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2006年诺贝尔生理学或医学奖获得者论文赏析
Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans
Experimental introduction of RNA into cells can be used incertain biological systems to interfere with the function of anendogenous gene 1,2 . Such effects have been proposed to resultfrom a simple antisense mechanism that depends on hybridiza-tion between the injected RNA and endogenous messenger RNAtranscripts. RNA interference has been used in the nematode Caenorhabditis elegans to manipulate gene expression 3,4 . Here weinvestigate the requirements for structure and delivery of theinterfering RNA. To our surprise, we found that double-strandedRNA was substantially more effective at producing interferencethan was either strand individually. After injection into adultanimals, purified single strands had at most a modest effect,whereas double-stranded mixtures caused potent and specificinterference. The effects of this interference were evident inboth the injected animals and their progeny. Only a few moleculesof injected double-stranded RNA were required per affected cell,arguing against stochiometric interference with endogenous mRNA and suggesting that there could be a catalytic or amplification component in the interference process.
下载全文：Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.pdf
Specific inhibition of gene expression by small double-stranded RNAs in invertebrate andvertebrate systems
Short interfering RNAs (siRNAs) are double-stranded RNAs of’21–25 nucleotides that have been shown to function as keyintermediaries in triggering sequence-specific RNA degradationduring posttranscriptional gene silencing in plants and RNA inter-ference in invertebrates. siRNAs have a characteristic structure,with 5'-phosphatey3'-hydroxyl ends and a 2-base 3' overhang oneach strand of the duplex. In this study, we present data thatsynthetic siRNAs can induce gene-specific inhibition of expressionin Caenorhabditis elegans and in cell lines from humans and mice.In each case, the interference by siRNAs was superior to theinhibition of gene expression mediated by single-stranded anti-sense oligonucleotides. The siRNAs seem to avoid the well docu-mented nonspecific effects triggered by longer double-strandedRNAs in mammalian cells. These observations may open a pathtoward the use of siRNAs as a reverse genetic and therapeutic toolin mammalian cells.
下载全文：Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.pdf
Genes and Mechanisms Related to RNA Interference Regulate Expression of the Small Temporal RNAsthat Control C. elegans Developmental Timing
RNAi is a gene-silencing phenomenon triggered by double-stranded (ds) RNA and involves the generation of 21 to 26 nt RNA segments that guide mRNA destruction. In Caenorhabditis elegans, lin-4 and let-7 encode small temporal RNAs (stRNAs) of 22 nt that regulate stage-specific development. Here we show that inactivation of genes related to RNAi pathway genes, a homolog of Drosophila Dicer (dcr-1), and two homologs of rde-1 (alg-1 and alg-2), cause heterochronic phenotypes similar to lin-4 and let-7 mutations. Further we show that dcr-1, alg-1, and alg-2 are necessary for the maturation and activity of the lin-4 and let-7 stRNAs. Our findings suggest that a common processing machinery generates guide RNAs that mediate both RNAi and endogenous gene regulation.
The rde-1 Gene, RNA Interference,and Transposon Silencing in C. elegans
Double-stranded (ds) RNA can induce sequence-specific inhibition of gene function in several organisms. However, both the mechanism and the physiological role of the interference process remain mysterious. In order to study the interference process, we have selected C. elegans mutants resistant to dsRNA-mediated interference (RNAi). Two loci, rde-1 and rde-4, are defined by mutants strongly resistant to RNAi but with no obvious defects in growth or development. We show that rde-1 is a member of the piwi/sting/argonaute/zwille/eIF2C gene family conserved from plants to vertebrates. Interestingly, several, but not all, RNAi-deficient strains exhibit mobilization of the endogenous transposons. We discuss implications for the mechanism of RNAi and the possibility that one natural function of RNAi is transposon silencing.
点击下载：The rde-1 Gene, RNA Interference,and Transposon Silencing in C_ elegans.pdf
Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans
Genetic interference mediated by double-stranded RNA (RNAi) has been a valuable tool in the analysis of gene function in Caenorhabditis elegans. Here we report an efficient induction of RNAi using bacteria to deliver double-stranded RNA. This method makes use of bacteria that are deficient in RNaseIII, an enzyme that normally degrades a majority of dsRNAs in the bacterial cell. Bacteria deficient for RNaseIII were engineered to produce high quantities of specific dsRNA segments. When fed to C. elegans, such engineered bacteria were found to produce populations of RNAi-affected animals with phenotypes that were comparable in expressivity to the corresponding loss-of-function mutants. We found the method to be most effective in inducing RNAi for non-neuronal tissue of late larval and adult hermaphrodites, with decreased effectiveness in the nervous system, in early larval stages, and in males. Bacteria-induced RNAi phenotypes could be maintained over the course of several generations with continuous feeding, allowing for convenient assessments of the biological consequences of specific genetic interference and of continuous exposure to dsRNAs.
On the Role of RNA Amplification in dsRNA-Triggered Gene Silencing
We have investigated the role of trigger RNA amplification during RNA interference (RNAi) in Caenorhabditis elegans. Analysis of small interfering RNAs (siRNAs) produced during RNAi in C. elegans revealed a substantial fraction that cannot derive directly from input dsRNA. Instead, a population of siRNAs (termed secondary siRNAs) appeared to derive from the action of a cellular RNA-directed RNA polymerase (RdRP) on mRNAs that are being targeted by the RNAi mechanism. The distribution of secondary siRNAs exhibited a distinct polarity (5'-->3' on the antisense strand), suggesting a cyclic amplification process in which RdRP is primed by existing siRNAs. This amplification mechanism substantially augments the potency of RNAi-based surveillance, while ensuring that the RNAi machinery will focus on expressed mRNAs.
点击下载：On the Role of RNA Amplification in dsRNA-Triggered Gene Silencing.pdf
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