Induction of gene silencing by hairpin RNA expression in Tetrahymena thermophila reveals a second small RNA pathway

Unlike in other eukaryotes, in which it causes gene silencing, RNA interference (RNAi) has been linked to programmed DNA deletion in the ciliate Tetrahymena thermophila. Here we have developed an efficient method to inducibly express double-stranded RNA hairpins and demonstrated that they cause gene silencing through targeted mRNA degradation in all phases of the life cycle, including growth, starvation, and mating. This technique offers a new tool for gene silencing in this model organism. Induction of RNA hairpins causes dramatic upregulation of Dicer and Argonaute family genes, revealing a system capable of rapidly responding to double-stranded RNA. These hairpins are processed into 23- to 24-nucleotide (nt) small RNAs, which are distinctly different from the 28- to 30-nt small RNAs known to be associated with DNA deletion. Thus, two different small RNA pathways appear to be responsible for gene silencing and DNA deletion. Surprisingly, expression of the RNA hairpin also causes targeted DNA deletion during conjugation, although at low efficiencies, which suggests a possible crossover of these two molecular paths.     

RNA interference in mammalian cells using siRNAs synthesized with T7 RNA polymerase

Methods that allow the specific silencing of a desired gene are invaluable tools for research. One of these is based on RNA interference (RNAi), a process by which double-stranded RNA (dsRNA) specifically suppresses the expression of a target mRNA. Recently, it has been reported that RNAi also works in mammalian cells if small interfering RNAs (siRNAs) are used to avoid activation of the interferon system by long dsRNA. Thus, RNAi could become a major tool for reverse genetics in mammalian systems. However, the high cost and the limited availability of the short synthetic RNAs and the lack of certainty that a designed siRNA will work present major drawbacks of the siRNA technology. Here we present an alternative method to obtain cheap and large amounts of siRNAs using T7 RNA polymerase. With multiple transfection procedures, including calcium phosphate co-precipitation, we demonstrate silencing of both exogenous and endogenous genes.     

Pol II-expressed shRNA knocks down Sod2 gene expression and causes phenotypes of the gene knockout in mice

RNA interference (RNAi) has been used increasingly for reverse genetics in invertebrates and mammalian cells, and has the potential to become an alternative to gene knockout technology in mammals. Thus far, only RNA polymerase III (Pol III)-expressed short hairpin RNA (shRNA) has been used to make shRNA-expressing transgenic mice. However, widespread knockdown and induction of phenotypes of gene knockout in postnatal mice have not been demonstrated. Previous studies have shown that Pol II synthesizes micro RNAs (miRNAs)-the endogenous shRNAs that carry out gene silencing function. To achieve efficient gene knockdown in mammals and to generate phenotypes of gene knockout, we designed a construct in which a Pol II (ubiquitin C) promoter drove the expression of an shRNA with a structure that mimics human miRNA miR-30a. Two transgenic lines showed widespread and sustained shRNA expression, and efficient knockdown of the target gene Sod2. These mice were viable but with phenotypes of SOD2 deficiency. Bigenic heterozygous mice generated by crossing these two lines showed nearly undetectable target gene expression and phenotypes consistent with the target gene knockout, including slow growth, fatty liver, dilated cardiomyopathy, and premature death. This approach opens the door of RNAi to a wide array of well-established Pol II transgenic strategies and offers a technically simpler, cheaper, and quicker alternative to gene knockout by homologous recombination for reverse genetics in mice and other mammalian species.     

RNA-mediated gene silencing in the phytopathogenic fungus Bipolaris oryzae

The Ascomycetous fungus Bipolaris oryzae is the causal agent of brown leaf spot disease in rice and is a model for studying photomorphogenetic responses by near-UV radiation. Targeted gene disruption (knockout) for functional analysis of photomorphogenesis-related genes in B. oryzae can be achieved by homologous recombination with low efficiency. Here, the applicability of RNA silencing (knockdown) as a tool for targeting endogenous genes in B. oryzae is reported. A polyketide synthase gene (PKS1), involved in fungal DHN melanin biosynthesis pathways, was targeted by gene silencing as a marker. The silencing vector encoding hairpin RNA of the PKS1 fragment was constructed in a two-step PCR-based cloning, and introduced into the B. oryzae genomic DNA. Silencing of the PKS1 gene resulted in albino phenotypes and reduction of PKS1 mRNA expression. These results demonstrate the applicability of targeted gene silencing as a useful reverse-genetics approach in B. oryzae.     

Pol II–Expressed shRNA Knocks Down Sod2 Gene Expression and Causes Phenotypes of the Gene Knockout in Mice

RNA interference (RNAi) has been used increasingly for reverse genetics in invertebrates and mammalian cells, and has the potential to become an alternative to gene knockout technology in mammals. Thus far, only RNA polymerase III (Pol III)–expressed short hairpin RNA (shRNA) has been used to make shRNA-expressing transgenic mice. However, widespread knockdown and induction of phenotypes of gene knockout in postnatal mice have not been demonstrated. Previous studies have shown that Pol II synthesizes micro RNAs (miRNAs)—the endogenous shRNAs that carry out gene silencing function. To achieve efficient gene knockdown in mammals and to generate phenotypes of gene knockout, we designed a construct in which a Pol II (ubiquitin C) promoter drove the expression of an shRNA with a structure that mimics human miRNA miR-30a. Two transgenic lines showed widespread and sustained shRNA expression, and efficient knockdown of the target gene Sod2. These mice were viable but with phenotypes of SOD2 deficiency. Bigenic heterozygous mice generated by crossing these two lines showed nearly undetectable target gene expression and phenotypes consistent with the target gene knockout, including slow growth, fatty liver, dilated cardiomyopathy, and premature death. This approach opens the door of RNAi to a wide array of well-established Pol II transgenic strategies and offers a technically simpler, cheaper, and quicker alternative to gene knockout by homologous recombination for reverse genetics in mice and other mammalian species.     

Lentivirus-delivered stable gene silencing by RNAi in primary cells

Genome-wide genetic approaches have proven useful for examining pathways of biological significance in model organisms such as Saccharomyces cerevisiae, Drosophila melanogastor, and Caenorhabditis elegans, but similar techniques have proven difficult to apply to mammalian systems. Although manipulation of the murine genome has led to identification of genes and their function, this approach is laborious, expensive, and often leads to lethal phenotypes. RNA interference (RNAi) is an evolutionarily conserved process of gene silencing that has become a powerful tool for investigating gene function by reverse genetics. Here we describe the delivery of cassettes expressing hairpin RNA targeting green fluorescent protein (GFP) using Moloney leukemia virus-based and lentivirus-based retroviral vectors. Both transformed cell lines and primary dendritic cells, normally refractory to transfection-based gene transfer, demonstrated stable silencing of targeted genes, including the tumor suppressor gene TP53 in normal human fibroblasts. This report demonstrates that both Moloney leukemia virus and lentivirus vector-mediated expression of RNAi can achieve effective, stable gene silencing in diverse biological systems and will assist in elucidating gene functions in numerous cell types including primary cells.     

Gene silencing by RNA interference in the koji mold Aspergillus oryzae

We found the orthologous genes required for RNA interference (RNAi) in the Aspergillus oryzae genome database, and constructed a set of tools for gene silencing using RNAi in A. oryzae. This system utilizes compatible restriction enzyme sites so that only a single target gene fragment is required to create the hairpin RNA cassette. For ease of handling, we also separated the construction of the hairpin RNA cassette for the target gene from its subsequent introduction into the expression vector. Using the brlA gene as a target for RNAi, we detected decreased mRNA levels and a delayed conidiation phenotype in the transformants. Furthermore, even though A. oryzae possesses three copies of the alpha-amylase gene, a single copy of an alpha-amylase RNAi construct was sufficient to downregulate the mRNA levels and decrease the enzymatic activity to 10% of control levels. Gene silencing by RNAi should provide a powerful genetic tool for post-genomic studies of the industrially important fungus A. oryzae.     

A technique to enzymatically construct libraries which express short hairpin RNA of arbitrary stem length

Short interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) usually used for RNA interference (RNAi) are double-stranded RNAs (dsRNAs) of 21 base pairs. However, siRNAs and shRNAs of longer stem length have been reported to show more potent gene silencing. Here, we report a new technique to enzymatically construct shRNA libraries containing clones from firefly luciferase cDNA and Jurkat cDNA. The technique allowed the efficacious generation of shRNAs of arbitrary stem length as desired, providing the clones which potently silenced the specified gene expression and presenting a high efficiency rate of gene silencing. Our results indicate that the technique permits the rapid, efficient, and low-cost preparation of genomewide shRNA expression libraries not only for humans and mice but also for sorts of biological species and that the relevant libraries are applicable for the search of genes related to phenotype changes and of new targets for drug discovery.     

Schistosoma japonicum: inhibition of Mago nashi gene expression by shRNA-mediated RNA interference

RNA interference (RNAi) mediated by short interfering RNA (siRNA) is a powerful reverse genetics tool and holds enormous therapeutic potential for various diseases, including parasite infections. siRNAs bind their complementary mRNA and lead to degradation of their specific mRNA targets. RNAi has been widely used for functional analysis of specific genes in various cells and organisms. In this paper, we tested the potential of silencing the expression of the Mago nashi gene in Schistosoma japonicum by siRNAs derived from shRNA expressed by mammalian Pol III promoter H1. Schistosomula, transformed from cercariae by mechanical shearing of the tails, were electroporated with Mago nashi shRNA expression vector. Aliquots of parasites were harvested at days 1, 3, and 5 after electroporation, respectively. Levels of Mago nashi mRNA and protein were determined by RT-PCR and Western blotting analysis. The results showed that shRNA expressed from mammalian Pol III promoter H1 specifically reduced the levels of Mago nashi mRNA and proteins in S. japonicum. Changes in testicular lobes were apparent when parasites were introduced into mammalian hosts. Thus, vector-mediated gene silencing is applicable to S. japonicum, which provides a means for the functional analysis of genes in this organism.     

RNA complementary to the 5' UTR of mRNA triggers effective silencing in Saccharomyces cerevisiae

Conditional silencing of target genes in Saccharomyces cerevisiae by antisense RNAs expressed in vivo has been challenged. The MFalpha1::lacZ fusion present in S. cerevisiae SF51-3 was chosen as a model target, and fragments of this gene were cloned in reverse orientation into the expression vector pYES2, bearing the GAL1 promoter. Among the different antisense constructs tested, only the one complementary to the 5' UTR of target mRNA featured effective silencing. Nevertheless, the expression in vivo of this antisense RNA could not be properly tuned by the absence or presence of galactose in the culture medium. Accordingly, conditional silencing could not be attained by this antisense hosted into pYES2. On the contrary, cloning the same antisense construct into the expression vector pSAL4 yielded a fully conditional silencing linked to the control of antisense expression by the absence or presence of Cu(2+) into the culture medium.     

Gene silencing using micro-RNA designed hairpins

During RNA interference (RNAi), long dsRNA is processed to approximately 21 nt duplexes, short interfering RNAs (siRNAs), which silence genes through a mRNA degradation pathway. Small temporal RNAs (stRNAs) and micro-RNAs (miRNAs) are approximately 21 nt RNAs that are processed from endogenously encoded hairpin-structured precursors, and function to silence genes via translational repression. Here we report that synthetic hairpin RNAs that mimic siRNAs and miRNA precursor molecules can target a gene for silencing, and the mechanism of silencing appears to be through mRNA degradation and not translational repression. The sequence and structural configuration of these RNAs are important, and even slight modification in structure can affect the silencing activity of the hairpins. Furthermore, these RNAs are active when expressed by DNA vectors containing polymerase III promoters, opening the possibility for new approaches in stable RNAi-based loss of function studies.     

RNA interference-inducing hairpin RNAs in plants act through the viral defence pathway

RNA interference (RNAi) is widely used to silence genes in plants and animals. It operates through the degradation of target mRNA by endonuclease complexes guided by approximately 21 nucleotide (nt) short interfering RNAs (siRNAs). A similar process regulates the expression of some developmental genes through approximately 21 nt microRNAs. Plants have four types of Dicer-like (DCL) enzyme, each producing small RNAs with different functions. Here, we show that DCL2, DCL3 and DCL4 in Arabidopsis process both replicating viral RNAs and RNAi-inducing hairpin RNAs (hpRNAs) into 22-, 24- and 21 nt siRNAs, respectively, and that loss of both DCL2 and DCL4 activities is required to negate RNAi and to release the plant's repression of viral replication. We also show that hpRNAs, similar to viral infection, can engender long-distance silencing signals and that hpRNA-induced silencing is suppressed by the expression of a virus-derived suppressor protein. These findings indicate that hpRNA-mediated RNAi in plants operates through the viral defence pathway.     

Artificial microRNA-mediated virus resistance in plants

RNA silencing in plants is a natural defense system against foreign genetic elements including viruses. This natural antiviral mechanism has been adopted to develop virus-resistant plants through expression of virus-derived double-stranded RNAs or hairpin RNAs, which in turn are processed into small interfering RNAs (siRNAs) by the host's RNA silencing machinery. While these virus-specific siRNAs were shown to be a hallmark of the acquired virus resistance, the functionality of another set of the RNA silencing-related small RNAs, microRNAs (miRNAs), in engineering plant virus resistance has not been extensively explored. Here we show that expression of an artificial miRNA, targeting sequences encoding the silencing suppressor 2b of Cucumber mosaic virus (CMV), can efficiently inhibit 2b gene expression and protein suppressor function in transient expression assays and confer on transgenic tobacco plants effective resistance to CMV infection. Moreover, the resistance level conferred by the transgenic miRNA is well correlated to the miRNA expression level. Comparison of the anti-CMV effect of the artificial miRNA to that of a short hairpin RNA-derived small RNA targeting the same site revealed that the miRNA approach is superior to the approach using short hairpin RNA both in transient assays and in transgenic plants. Together, our data demonstrate that expression of virus-specific artificial miRNAs is an effective and predictable new approach to engineering resistance to CMV and, possibly, to other plant viruses as well.