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.     

Gene silencing by double-stranded RNA

Thanks to the Nobel Foundation for permission to publish this Lecture. We report here the Nobel Lecture delivered by Professor Andrew Z Fire. Together with the accompanying lecture by Professor Mello this lecture describes the exciting years leading to the discovery of RNA interference (RNAi) and some of the underlying molecular mechanisms. Professor Fire nicely points out his own contribution and the contribution of other research groups to the development of this field. He also presents an interesting discussion on the role of RNAi in immunity and challenges us with a number of open questions. The lecture ends presenting the great potential of exploiting RNAi for therapeutical purposes.     

Local RNA conformational dynamics revealed by 2-aminopurine solvent accessibility

Acrylamide quenching is widely used to monitor the solvent exposure of fluorescent probes in vitro. Here, we tested the utility of this technique to discriminate local RNA secondary structures using the fluorescent adenine analogue 2-aminopurine (2-AP). Under native conditions, the solvent accessibilities of most 2-AP-labeled RNA substrates were poorly resolved by classical single-population models; rather, a two-state quencher accessibility algorithm was required to model acrylamide-dependent changes in 2-AP fluorescence in structured RNA contexts. Comparing 2-AP quenching parameters between structured and unstructured RNA substrates permitted the effects of local RNA structure on 2-AP solvent exposure to be distinguished from nearest neighbor effects or environmental influences on intrinsic 2-AP photophysics. Using this strategy, the fractional accessibility of 2-AP for acrylamide ( f a) was found to be highly sensitive to local RNA structure. Base-paired 2-AP exhibited relatively poor accessibility, consistent with extensive shielding by adjacent bases. 2-AP in a single-base bulge was uniformly accessible to solvent, whereas the fractional accessibility of 2-AP in a hexanucleotide loop was indistinguishable from that of an unstructured RNA. However, these studies also provided evidence that the f a parameter reflects local conformational dynamics in base-paired RNA. Enhanced base pair dynamics at elevated temperatures were accompanied by increased f a values, while restricting local RNA breathing by adding a C-G base pair clamp or positioning 2-AP within extended RNA duplexes significantly decreased this parameter. Together, these studies show that 2-AP quenching studies can reveal local RNA structural and dynamic features beyond those that can be measured by conventional spectroscopic approaches.     

Gene silencing：Double－stranded RNA mediated mRNA degradation and gene inactivation

INTRODUCTIONThe genome structure of plants can be alteredby genetic transformation. During the process ofgene transfer, Agrobacterium tumefaCJens integratepart of their genome into the genome of susceptiblespecies. Recently, genetic transfOrmation techniqueshave been used to modify significantly the organi-zation of the genome. Introducing transgenes intop1ants can both modify the number of copies of agiven sequence and affect gene expression. Becausethe expression of a transgene cannot…     

Site-specific variations in RNA folding thermodynamics visualized by 2-aminopurine fluorescence

The fluorescent base analogue 2-aminopurine (2-AP) is commonly used to study specific conformational and protein binding events involving nucleic acids. Here, combinations of steady-state and time-resolved fluorescence spectroscopy of 2-AP were employed to monitor conformational transitions within a model hairpin RNA from diverse structural perspectives. RNA substrates adopting stable, unambiguous secondary structures were labeled with 2-AP at an unpaired base, within the loop, or inside the base-paired stem. Steady-state fluorescence was monitored as the RNA hairpins made the transitions between folded and unfolded conformations using thermal denaturation, urea titration, and cation-mediated folding. Unstructured control RNA substrates permitted the effects of higher-order RNA structures on 2-AP fluorescence to be distinguished from stimulus-dependent changes in intrinsic 2-AP photophysics and/or interactions with adjacent residues. Thermodynamic parameters describing local conformational changes were thus resolved from multiple perspectives within the model RNA hairpin. These data provided energetic bases for construction of folding mechanisms, which varied among different folding-unfolding stimuli. Time-resolved fluorescence studies further revealed that 2-AP exhibits characteristic signatures of component fluorescence lifetimes and respective fractional contributions in different RNA structural contexts. Together, these studies demonstrate localized conformational events contributing to RNA folding and unfolding that could not be observed by approaches monitoring only global structural transitions.     

Antigenic structure of double-stranded RNA analogues having varying activity in interferon induction.

Antibodies were induced by immunization of rabbits with methylated bovine serum albumin complexes of: poly(I).poly(BC), an effective interferon inducer; poly(c7A).poly(rT), a noninducer that can block induction by active poly(A).poly(rT); and poly(A).poly(Um), which has neither inducing nor blocking activity. Similar complexes of f2 phage RNA or tRNA did not induce anti-nucleic acid antibodies. Each anti-polynucleotide serum contained some antibodies specific for double-stranded structure. Antibodies were immunospecifically purified from precipitates made with each serum and homologous or cross-reacting double-stranded polynucleotides. The purified antibodies distinguished among varying helices bearing base or ribose modifications. Antipoly(I).poly(BC) specificity paralleled that of the interferon induction system. Anti-poly(A).poly(Um) specificity favored the 2'-modified polymers. Anti-poly(c7A).poly(rT) antibodies were the least discriminating. Cross-reaction results indicated that some antibodies reacted with determinants that included both sugar-phosphate backbones. In far antibody excess, antigen:antibody ratios in precipitating complexes reached a minimum of 7 to 12 base pairs per bivalent IgG molecule. Single antigenic determinants may span about 4 base pairs, with primary contact sites including the phosphate groups and the furanose.     

Growth inhibition of mitogen-stimulated fibroblasts induced by double-stranded RNA depends on cell density

Polyinosinic-polycytidylic acid [poly(I:C)], a synthetic double-stranded RNA, is an inhibitor of mitogen-induced proliferation of normal fibroblasts. We show that this inhibition depends strongly on cell density. While cultures with densities at or above confluence are completely inhibited by poly(I:C) in their proliferative response to epidermal growth factor (EGF), the proliferation of sparse (subconfluent) cultures is only delayed. Conditioned medium from dense fibroblasts exposed to poly(I:C) inhibits EGF stimulation of sparse cells, indicating that the inhibition is, at least in part, mediated by a factor released from the cells. Preincubation of quiescent cultures with poly(I:C) renders the cells refractory to the inhibitory effects of poly(I:C). This desensitization correlates with a decreased production of the inhibitor. Since the inhibition of mitogenic stimulation by poly(I:C) is completely overcome by antisera recognizing interferon-beta (IFN-beta) and interleukin-6 (IL-6), we tested the effect of IL-6 and IFN-beta on EGF mitogenicity. None of the available IL-6 preparations had any effect on cell cycle entry. IFN-beta caused a dose-dependent delay of cell division but did not affect the density-dependent proportion of cells entering the cell cycle in response to EGF. Thus, IFN-beta cannot be the sole mediator of the poly(I:C)-induced inhibition. In the presence of dexamethasone, poly(I:C) did not inhibit EGF mitogenis. Indeed, the combined presence of poly(I:C) and dexamethasone did more than just restore the density-dependent control levels of EGF stimulation; most cells entered the cell cycle even at extremely high cell densities. Thus, poly(I:C) in combination with dexamethasone could deactivate the cell density-dependent negative control of proliferation.     

Post-transcriptional gene silencing by double-stranded RNA

Imagine being able to knock out your favourite gene with only a day's work. Not just in one model system, but in virtually any organism: plants, flies, mice or cultured cells. This sort of experimental dream might one day become reality as we learn to harness the power of RNA interference, the process by which double-stranded RNA induces the silencing of homologous endogenous genes. How this phenomenon works is slowly becoming clear, and might help us to develop an effortless tool to probe gene function in cells and animals.     

A deletion mutant of L-A double-stranded RNA replicates like M1 double-stranded RNA.

X double-stranded RNA (dsRNA) is a 0.52-kilobase dsRNA molecule that arose spontaneously in a nonkiller strain of Saccharomyces cerevisiae originally containing L-A and L-BC dsRNAs (L-BC is the same size as L-A but shares no homology with it). X hybridized with L-A, and direct RNA sequencing of X showed that the first 5' 25 base pairs (of the X positive strand) and at least the last 110 base pairs of the 3' end were identical to the ends of L-A dsRNA. X showed cytoplasmic inheritance and, like M1, was dependent on L-A for its maintenance. X was encapsidated in viruslike particles whose major coat protein was provided by L-A (as is true for M1), and X was found in viruslike particles with one to eight X molecules per particle. This finding confirms our "head-full replication" model originally proposed for M1 and M2. Like M1 or M2, X lowers the copy number of L-A, especially in a ski host. Surprisingly, X requires many chromosomal MAK genes that are necessary for M1 but not for L-A.     

Poliovirus single-stranded RNA and double-stranded RNA: differential infectivity in enucleate cells.

The ability of poliovirus virion RNA and double-stranded RNA (replicative form) to replicate in enucleate mouse L cells was investigated. Virion RNA replicated successfully in the absence of the cell nucleus, whereas replicative form infection did not produce any detectable progeny in enucleate cells. The results provide direct evidence of a nuclear requirement early in the infection initiated by replicative form RNA.     

Interferon-beta induction through toll-like receptor 3 depends on double-stranded RNA structure

Type I interferons (IFN-alpha/beta) play an essential role in both innate and adaptive antiviral immune responses. IFN- beta is produced by fibroblasts and myeloid dendritic cells (DCs) upon viral infection or in response to doublestranded RNA (dsRNA). Several intracellular molecules having a dsRNA-binding motif such as dsRNA-dependent protein kinase recognize dsRNA in a sequence-independent manner and induce antiviral innate responses. Toll-like receptor (TLR) 3, a member of TLR family proteins, recognizes extracellular dsRNA and activates NF- kappaB and the IFN-beta promoter leading to the induction of IFN-beta production. Here we analyzed the dsRNA structure capable of inducing TLR3-mediated IFN-beta production using various synthetic RNA duplexes. In contrast to the recognition of dsRNA by intracellular molecules, TLR3 preferentially recognizes polyriboinocinic:polyribocytidylic acid (poly(I:C)) rather than synthetic virus-derived dsRNAs. 2'-O-methyl or 2'-fluoro modification of cytidylic acid abolished the IFN-beta-inducing ability of the poly(I:C) duplex, and these modified dsRNAs inhibited poly(I:C)-induced TLR3-mediated IFN-beta production by fibroblasts and DCs. In addition, poly(dI:dC), a non-IFN inducer, also blocked poly(I:C)-induced IFN-beta induction. Since TLR3 is localized in the intracellular compartment of DCs where signaling occurs, modified dsRNAs may compete with poly(I:C) for binding to the cell-surface receptor that transfers dsRNA into TLR3-enriched vesicles. Thus, TLR3 recognizes a unique dsRNA structure that largely differs from those recognized by other dsRNA-binding proteins.     

Double-stranded RNAs that encode killer toxins in Saccharomyces cerevisiae: unstable size of M double-stranded RNA and inhibition of M2 replication by M1.

The sizes of M1 and M2 (but not L) change rapidly with growth, varying by perhaps as much as 33%. Size variation is seen within 76 generations. In addition, the exclusion of M2 by M1 or L-A-E [( EXL]) is mediated by inhibition of replication or segregation, not by enhanced degradation of preexisting molecules.