type siRNA is almost as effective as the siRNA duplex, while the corresponding methylated M2+4 version of the antisense had reduced activity. Most of the chemically modified versions tested had near-wild-type initial activity, while the long-term activity was increased for certain siRNA species. Our results may improve the design of siRNAs for in vivo experiments.
Assuntos
RNA Interferente Pequeno/genética , Linhagem Celular , Humanos , Mutação , Oligorribonucleotídeos/síntese química , Oligorribonucleotídeos/genética , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/síntese química , Tromboplastina/genética , Tromboplastina/metabolismo , TransfecçãoRESUMO
RNA interference (RNAi), mediated by either long double-stranded RNA (dsRNA) or short interfering RNA (siRNA), has become a routine tool for transient knockdown of gene expression in a wide range of organisms. The antisense strand of the siRNA duplex (antisense siRNA) was recently shown to have substantial mRNA depleting activity of its own. Here, targeting human Tissue Factor mRNA in HaCaT cells, we perform a systematic comparison of the activity of antisense siRNA and double-strand siRNA, and find almost identical target position effects, appearance of mRNA cleavage fragments and tolerance for mutational and chemical backbone modifications. These observations, together with the demonstration that excess inactive double-strand siRNA blocks antisense siRNA activity, i.e. shows sequence-independent competition, indicate that the two types of effector molecules share the same RNAi pathway. Interest ingly, both FITC-tagged and 3'-deoxy antisense siRNA display severely limited activity, despite having practically wild-type activity in a siRNA duplex. Finally, we find that maximum depletion of target mRNA expression occurs significantly faster with antisense siRNA than with double-strand siRNA, suggesting that the former enters the RNAi pathway at a later stage than double-strand siRNA, thereby requiring less time to exert its activity.
Assuntos
Interferência de RNA/fisiologia , RNA Interferente Pequeno/fisiologia , Linhagem Celular , Regulação da Expressão Gênica , Humanos , RNA Antissenso/genética , RNA Antissenso/fisiologia , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Tromboplastina/genética , TransfecçãoRESUMO
Chemically synthesised 21-23 bp double-stranded short interfering RNAs (siRNA) can induce sequence-specific post-transcriptional gene silencing, in a process termed RNA interference (RNAi). In the present study, several siRNAs synthesised against different sites on the same target mRNA (human Tissue Factor) demonstrated striking differences in silencing efficiency. Only a few of the siRNAs resulted in a significant reduction in expression, suggesting that accessible siRNA target sites may be rare in some human mRNAs. Blocking of the 3'-OH with FITC did not reduce the effect on target mRNA. Mutations in the siRNAs relative to target mRNA sequence gradually reduced, but did not abolish mRNA depletion. Inactive siRNAs competed reversibly with active siRNAs in a sequence-independent manner. Several lines of evidence suggest the existence of a near equilibrium kinetic balance between mRNA production and siRNA-mediated mRNA depletion. The silencing effect was transient, with the level of mRNA recovering fully within 4-5 days, suggesting absence of a propagative system for RNAi in humans. Finally, we observed 3' mRNA cleavage fragments resulting from the action of the most effective siRNAs. The depletion rate-dependent appearance of these fragments argues for the existence of a two-step mRNA degradation mechanism.
Assuntos
Inativação Gênica , RNA não Traduzido/farmacologia , Tromboplastina/genética , Animais , Pareamento Incorreto de Bases , Sequência de Bases , Células COS , Linhagem Celular , Marcação de Genes , Células HeLa , Humanos , Cinética , Processamento Pós-Transcricional do RNA , RNA Mensageiro/análise , RNA Interferente Pequeno , RNA não Traduzido/química , RNA não Traduzido/metabolismo , Tromboplastina/antagonistas & inibidores , Tromboplastina/biossíntese , TransfecçãoRESUMO
Successful applications of RNAi in mammalian cells depend upon effective knockdown of targeted transcripts and efficient intracellular delivery of either preformed si/shRNAs or vector expressed si/shRNAs. We have previously demonstrated that 27 base pair double stranded RNAs which are substrates for Dicer can be up to 100 times more potent than 21mer siRNAs. In this mini-review we elaborate upon the rationale and design strategies for creating Dicer substrate RNAs that provide enhanced knockdown of targeted RNAs and minimize the utilization of the sense strand as RNAi effectors. Expression of shRNAs or siRNAs in mammalian cells can be achieved via transcription from either Pol II or Pol III promoters. There are certain constrictions in designing such vectors, and these are described here. Additionally, we review strategies for inducible shRNA expression and the various viral vectors that can be used to transduce shRNA genes into a variety of cells and tissues. The overall goal of this mini-review is to provide an overview of available approaches for optimizing RNAi mediated down regulation of gene expression in mammalian cells via RNA interference. Although the primary focus is the use of RNAi mediated cleavage of targeted transcripts, it is highly probable that some of the approaches described herein will be applicable to RNAi mediated inhibition of translation and transcriptional gene silencing.
Assuntos
Interferência de RNA/fisiologia , RNA Interferente Pequeno/metabolismo , Animais , Sequência de Bases , Inativação Gênica , Técnicas de Transferência de Genes , Técnicas Genéticas , Vetores Genéticos , Proteínas de Fluorescência Verde/metabolismo , Humanos , MicroRNAs/química , Modelos Genéticos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , RNA/metabolismo , RNA Polimerase III/química , RNA Mensageiro/metabolismo , Ribonuclease III/genética , Transcrição GênicaRESUMO
RNA interference (RNAi) is a powerful genetic tool for loss-of-function studies in mammalian cells and is also considered a potentially powerful therapeutic modality for the treatment of a variety of human diseases. During the past 3 years a number of systems for conditional RNAi have been developed that allow controlled expression of short hairpin RNA (shRNA) triggers of RNAi. The simplest strategy relies on tet-operable polymerase III-promoted shRNAs and co-expression of the tetracycline regulatory protein, TetR. In this study we have combined these features into a single lentiviral vector that upon delivery to target cells allows robust induction of shRNAs, even with low levels of doxycycline; importantly, we show minimal leakiness in the absence of inducer. We have exploited the regulatory properties of our system by targeting an essential cellular gene, the nuclear RNaseIII endonuclease Drosha. Drosha is the core catalytic component of the "microprocessor complex" and cleaves the primary microRNA (miRNA) transcripts into their pre-miRNA hairpin intermediates. We anticipate that our vector will facilitate functional studies of miRNA biogenesis.
Assuntos
Doxiciclina/farmacologia , Lentivirus/genética , RNA Interferente Pequeno/genética , Ribonuclease III/genética , Sequência de Bases , Northern Blotting , Western Blotting , Linhagem Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Vetores Genéticos/genética , Humanos , Luciferases/genética , Luciferases/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Reação em Cadeia da Polimerase , Interferência de RNA , Precursores de RNA/genética , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , Ribonuclease III/metabolismo , Transdução GenéticaRESUMO
RNA interference is a powerful tool for target-specific knockdown of gene expression. The triggers for this process are duplex small interfering RNAs (siRNAs) of 21-25 nt with 2-bp 3' overhangs produced in cells by the RNase III family member Dicer. We have observed that short RNAs that are long enough to serve as Dicer substrates (D-siRNA) can often evoke more potent RNA interference than the corresponding 21-nt siRNAs; this is probably a consequence of the physical handoff of the Dicer-produced siRNAs to the RNA-induced silencing complex. Here we describe the design parameters for D-siRNAs and a protocol for in vitro and in vivo intraperitoneal delivery of D-siRNAs and siRNAs to macrophages. siRNA delivery and transfection and analysis of macrophages in vivo can be accomplished within 36 h.
Assuntos
Macrófagos/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ribonuclease III/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Camundongos , RNA Interferente Pequeno/química , Especificidade por Substrato , Fatores de Tempo , TransfecçãoRESUMO
To determine mechanistically how siRNAs mediate transcriptional gene silencing (TGS) in human cells, we have measured histone methylation at targeted promoters, the dependency on active transcription, and whether or not both strands of the siRNA are required for siRNA-mediated TGS. We report here that siRNA treatment increases both H3K9 and H3K27 methylation of the targeted EF1A promoter and that this increase is dependent on nuclear specific delivery of the siRNA. We also find that TGS can be directed by the antisense strand alone, and requires active transcription by RNA polymerase II in human cells as evidenced by sensitivity to alpha-amanatin. The observation of antisense strand-specific siRNA-mediated TGS of EF1A was substantiated by targeting the U3 region of the HIV-1 LTR/promoter. Furthermore, we show that the antisense strand of siRNA EF52 associates with the transiently expressed Flag-tagged DNMT3A, the targeted EF1A promoter, and trimethylated H3K27. The observations reported here implicate a functional link between siRNA-mediated targeting of genomic regions (promoters), RNA Pol II function, histone methylation, and DNMT3A and support a paradigm in which the antisense strands of siRNAs alone can direct sequence-specific transcriptional gene silencing in human cells.
Assuntos
Histonas/metabolismo , Interferência de RNA , RNA Antissenso/metabolismo , RNA Interferente Pequeno/metabolismo , Amanitinas/farmacologia , Sequência de Bases , Células Cultivadas , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA Metiltransferase 3A , Repetição Terminal Longa de HIV , Histonas/genética , Humanos , Metilação , Dados de Sequência Molecular , Proteínas Nucleares/genética , Inibidores da Síntese de Ácido Nucleico/farmacologia , Regiões Promotoras Genéticas , Interferência de RNA/efeitos dos fármacos , RNA Polimerase II/efeitos dos fármacos , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , RNA Antissenso/genética , RNA Interferente Pequeno/genética , Fatores de Transcrição/genéticaRESUMO
The Src family kinase Lck is essential for T cell Ag receptor-mediated signaling. In this study, we report the effects of acute elimination of Lck in Jurkat TAg and primary T cells using RNA interference mediated by short-interfering RNAs. In cells with Lck knockdown (kd), proximal TCR signaling was strongly suppressed as indicated by reduced zeta-chain phosphorylation and intracellular calcium mobilization. However, we observed sustained and elevated phosphorylation of ERK1/2 in Lck kd cells 30 min to 2 h after stimulation. Downstream effects on immune function as determined by activation of a NFAT-AP-1 reporter, and TCR/CD28-stimulated IL-2 secretion were strongly augmented in Jurkat and primary T cells, respectively. As expected, overexpression of SHP-1 in Jurkat cells inhibited TCR-induced NFAT-AP-1 activation, but this effect could be overcome by simultaneous kd of Lck. Furthermore, acute elimination of Lck also suppressed TCR-mediated activation of SHP-1, suggesting the possible role of SHP-1 in a negative feedback loop originating from Lck. This report underscores Lck as an important mediator of proximal TCR signaling, but also indicates a suppressive role on downstream immune function.
Assuntos
Proteína Tirosina Quinase p56(lck) Linfócito-Específica/deficiência , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Northern Blotting , Antígenos CD28/imunologia , Antígenos CD28/metabolismo , Cálcio/metabolismo , Linhagem Celular Tumoral , Ativação Enzimática/imunologia , Humanos , Imunoprecipitação , Interleucina-2/imunologia , Interleucina-2/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Células Jurkat , Proteína Quinase 1 Ativada por Mitógeno/imunologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/imunologia , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fatores de Transcrição NFATC/imunologia , Fatores de Transcrição NFATC/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Tirosina Fosfatases/imunologia , Proteínas Tirosina Fosfatases/metabolismo , RNA Interferente Pequeno , TransfecçãoRESUMO
Randomly designed siRNA targeting different positions within the same mRNA display widely differing activities. We have performed a statistical analysis of 46 siRNA, identifying various features of the 19bp duplex that correlate significantly with functionality at the 70% knockdown level and verified these results against an independent data set of 34 siRNA recently reported by others. Features that consistently correlated positively with functionality across the two data sets included an asymmetry in the stability of the duplex ends (measured as the A/U differential of the three terminal basepairs at either end of the duplex) and the motifs S1, A6, and W19. The presence of the motifs U1 or G19 was associated with lack of functionality. A selection algorithm based on these findings strongly differentiated between the two functional groups of siRNA in both data sets and proved highly effective when used to design siRNA targeting new endogenous human genes.
Assuntos
Algoritmos , Inativação Gênica , Modelos Genéticos , Modelos Estatísticos , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , Análise de Sequência de RNA/métodos , Relação Estrutura-Atividade , Animais , Composição de Bases/genética , Sequência de Bases , Células COS , Chlorocebus aethiops , Perfilação da Expressão Gênica/métodos , Dados de Sequência Molecular , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Alinhamento de Sequência/métodos , Homologia de Sequência do Ácido NucleicoRESUMO
Tissue factor (TF) is the membrane receptor of the serine protease coagulation factor VIIa (FVIIa). Formation of the TF/FVIIa complex initiates the coagulation cascade. We used short hairpin RNA (shRNA)-mediated RNA interference to knock down TF expression in the human metastatic melanoma cell line LOX-L. After transfection with the shRNA construct, 3 stable clones with significantly downregulated TF expression were established. They exhibited decreased proliferation in vitro as determined by (14)C thymidine incorporation and soft agar assay. The in vivo metastatic potential was assessed in an experimental pulmonary metastasis model in which cells from different clones were injected into the tail vein of nude mice. The incidence of pulmonary tumors was significantly lower in mice receiving shRNA-expressing cells (33% +/- 15%) than in control mice injected with wild-type cells or cells stably transfected with empty expression vector (90% +/- 10%). The mice injected with TF-downregulated cells had markedly longer survival time (69 +/- 17 days) compared to the control mice (35.6 +/- 5 days; p = 0.03). Thus, reduction of TF levels in LOX-L cells significantly delayed and reduced lung tumor formation. As a first step in elucidating the molecular basis for this effect, we compared the global gene expression profile in TF-downregulated cells and control cells by using cDNA microarray analysis. Forty-four known human genes were found to be significantly upregulated (> 2-fold; p < 0.05) and 228 genes significantly downregulated (>or= 3-fold; p < 0.05) in TF-downregulated cells compared to control cells. The differentially expressed genes encode proteins functioning in transcription, translation, cell communication and cell growth/death. The results provide a basis for investigating molecular mechanisms underlying the effects of TF on the metastatic capacity of LOX-L melanoma cells.
Assuntos
Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Melanoma/metabolismo , Melanoma/secundário , Tromboplastina/metabolismo , Animais , Linhagem Celular Tumoral , DNA de Neoplasias/análise , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Humanos , Incidência , Camundongos , Camundongos Nus , Análise de Sequência com Séries de Oligonucleotídeos , RNA Neoplásico/metabolismo , Serina Endopeptidases/metabolismo , Taxa de Sobrevida , Tromboplastina/genética , TransfecçãoRESUMO
C-terminal Src kinase (Csk) controls the Src family kinase Lck, which is essential for T cell antigen receptor (TCR)-mediated signaling. For the first time, we here report the effects of acute elimination of Csk in Jurkat T cells and primary T cells using short interfering (si) RNA. In both cell types, 70-85% knockdown of Csk was achieved within 48 h. No alterations in surface expression of CD3, CD4 or CD8, or in Lck protein level were observed. Phosphorylation of Y505 in Lck was markedly reduced and a concomitant 4-5-fold increase in Lck Y394 phosphorylation was observed both in normal and Jurkat T cells. Kinase assays revealed 2-3-fold higher Lck activity. In Jurkat cells, basal levels of zeta chain phosphorylation were elevated, and spontaneous NFAT-AP-1 activation occurred, indicating aberrant Lck kinase activity. After TCR triggering, Csk knockdown cells revealed faster and stronger, but not sustained, phosphorylation of Lck Y394 and zeta chains compared to control. TCR-induced activation of NFAT-AP-1 and TCR/CD28-stimulated IL-2 secretion occurred at weaker stimuli and with augmented responses in Csk knockdown Jurkat and primary T cells, respectively. Altogether, these data suggest that acute elimination of Csk in T cells without evolution of compensatory mechanisms results in aberrant Lck activity and augmented TCR-stimulated responses.