Evidence that processed small dsRNAs may mediate sequence-specific mRNA degradation during RNAi in Drosophila embryos

BACKGROUND: RNA interference (RNAi) is a phenomenon in which introduced double-stranded RNAs (dsRNAs) silence gene expression through specific degradation of their cognate mRNAs. Recent analyses in vitro suggest that dsRNAs may be copied, or converted, into 21-23 nucleotide (nt) guide RNAs that direct the nucleases responsible for RNAi to their homologous mRNA targets. Such small RNAs are also associated with gene silencing in plants. RESULTS: We developed a quantitative single-embryo assay to examine the mechanism of RNAi in vivo. We found that dsRNA rapidly induced mRNA degradation. A fraction of dsRNAs were converted into 21-23 nt RNAs, and their time of appearance and persistence correlated precisely with inhibition of expression. The strength of RNAi increased disproportionately with increasing dsRNA length, but an 80bp dsRNA was capable of effective gene silencing. RNAi was saturated at low dsRNA concentration and inhibited by excess unrelated dsRNA. The antisense strand of the dsRNA determined target specificity, and excess complementary sense or antisense single-stranded RNAs (ssRNAs) competed with the RNAi reaction. CONCLUSIONS: Processed dsRNAs can act directly to mediate RNAi, with the antisense strand determining mRNA target specificity. The involvement of 21-23 nt RNAs is supported by the kinetics of the processing reaction and the observed size dependence. RNAi depends on a limiting factor, possibly the nuclease that generates the 21-23 mer species. The active moiety appears to contain both sense and antisense RNA strands.     

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.     

Gene silencing by double-stranded RNA

Eukaryotes silence gene expression in the presence of double-stranded RNA homologous to the silenced gene. Silencing occurs by the targeted degradation of mRNA. Biochemical reactions that recapitulate this phenomenon generate RNA fragments of 21--23 nucleotides from the double-stranded RNA. These stably associate with an RNA endonuclease and probably serve as a discriminator to select mRNAs. Once selected, mRNAs are cleaved at sites 21--23 nucleotides apart. This mechanism, termed RNAi, has functional links to viral defense and silencing phenomena, such as cosuppression. It also functions to repress the hopping of transposable elements.     

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.     

RNA干扰抑制乙型肝炎病毒复制及基因表达研究进展

RNA干扰(RNAinterference,RNAi)是指由21～23个核苷酸组成的双链RNA(dsRNA)所引发的生物细胞内同源基因转录后沉默的现象,是生物体在进化过程中普遍存在的一种基因调控机制。目前对由乙型肝炎病毒(HBV)引起的病毒性肝炎尚无令人满意的治疗效果,而RNA干扰技术的出现为各类慢性HBV感染的治疗开辟了新的途径。本文对RNA干扰抑制HBV复制及基因表达的研究现状、存在问题及应用前景进行了综述。     

Decay of mRNAs targeted by RISC requires XRN1, the Ski complex, and the exosome

RNA interference (RNAi) is a conserved RNA silencing pathway that leads to sequence-specific mRNA decay in response to the presence of double-stranded RNA (dsRNA). Long dsRNA molecules are first processed by Dicer into 21-22-nucleotide small interfering RNAs (siRNAs). The siRNAs are incorporated into a multimeric RNA-induced silencing complex (RISC) that cleaves mRNAs at a site determined by complementarity with the siRNAs. Following this initial endonucleolytic cleavage, the mRNA is degraded by a mechanism that is not completely understood. We investigated the decay pathway of mRNAs targeted by RISC in Drosophila cells. We show that 5' mRNA fragments generated by RISC cleavage are rapidly degraded from their 3' ends by the exosome, whereas the 3' fragments are degraded from their 5' ends by XRN1. Exosome-mediated decay of the 5' fragments requires the Drosophila homologs of yeast Ski2p, Ski3p, and Ski8p, suggesting that their role as regulators of exosome activity is conserved. Our findings indicate that mRNAs targeted by siRNAs are degraded from the ends generated by RISC cleavage, without undergoing decapping or deadenylation.     

Long dsRNA and silent genes strike back:RNAi in mouse oocytes and early embryos

RNA interference (RNAi) refers to the selective degradation of mRNA induced by double-stranded RNA (dsRNA), first discovered in Caenorhabditis elegans. Homology-dependent silencing phenomena related to RNAi have been observed in many species from all eukaryotic kingdoms. RNAi and related mechanisms share several conserved components. The hallmark of these phenomena is the presence of short dsRNA molecules (21-25 bp long), termed short interfering RNA (siRNA), which are generated from dsRNA by the activity of Dicer, a specific type III RNAse. These molecules serve as a template for the recognition and cleavage of the cognate mRNA. As it is beyond the scope of a single review to cover all aspects of RNAi, this review will focus on certain steps of the pathway relevant to mammals and on the use of long dsRNA to specifically silence genes in mammalian cells permissive to this technique, such as oocytes and early embryos.     

Enzymatic production and expression of shRNAmir30 from cDNAs

RNA interference (RNAi) is an important tool for studying gene function and genetic networks. Double-stranded RNA (dsRNA) triggers RNAi that selectively silences gene expression mainly by degrading target mRNA sequences. Short interfering RNA, short hairpin RNA (shRNA), long dsRNA, and microRNA-based shRNA (shRNAmir) are four different types of dsRNA that have been widely used to silence gene expression in cultured cells, tissues, organs, and organisms. Long dsRNAs are usually 200–500 nucleotides in length and can selectively suppress expression of target genes in Caenorhabditis elegans and Drosophila but not in mammals due to unwanted non-specific knockdown. Thus, multiple attempts have been made to synthesize, express, and deliver short dsRNAs that specifically silence target genes in mammals. We describe a method for constructing an RNAi library by converting cDNAs into shRNAmir30 sequences by sequential treatment with different enzymes and affinity purification of biotin- or digoxygenin-labeled DNA fragments. We also developed a system to generate stable cell lines that uniformly express shRNAmir30s and fluorescence reporters by Cre recombinase-dependent site-specific recombination. Thus, combined with the RNAi library, this system facilitates screening for potent RNAi sequences that strongly suppress expression of target genes.