Drosophila U6 promoter-driven short hairpin RNAs effectively induce RNA interference in Schneider 2 cells

The effect of RNA interference (RNAi) is generally more potent in Drosophila Schneider 2 (S2) cells than in mammalian cells. In mammalian cells, PolIII promoter-based DNA vectors can be used to express small interfering RNA (siRNA) or short hairpin RNA (shRNA); however, this has not been demonstrated in cultured Drosophila cells. Here we show that shRNAs transcribed from the Drosophila U6 promoter can efficiently trigger gene silencing in S2 cells. By targeting firefly luciferase mRNA, we assessed the efficacy of the shRNAs and examined the structural requirements for highly effective shRNAs. The silencing effect was dependent on the length of the stem region and the sequence of the loop region. Furthermore, we demonstrate that the expression of the endogenous cyclin E protein can be repressed by the U6 promoter-driven shRNAs. Drosophila U6 promoter-based shRNA expression systems may permit stable gene silencing in S2 cells.     

Potent RNAi by short RNA triggers

RNA interference (RNAi) is a gene-silencing mechanism by which a ribonucleoprotein complex, the RNA-induced silencing complex (RISC) and a double-stranded (ds) short-interfering RNA (siRNA), targets a complementary mRNA for site-specific cleavage and subsequent degradation. While longer dsRNA are endogenously processed into 21- to 24-nucleotide (nt) siRNAs or miRNAs to induce gene silencing, RNAi studies in human cells typically use synthetic 19- to 20-nt siRNA duplexes with 2-nt overhangs at the 3′-end of both strands. Here, we report that systematic synthesis and analysis of siRNAs with deletions at the passenger and/or guide strand revealed a short RNAi trigger, 16-nt siRNA, which induces potent RNAi in human cells. Our results indicate that the minimal requirement for dsRNA to trigger RNAi is an ~42 Å A-form helix with ~1.5 helical turns. The 16-nt siRNA more effectively knocked down mRNA and protein levels than 19-nt siRNA when targeting the endogenous CDK9 gene, suggesting that 16-nt siRNA is a more potent RNAi trigger. In vitro kinetic analysis of RNA-induced silencing complex (RISC) programmed in HeLa cells indicates that 16-nt siRNA has a higher RISC-loading capacity than 19-nt siRNA. These results suggest that RISC assembly and activation during RNAi does not necessarily require a 19-nt duplex siRNA and that 16-nt duplexes can be designed as more potent triggers to induce RNAi.     

U6 promoter-driven siRNAs with four uridine 3' overhangs efficiently suppress targeted gene expression in mammalian cells

The first evidence for gene disruption by double-stranded RNA (dsRNA) came from careful analysis in Caenorhabditis elegans. This phenomenon, called RNA interference (RNAi), was observed subsequently in various organisms, including plants, nematodes, Drosophila, and protozoans. Very recently, it has been reported that in mammalian cells, 21- or 22-nucleotide (nt) RNAs with 2-nt 3' overhangs (small inhibitory RNAs, siRNAs) exhibit an RNAi effect. This is because siRNAs are not recognized by the well-characterized host defense system against viral infections, involving dsRNA-dependent inhibition of protein synthesis. However, the current method for introducing synthetic siRNA into cells by lipofection restricts the range of applications of RNAi as a result of the low transfection efficiencies in some cell types and/or short-term persistence of silencing effects. Here, we report a vector-based siRNA expression system that can induce RNAi in mammalian cells. This technical advance for silencing gene expression not only facilitates a wide range of functional analysis of mammalian genes but might also allow therapeutic applications by means of vector-mediated RNAi.     

Hairpin RNAs derived from RNA polymerase II and polymerase III promoter-directed transgenes are processed differently in plants

RNA polymerase III (Pol III) as well as Pol II (35S) promoters are able to drive hairpin RNA (hpRNA) expression and induce target gene silencing in plants. siRNAs of 21 nt are the predominant species in a 35S Pol II line, whereas 24- and/or 22-nucleotide (nt) siRNAs are produced by a Pol III line. The 35S line accumulated the loop of the hpRNA, in contrast to full-length hpRNA in the Pol III line. These suggest that Pol II and Pol III-transcribed hpRNAs are processed by different pathways. One Pol III transgene produced only 24-nt siRNAs but silenced the target gene efficiently, indicating that the 24-nt siRNAs can direct mRNA degradation; specific cleavage was confirmed by 5' rapid amplification of cDNA ends (RACE). Both Pol II- and Pol III-directed hpRNA transgenes induced cytosine methylation in the target DNA. The extent of methylation is not correlated with the level of 21-nt siRNAs, suggesting that they are not effective inducers of DNA methylation. The promoter of a U6 transgene was significantly methylated, whereas the promoter of the endogenous U6 gene was almost free of cytosine methylation, suggesting that endogenous sequences are more resistant to de novo DNA methylation than are transgene constructs.     

In vitro analysis of RNA interference in Drosophila melanogaster

Double-stranded RNA (dsRNA) triggers the destruction of mRNA sharing sequence with the dsRNA, a phenomenon termed RNA interference (RNAi). The dsRNA is converted by endonucleolytic cleavage into 21- to 23-nt small interfering RNAs (siRNAs), which direct a multiprotein complex, the RNA-induced silencing complex to cleave RNA complementary to the siRNA. RNAi can be recapitulated in vitro in lysates of syncytial blastoderm Drosophila embryos. These lysates reproduce all of the known steps in the RNAi pathway in flies and mammals. Here we explain how to prepare and use Drosophila embryo lysates to dissect the mechanism of RNAi.     

Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference

In the present study, the relationship between short interfering RNA (siRNA) sequence and RNA interference (RNAi) effect was extensively analyzed using 62 targets of four exogenous and two endogenous genes and three mammalian and Drosophila cells. We present the rules that may govern siRNA sequence preference and in accordance with which highly effective siRNAs essential for systematic mammalian functional genomics can be readily designed. These rules indicate that siRNAs which simultaneously satisfy all four of the following sequence conditions are capable of inducing highly effective gene silencing in mammalian cells: (i) A/U at the 5′ end of the antisense strand; (ii) G/C at the 5′ end of the sense strand; (iii) at least five A/U residues in the 5′ terminal one-third of the antisense strand; and (iv) the absence of any GC stretch of more than 9 nt in length. siRNAs opposite in features with respect to the first three conditions give rise to little or no gene silencing in mammalian cells. Essentially the same rules for siRNA sequence preference were found applicable to DNA-based RNAi in mammalian cells and in ovo RNAi using chick embryos. In contrast to mammalian and chick cells, little siRNA sequence preference could be detected in Drosophila in vivo RNAi.     

In vitro analysis of RNA interference in Drosophila melanogaster

Double-stranded RNA (dsRNA) triggers the destruction of mRNA sharing sequence with the dsRNA, a phenomenon termed RNA interference (RNAi). The dsRNA is converted by endonucleolytic cleavage into 21- to 23-nt small interfering RNAs (siRNAs), which direct a multiprotein complex, the RNA-induced silencing complex to cleave RNA complementary to the siRNA. RNAi can be recapitulated in vitro in lysates of syncytial blastoderm Drosophila embryos. These lysates reproduce all of the known steps in the RNAi pathway in flies and mammals. Here we explain how to prepare and use Drosophila embryo lysates to dissect the mechanism of RNAi.     

Modified 27-nt dsRNAs with dramatically enhanced stability in serum and long-term RNAi activity

The present study describes improved properties of 27-nt dsRNAs over 21-nt siRNAs, and accents on the possibility to use their modifications and conjugates for direct long-term gene silencing in viable cells and animals, avoiding conventional transfectants. Using a Renilla Luciferase gene-silencing system and cultured cell lines, we established that 27-nt dsRNAs possessed about three to five times higher "long-term" RNAi activity than 21-nt siRNAs and 21-nt dsRNAs. Moreover, if RNA duplexes were preincubated with cell-cultured medium for several hours before their transfection in cells, 21-mer completely lost its RNAi effect, while 27-mer, its amino modifications, thiol modifications, and cholesterol conjugates manifested a strong gene silencing. In attempts to clarify the reason(s) for the higher RNAi activity of 27-nt dsRNAs, we found that they were approximately 100 times more stable than 21-nt siRNA and 21-nt dsRNA in cell-cultured medium supplemented with 10% inactivated serum, approximately 50 times more stable in 90% inactivated serum, and approximately six times more stable in active serum. The 5' sense modification was selected as the most stable, accessible to Dicer, and with highest RNAi potential. The RNAi activity of 5' sense modifications was higher even than the activity of nonmodified 27-nt dsRNA. The 5' sense amino modification also did not influence the activity of 21-nt siRNA, right overhang 25/27-nt (R25D/27), and 25D/27-nt RNAs. The stability of 5' sense modified R25D/27-nt and 25D/27-nt RNAs in serum was lower than that of blunt 27-nt dsRNA. However, these asymmetric RNAs were more active than modified and nonmodified blunt 27-nt dsRNAs, which demonstrates the superiority of the asymmetric design. The 5' sense modifications were considered as most appropriate for conjugation with small signal molecules to facilitate the intracellular delivery of RNA duplex, to preserve its RNAi capacity, and to ensure a possibility for rapid long-term gene silencing in viable cells and animals. The 5' sense conjugation with cholesterol approved this assumption.     

Analysis of gene function in somatic mammalian cells using small interfering RNAs

RNA interference (RNAi) is a highly conserved gene silencing mechanism that uses double-stranded RNA (dsRNA) as a signal to trigger the degradation of homologous mRNA. The mediators of sequence-specific mRNA degradation are 21- to 23-nt small interfering RNAs (siRNAs) generated by ribonuclease III cleavage from longer dsRNAs. Twenty-one-nucleotide siRNA duplexes trigger specific gene silencing in mammalian somatic cells without activation of the unspecific interferon response. Here we provide a collection of protocols for siRNA-mediated knockdown of mammalian gene expression. Because of the robustness of the siRNA knockdown technology, genomewide analysis of human gene function in cultured cells has now become possible.     

A novel siRNA validation system for functional screening and identification of effective RNAi probes in mammalian cells

Small interfering RNAs (siRNAs) have become the most powerful and widely used gene silencing reagents for reverse functional genomics and molecular therapeutics. The key challenge for achieving effective gene silencing in particular for the purpose of the therapeutics is primarily dependent on the effectiveness and specificity of the RNAi targeting sequence. However, only a limited number of siRNAs is capable of inducing highly effective and sequence-specific gene silencing by RNA interference (RNAi) mechanism. In addition, the efficacy of siRNA-induced gene silencing can only be experimentally measured based on inhibition of the target gene expression. Therefore, it is important to establish a fully robust and comparative validating system for determining the efficacy of designed siRNAs. In this study, we have developed a reliable and quantitative reporter-based siRNA validation system that consists of a short synthetic DNA fragment containing an RNAi targeting sequence of interest and two expression vectors for targeting reporter and triggering siRNA expression. The efficacy of the siRNAs is measured by their abilities to inhibit expression of the targeting reporter gene with easily quantified readouts including enhanced green fluorescence protein (EGFP) and firefly luciferase. Using fully analyzed siRNAs against human hepatitis B virus (HBV) surface antigen (HBsAg) and tumor suppressor protein p53, we have demonstrated that this system could effectively and faithfully report the efficacy of the corresponding siRNAs. In addition, we have further applied this system for screening and identification of the highly effective siRNAs that could specifically inhibit expression of mouse matrix metalloproteinase-7 (MMP-7), Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1), and human serine/threonine kinase AKT1. Since only a readily available short synthetic DNA fragment is needed for constructing this novel reporter-based siRNA validation system, this system not only provides a powerful strategy for screening highly effective siRNAs but also implicates in the use of RNAi for studying novel gene function in mammals.     

Silencing of HIV-1 gene expression by siRNAs in transduced cells

The RNA interference (RNAi) phenomenon is a recently observed process in which the introduction of a double-stranded RNA (dsRNA) into cells causes the specific degradation of an mRNA containing the same sequence. To study dsRNA-mediated gene interference targeted to the env gene (NL4-3: 7490-7508) in HIV-1 infected cells, we constructed tandem-type and hairpin-type siRNA expression vectors, which were under the control of two U6 promoters. We also constructed lentiviral-based siRNA expression vectors for further assessment of their antiviral activity in transduced cells. At both the transient plasmid and lentiviral-mediated RNA expression levels, the siRNA encoding the env fragment exhibited sequence-specific suppression of target gene expression and strongly inhibited (> or = 90%) HIV-1 infection in the cells, as compared to the antisense RNA expression vector. Targeting the HIV-1 env gene with siRNAs encoding the env gene fragment (7490-7508) might be an effective strategy for gene therapy applications in HIV-1/AIDS treatment and management.     

Silencing of HIV-1 Gene Expression by Sirnas in Transduced Cells

The RNA interference (RNAi) phenomenon is a recently observed process in which the introduction of a double-stranded RNA (dsRNA) into cells causes the specific degradation of an mRNA containing the same sequence. To study dsRNA-mediated gene interference targeted to the env gene (NL4-3: 7490-7508) in HIV-1 infected cells, we constructed tandem-type and hairpin-type siRNA expression vectors, which were under the control of two U6 promoters. We also constructed lentiviral-based siRNA expression vectors for further assessment of their antiviral activity in transduced cells. At both the transient plasmid and lentiviral-mediated RNA expression levels, the siRNA encoding the env fragment exhibited sequence-specific suppression of target gene expression and strongly inhibited (≥90%) HIV-1 infection in the cells, as compared to the antisense RNA expression vector. Targeting the HIV-1 env gene with siRNAs encoding the env gene fragment (7490–7508) might be an effective strategy for gene therapy applications in HIV-1/AIDS treatment and management.     

Transcriptional targeting of small interfering RNAs into cancer cells

Small interfering RNAs (siRNAs) are widely used for analyzing gene function and have the potential to be developed into human therapeutics. However, persistent siRNA expression in normal cells may cause toxic side effects. Therefore, the therapeutic applications of RNAi in cancer require either the specific delivery of synthetic siRNAs into cancer cells or the control of siRNA expression. Accordingly, we have developed a cancer-specific vector that expresses siRNAs from the human survivin promoter. A plasmid vector expressing siRNAs under this promoter enabled efficient gene silencing of gene expression in different cancer cell lines. The levels of inhibition were comparable to that obtained with the constitutively active U6 promoter. By contrast to U6 promoter, no significant gene silencing was obtained with the Survivin promoter in normal mammary epithelial cells. Collectively, these data indicate that the survivin promoter is suitable for directing siRNA expression in cancer cells, but not normal cells.