A virus-derived short hairpin RNA confers resistance against sugarcane mosaic virus in transgenic sugarcane

RNA interference (RNAi) is commonly used to produce virus tolerant transgenic plants. The objective of the current study was to generate transgenic sugarcane plants expressing a short hairpin RNAs (shRNA) targeting the coat protein (CP) gene of sugarcane mosaic virus (SCMV). Based on multiple sequence alignment, including genomic sequences of four SCMV strains, a conserved region of ~ 456 bp coat protein (CP) gene was selected as target gene and amplified through polymerase chain reaction (PCR). Subsequently, siRNAs2 and siRNA4 were engineered as stable short hairpin (shRNA) transgenes of 110 bp with stem and loop sequences derived from microRNA (sof-MIR168a; an active regulatory miRNA in sugarcane). These transgenes were cloned in independent RNAi constructs under the control of the polyubiquitin promoter. The RNAi constructs were delivered into two sugarcane cultivars ‘SPF-234 and NSG-311 in independent experiments using particle bombardment. Molecular identification through PCR and Southern blot revealed anti-SCMV positive transgenic lines. Upon mechanical inoculation of transgenic and non-transgenic sugarcane lines with SCMV, the degree of resistance was found variable among the two sugarcane cultivars. For sugarcane cultivar NSG-311, the mRNA expression of the CP–SCMV was reduced to 10% in shRNA2-transgenic lines and 80% in shRNA4-transgenic lines. In sugarcane cultivar SPF-234, the mRNA expression of the CP–SCMV was reduced to 20% in shRNA2-transgenic lines and 90% in shRNA4 transgenic lines, revealing that transgenic plants expressing shRNA4 were almost immune to SCMV infection.     

Coexpression of Escherichia coli RNase III in silkworm cells improves the efficiency of RNA interference induced by long hairpin dsRNAs

Abstract Long hairpin dsRNA transcribed from chromosomal DNA can induce RNA interference in Bombyx mori cells, although its gene silencing efficiency is lower than that of exogenously introduced double‐stranded RNAs (dsRNAs). To solve this problem, we monitored the nuclear cytoplasmic translocation of the transcribed hairpin dsRNA and analyzed the processing efficiency into mature small interfering RNA (siRNA). Northern blot analysis revealed that the transcribed hairpin dsRNAs were spliced and transported into the cytoplasm, but were not effectively diced into siRNAs. Interestingly, RNAi with hairpin dsRNAs from genome‐integrated IR transgene was stimulated by the coexpression of Escherichia coli RNase III, although this exogenous enzyme seemed to bring about nonspecific cleavage of cellular mRNA.     

pWormgatePro enables promoter-driven knockdown by hairpin RNA interference of muscle and neuronal gene products in Caenorhabditis elegans

Recent advances in genome research and RNA interference (RNAi) technology have accelerated the adoption of genome-wide experimental approaches for determining gene function in the model organism Caenorhabditis elegans. Despite recent successes, the application of RNAi is limited when gene knockdown causes complex phenotypes or embryonic lethality. Recently, the high-throughput pWormgate cloning system has been introduced as a tool to efficiently generate heat-shock-inducible hairpin RNA constructs using the Gateway recombination technology. We have modified pWormgate into a versatile hairpin cloning plasmid, pWormgatePro, which facilitates temporally and spatially inducible hairpin RNAi using constitutively active, tissue-specific promoters. To demonstrate its utility we knocked down unc-22 in body wall muscles as well as the axon guidance gene unc-5 in the nervous system indicating that promoter-driven hairpins can overcome the neuronal resistance to RNAi. Using pWormgatePro we also show that RNAi in the nervous system of C. elegans is non-autonomous and that spreading of the RNAi signal from neurons to muscle is substantially reduced but not abolished in spreading-defective sid-1 mutant animals. Our findings illustrate the effectiveness of pWormgatePro for gene silencing in muscle cells and neurons and bring forward the possibility of applying tissue-specific RNAi on a genome-wide scale.     

Comparison of bovine RNA polymerase III promoters for short hairpin RNA expression

RNA interference (RNAi) mediated by DNA-based expression of short hairpin RNA (shRNA) is a powerful method of sequence-specific gene knockdown. A number of vectors for expression of shRNA have been developed that feature promoters from RNA polymerase III (pol III)-transcribed genes of mouse or human origin. To advance the use of RNAi as a tool for functional genomic research and for future development of specific therapeutics in the bovine species, we have developed shRNA expression vectors that feature novel bovine RNA pol III promoters. We characterized two bovine U6 small nuclear RNA (snRNA) promoters (bU6-2 and bU6-3) and a bovine 7SK snRNA promoter (b7SK). We compared the efficiency of each of these promoters to express shRNA molecules. Promoter activity was measured in the context of RNAi by targeting and suppressing the reporter gene encoding enhanced green fluorescent protein. Results show that the b7SK promoter induced the greatest level of suppression in a range of cell lines. The comparison of these bovine promoters in shRNA expression is an important component for the future development of bovine-specific RNAi-based research.     

Delivery of short hairpin RNAs by transkingdom RNA interference modulates the classical ABCB1-mediated multidrug-resistant phenotype of cancer cells

Delivery of RNA interference (RNAi)-mediating agents to target cells is one of the major obstacles for the development of RNAi-based therapies. One strategy to overcome this barrier is transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells to induce RNAi. In this study, the tkRNAi approach was used for modulation of the “classical” ABCB1-mediated multidrug resistance (MDR) in human cancer cells. Subsequent to treatment with anti-ABCB1 shRNA expression vector bearing E. coli, MDR cancer cells (EPG85 257RDB) showed 45% less ABCB1 mRNA expression. ABCB1 protein expression levels were reduced to a point at which merely a weak band could be detected. Drug accumulation was enhanced 11-fold, to an extent that it reached 45% of the levels in non-resistant cells and resistance to daunorubicin was decreased by 40%. The data provide the proof-of-concept that tkRNAi is suitable for modulation of “classical” MDR in human cancer cells. Overall, the prototype tkRNAi system tested here did not yet attain the levels of gene silencing seen with conventional siRNAs nor virally delivered shRNAs; but the tkRNAi system for gene-silencing of ABCB1 is still being optimized, and may become a powerful tool for delivery of RNAi effectors for the reversal of cancer MDR in future.     

Two-step cleavage of hairpin RNA with 5' overhangs by human DICER

Background  

DICER is an RNase III family endoribonuclease that processes precursor microRNAs (pre-miRNAs) and long double-stranded RNAs, generating microRNA (miRNA) duplexes and short interfering RNA duplexes with 20~23 nucleotides (nts) in length. The typical form of pre-miRNA processed by the Drosha protein is a hairpin RNA with 2-nt 3' overhangs. On the other hand, production of mature miRNA from an endogenous hairpin RNA with 5' overhangs has also been reported, although the mechanism for this process is unknown.     

Detection of exogenous double‐stranded RNA movement in in vitro peanut plants

• New technologies are needed to eliminate mycotoxins and/or fungal pathogens from agricultural products. RNA interference (RNAi) has shown potential to control fungi associated with crops. In RNAi, double‐stranded RNA (dsRNA) targets homologous mRNA for cleavage, and can reach the mRNA of pathogens in contact with the plant. The key element in this process is the movement of RNA signals cell‐to‐cell and over long distances within the plant, and between host plants and parasites.
• In this study, we selected a regulatory gene in the aflatoxin biosynthesis pathway, aflS/aflR, necessary for the production of aflatoxins in Aspergillus spp. We designed a Dicer‐substrate RNA (DsiRNA) to study the movement and stability of the duplex over time in in vitro peanut plants using stem‐loop primers and RT‐PCR for DsiRNA detection.
• The preliminary results demonstrated that DsiRNA was absorbed and moved away from the point of application, spread systemically and was transported rapidly, most likely through the phloem of the shoot, to the sink tissues, such as new auxiliary shoots, flowers and newly formed pegs. The DsiRNA remained detectable for at least 30 days after treatment.
• This is the first time that movement of exogenous DsiRNA in in vitro peanut plants has been described. Since DsiRNA was detectable in the pegs 15 days after treatment, aflatoxin reduction may be possible if the duplexes containing part of the aflatoxin biosynthesis pathogen gene induce silencing in the peanut seeds colonised by Aspergillus spp. The application of small RNAs could be a non‐transformative option for mycotoxin contamination control.

     

Properties of gene knockdown system by vector‐based siRNA in zebrafish

RNA interference (RNAi) has emerged as a powerful tool to silence specific genes. Vector‐based RNAi systems have been developed to downregulate targeted genes in a spatially and temporally regulated fashion both in vitro and in vivo. The zebrafish (Danio rerio) is a model animal that has been examined based on a wide variety of biological techniques, including embryonic manipulations, forward and reverse genetics, and molecular biology. However, a heritable and tissue‐specific knockdown of gene expression has not yet been developed in zebrafish. We examined two types of vector, which produce small interfering RNA (siRNA), the direct effector in RNAi system; microRNA (miRNA) process mimicking vectors with a promoter for RNA polymerase II and short hairpin RNA (shRNA) expressing vector through a promoter for RNA polymerase III. Though gene‐silencing phenotypes were not observed in the miRNA process mimicking vectors, the transgenic embryos of the second vector (Tg(zU6‐shGFP)), shRNA expressing vector for enhanced green fluorescence protein, revealed knockdown of the targeted gene. Interestingly, only the embryos from Tg(zU6‐shGFP) female but not from the male fish showed the downregulation. Comparison of the quantity of siRNA produced by each vector indicates that the vectors tested here induced siRNA, but at low levels barely sufficient to silence the targeted gene.     

RNA interference by small hairpin RNAs synthesised under control of the human 7S K RNA promoter

Small interfering RNAs (siRNAs) represent RNA duplexes of 21 nucleotides in length that inhibit gene expression. We have used the human gene-external 7S K RNA promoter for synthesis of short hairpin RNAs (shRNAs) which efficiently target human lamin mRNA via RNA interference (RNAi). Here we demonstrate that orientation of the target sequence within the shRNA construct is important for interference. Furthermore, effective interference also depends on the length and/or structure of the shRNA. Evidence is presented that the human 7S K promoter is more active in vivo than other gene-external promoters, such as the human U6 small nuclear RNA (snRNA) gene promoter.