Double-Stranded RNA Minireview as a Template for Gene Silencing

relieve the inhibition, suggesting micrococcal nuclease When double-stranded RNA (dsRNA) corresponding to was not merely competing for nucleic acid binding.) a sense and antisense sequence of an endogenous Further pieces of the puzzle are provided by beautiful mRNA is introduced into a cell, in organisms ranging work from a collaborative effort of Phil Zamore, Tom from trypanosomes to mice, the cognate mRNA is deTuschl, Phil Sharp, and David Bartel, published in the graded and the gene is silenced (reviewed in Fire, 1999; March 31, 2000, issue of Cell (Zamore et al., 2000). It Bosher and Labouesse, 2000). This type of posttranwas these authors who first reported RNAi could work scriptional gene silencing (PTGS) was first discovered in vitro (Tuschl et al., 1999), and this more recent study in C. elegans (Fire et al., 1998) and is called RNA interfercontinues the characterization of their system, a cellence, or RNAi. RNAi shows many similarities to the PTGS free extract made from syncytial blastoderm Drosophila that is sometimes observed when a transgene is introembryos. In contrast to the S2 extracts, these extracts duced into a cell, and the processes seem to require are prepared from cells that have not been previously some of the same gene products (Catalanotto et al., treated with dsRNA. Rather, RNAi is performed from 2000; Ketting and Plasterk, 2000). If transgene-induced start to finish in a cell-free system, allowing the authors silencing of an endogenous gene, or cosuppression, to radioactively label the dsRNA and mRNA and monitor also involves dsRNA, somehow the cell must make both the fate of both molecules. sense and antisense copies of the transgene sequence. In the presence or absence of the targeted mRNA, PTGS has captured the interest (and imagination) of the authors find that a portion of the dsRNA is cleaved geneticists and molecular biologists alike, and now the to the small pieces, and here the length of the molecules first clues about its mechanism will certainly bring the is mapped to 21–23 nucleotides. Radioactive 21to 23biochemists into the fold. As is often the case for biologimers are observed when either the sense or antisense cal processes, the first hint about the mechanism comes strand of the dsRNA is radiolabeled, verifying that both from the identification of molecules that appear to be strands are cleaved, and implicating a dsRNA nuclease reaction intermediates. In particular, several recent pain the process. pers report the identification of small RNA molecules, The most exciting observations are made when the 21–25 nucleotides in length (21to 25-mers), that correauthors monitor the fate of the mRNA in the presence spond to sense and antisense pieces of the dsRNA or or absence of cognate dsRNA. The mRNA is degraded transgene introduced into the cell. only in the presence of the dsRNA and only within the Evidence that Small RNAs Are Required for Certain sequences spanned by the dsRNA. Remarkably, cleavTypes of PTGS age sites within the mRNA occur at specific sites, Consistent with the idea that the sense and antisense spaced 21–23 nucleotides apart, again suggesting 21to 25-mers are important for transgene-induced cleavage was somehow templated by the small pieces PTGS, they are observed in plants containing transof the dsRNA. After electrophoresis on a sequencing genes that induce silencing but are notably absent from gel, a 59 end-labeled mRNA appears as a ladder of bands plants whose transgenes are expressed normally (Hamat 21–23 nucleotide intervals, suggesting that each ilton and Baulcombe, 1999). A correlation with dsRNAmRNA in the population is cleaved only once or twice. induced silencing is provided by the recent report of (Since the mRNA is radiolabeled only at its 59 end, cleava nuclease activity isolated from cultured Drosophila age of each mRNA at every 21–23 nucleotide interval S2 cells that had been transfected with dsRNA to initiwould result in an autoradiogram showing only the most ate RNAi (Hammond et al., 2000). The partially purified 59 21–23 nucleotide piece.) The cleavage of the mRNA nuclease degrades RNA in a manner consistent with the is unaffected by several translation inhibitors but is ATP degradation known to occur during RNAi—it is sedependent. quence specific and will only degrade RNAs matching A Model for mRNA Degradation by RNAi one of the strands of the dsRNA used to transfect the Figure 1 presents a model for how mRNA is degraded S2 cells. The nuclease activity was partially purified from during RNAi. The model is based on the recent observa-