RNAi

Background RNAi in other systems Mechanism of RNAi Current RNAi studies RNAi protocols from the FireLab at the Carnegie Institution of WashingtonBackgroundRNAi (RNA interference) refers to the introduction of homologous double stranded RNA (dsRNA)
Nội dung trích xuất từ tài liệu:
RNAiBackgroundRNAi in other systemsMechanism of RNAiCurrent RNAi studiesRNAi protocols from the FireLab at the Carnegie Institution of WashingtonBackgroundRNAi (RNA interference) refers to the introduction of homologous double stranded RNA(dsRNA) to specifically target a genes product, resulting in null or hypomorphicphenotypes. The use of antisense RNA to interfere with a genes activity in C. eleganswas first utilised by Su Guo and Ken Kemphues to study par-1 ; however, it was reportedthat control sense RNA also produced a par-1 mutant phenotype (Cell 81: 611-20, 1995).Subsequently, it was discovered by Fire et al. 98 that it is the presence of dsRNA,formed from the annealing of sense and antisense strands present in the in vitro RNApreps, that is responsible for producing the interfering activity. Introduction of dsRNAinto an adult worm results in the loss of the targeted endogenous mRNA from both theadult and its progeny. This phenomenon has been effectively harnessed to study an everincreasing number of maternal and zygotic genes in C. elegans.The most interesting aspects of RNAi are the following: dsRNA, rather than single-stranded antisense RNA, is the interfering agent • it is highly specific • it is remarkably potent (only a few dsRNA molecules per cell are required for • effective interference) the interfering activity (and presumably the dsRNA) can cause interference in • cells and tissues far removed from the site of introductionFigure 1. Effects of mex-3 RNA interference on levels of the endogenous mRNA.Nomarski DIC micrographs show in situ hybridization of 4-cell stage embryos. (A)Negative control showing lack of staining in the absence of the hybridization probe. (B)Embryo from uninjected parent showing normal pattern of endogenous mex-3 RNA(purple staining). (C) Embryo from parent injected with purified mex-3 antisense RNA.These embryos (and the parent animals) retain mex-3 mRNA, although levels may besomewhat less than wild type. (D) Late 4-cell stage embryo from a parent injected withdsRNA corresponding to mex-3 ; no mex-3 RNA is detected. (Templates used forinterfering RNA and in situ probes were largely non-overlapping.)Each embryo is approximately 50 µm in length.(For details see: Fire et al. 98 Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans Nature 391: 806-11)RNAi in Other SystemsMore surprisingly, introduction of dsRNA has been recently shown to produce specificphenocopies of null mutations in such phylogenetically diverse organisms as Drosophila (Kennerdell, JR and RW Carthew 98 Development 95: 1017-26; • Misquitta, L and BM Paterson 99 PNAS 96: 1451-56) trypanosomes (Ngo, H et al. 98 PNAS 95: 14687-92) • planaria (Newmark, P and A. Sanchez 99 PNAS 96: 5049-54 ) •The phenomenon of post-transcriptional gene silencing observed in PLANTS may alsobe due to a related RNAi mechanism. See Waterhouse et al. 98 Virus resistance andgene silencing in plants can be induced by simulataneous expression of sense andantisense RNA PNAS 95: 13959-64.(Also, for recent review of the field, see Sharp, P 99 Genes & Development 13: 139-41)Mechanism of RNAiIn recent years several studies have shed light on the underlying mechanisms of howdsRNA results in the loss of the targeted homologous mRNA. Early observationsindicated that the primary interference effects are post-transcriptional. First it wasobserved by Craig Mello, and reported in Fire et al. (98), that only dsRNA targeting exonsequences was effective (promoter and intron sequences could not produce an RNAieffect). Additional evidence supporting mature messages as the most likely target ofRNA-mediated interference is summarised below (from Montgomery et al. ‘98, PNAS95: 15502-07):* primary DNA sequence of target appears unaltered* initiation and elongation of transcription appear unaffected* nascent transcripts can be detected but are apparently degraded before leaving thenucleusRNAi is remarkably potent (i.e., only a few dsRNA molecules per cell are required toproduce effective interference). This observation suggested that the dsRNA must beeither replicated and/or function catalytically; models that have been mutually supportedby further studies. Genetic and biochemical studies involving plants and flies as well asworms have uncovered similar processes in which the dsRNA is cleaved into ~23 bpshort interfering RNAs (siRNAs) by an enzyme called Dicer (Bernstein et al., 2001;Hamilton & Baulcombe, 1999, Science 286: 950), thus producing multiple “trigger”molecules from the original single dsRNA. The siRNA-Dicer complex recruitsadditional components to form an RNA-induced Silencing Complex (RISC) in which theunwound siRNA base pairs with complementary mRNA, thus guiding the RNAimachinery to the target mRNA resulting in the effective cleavage and subsequentdegradation of the mRNA (Hammond et al., 2000, Nature 404: 293-96, Zamore et al.,2000, Cell 101: 25-33; Pham et al., 2004, Cell 117: 83-94). In this way, the activatedRISC could potentially target multiple mRNAs, and thus function catalytically.In addition, a role for RNA-dependent RNA polymerases (RdRP) has been found forsome species; mutations have been shown to effect the RNAi response as well as result inincreased viral susceptibility, and/or developmental defects (reviewed in Hutvagner &Zamore, 2002, Curr. Opin. Genet. Dev. 12: 225-32). This ability to generate dsRNA denovo supports the replication hypothesis.Current RNAi StudiesDIFFERING SUSCEPTIBILITIES TO SYSTEMIC RNA INTERFERENCE WITHINSOIL NEMATODES OF THE GENUS CAENORHABDITISVeronic Descotte and Mary K. MontgomeryAlthough certain features of the mechanism responsible for the RNAi response appearevolutionarily conserved (e.g. Dicer), the phe ...