A Remarkable History

RNAi: A Guide to Gene Silencing Edited by G.J. Hannon Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press (2003). 436 pp. $130.00“Because the mechanism is not known, we will refer to this technique as RNAi, for RNA-mediated interference.”(Rocheleau et al., Cell 90, 707–716, 1997). RNAi was coined half a dozen years ago to describe a completely mysterious phenomenon in C. elegans whereby injection of either sense or antisense RNA efficiently inhibited gene function. The following year Andy Fire, Craig Mello, and colleagues showed that the initiating agent was actually double-stranded RNA (dsRNA), that it acted posttranscriptionally, and that the silencing effect could spread between cells (Fire et al., Nature 391, 806, 1998; Montgomery et al., Proc. Natl. Acad. Sci. USA 95, 15502–15507, 1998). A natural role for RNAi in C. elegans has not been discovered, although mutation of some genes required for RNAi results in elevated levels of transposition and desilencing of repetitive transgenes (reviewed in Tijsterman et al., Ann. Rev. Genet. 36, 489–519, 2002). A similar process called posttranscriptional gene silencing (PTGS) that also spreads systemically had been carefully documented in transgenic and virus-infected plants and is now recognized to be RNAi (reviewed in Waterhouse et al., Nature 411, 834–842, 2001). PTGS defective mutants are sensitive to viral infection and plant viruses have activities that hinder PTGS, providing compelling evidence that the systemic spread of silencing information provides sequence-specific immunity to uninfected cells. A similarly satisfactory explanation for systemic silencing in C. elegans has not yet been proposed, although analysis of the process has identified a membrane protein that transports dsRNA into cells (Winston et al., Science 295, 2456–2459, 2002; Feinberg and Hunter, Science 301, 1545–1547, 2003). At the close of 1998, RNAi was demonstrated in Drosophila and Trypanosomes (Kennerdell and Carthew, Cell 95, 1017–1026, 1998; Ngo et al., Proc. Natl. Acad. Sci. USA 95, 14687–14692, 1998), initiating a deluge of reports as group after group showed that RNAi was effective in a wide range of invertebrates and even early mammalian embryos (reviewed in Hannon, Nature 418, 244–251, 2002). Genetic studies in fungi, plants, and nematodes identified requirements for RNA-dependent RNA polymerase and members of the Argonaute family, providing evidence for an ancient and singular origin for RNA-mediated gene silencing (RMGS).Nearly as remarkable as the discovery and ubiquitousness of RNAi is the rapidity with which the mystery has given way to mechanistic insight, including the discovery of small interfering RNAs (siRNAs) and their use to initiate effective RNAi in mammalian cells. The long dsRNA was found to be diced into 21–25 nucleotide-long siRNA, which then act as guide sequences to target an unknown nuclease to the target mRNA. The target mRNA is then sliced 10 nucleotides from the 5′ end of the siRNA. The observation that introducing siRNAs directly into C. elegans or Drosophila cells initiated RNAi suggested that RNAi might be feasible in mammalian cells. Mammalian cells are exquisitely sensitive to the presence of cytoplasmic dsRNA: a single molecule of dsRNA is sufficient to induce a stringent antiviral response and lead to nonspecific inhibition of gene function. However, siRNA is too short to initiate the antiviral response! The demonstration of siRNAi-mediated RNAi in mammalian cells has revolutionized somatic cell genetics and the ability to rapidly generate transgenic animals that express constructs that produce short hairpin RNA (shRNA) suggests the onset of a second revolution. The efficacy of RNAi in mammalian cells has naturally led to the successful demonstration that the specificity of RNAi can be exploited to inhibit dominant oncogenes while the potency of RNAi can inhibit virus replication and function in cultured cells and mice. These observations bode well for effective gene-specific therapeutic applications.The structural similarity between siRNAs and microRNAs (miRNA), small single-stranded RNAs imperfectly complementary to their target mRNAs that repress mRNA translation (Ambros, Cell 107, 823–826, 2001), led to the finding that these two gene regulatory processes share a common dsRNA processing enzyme and associate with homologous proteins. The similarity in plants is even more extensive as miRNAs can act by an RNAi-like slicing of the target mRNA rather than repressing translation (Llave et al., Science 297, 2053–2055, 2002). Experiments with human cell extracts indicate that the perfect versus mismatched complementarity between the guide RNA and the mRNA target determines whether the mRNA is sliced or repressed, suggesting overlap between the siRNA and miRNA-associated machinery (Hutvagner and Zamore, Science 297, 2056–2060, 2002). siRNAs and miRNAs, along with other less homogenous noncoding RNAs, are implicated in a wide-range and growing number of RMGS phenomena including diverse aspects of RNA metabolism, alterations in chromatin structure and function, programmed genome rearrangement, imprinting, X inactivation, and inhibiting expression of repetitive DNA sequences including transposons. Despite the tremendous achievements and progress of the last five years there remains much to be discovered.It is in this regard that publication of RNAi: A Guide to Gene Silencing, edited by Gregory Hannon, is timely. This book is a collection of essays, contributed by members of the community, that describe the historical context and current understanding of RNA silencing, along with a healthy dose of practical information. However, the quality of the experimental procedures sections is quite uneven; some provide insufficient detail for the non-expert or provide information readily accessible in a single source. For those new to the field the first few chapters provide an introduction to the spooky biology behind gene silencing. For those of us that suffered through the dark years as participants and observers, it is a memento of progress.Each chapter contains sufficient background material to be read autonomously. The first third of the book introduces the biology of RNA silencing, including cosuppression, DNA methylation, formation and maintenance of heterochromatin, as well as historical views of the major RNAi discoveries in C. elegans and biochemical analysis of RNAi competent extracts. The first chapter captures the intellectual dissonance that accompanied the early descriptions and investigation of cosuppression in plants. The initial experiments were innocent enough. With the goal of producing an ever more purple petunia, transgenes for anthocyanin synthesis were introduced into plants. The resulting unexpectedly white flowers lacked expression of even the endogenous gene. This homology-dependent gene silencing is called “cosuppression”. The second chapter is an incredibly lucid description of cosuppression in animals and is in competition with the fifth and thirteenth chapters on the biochemistry and enzymology of RNAi as the most informative and enjoyable chapter. There are also well placed chapters devoted to RNase III and RNA-dependent RNA polymerase enzymology.The remaining chapters cover organism-specific details for achieving and using RNAi. There is a comprehensive chapter on the ins and outs of whole-genome RNAi screening using the remarkable ability to initiate RNAi in C. elegans by soaking them in dsRNA or feeding them bacteria that express gene-specific dsRNA. There are two separate chapters on RNAi in Drosophila; one describing diverse strategies for exploiting RNAi in Drosophila cell lines and the other in whole flies. The thirteenth chapter on mammalian RNAi, along with an excellent comparative review of RNAi enzymology, provides a summary of documented best practices. RNAi in avian embryos is poised to revitalize this decades old model for embryology and this “shocking” chapter, focused on initiation of RNAi by electroporation of long dsRNA in chick, causes me to wonder how truly wide-spread the antiviral response in vertebrates is. The final chapter introduces what I believe is the brightest future for RNAi: the use of RNAi in nontraditional model and nonmodel organisms. First, it is in the odd little creatures that unexpected phenomena like catalytic RNA, RNA editing, genome reorganization, or RNAi have been and will continue to be discovered. This is exciting and gloriously unpredictable. The second exciting but predictable avenue is the ability to pursue functional analysis among diverse and closely related animals. This will be extremely informative for inferring the paths of evolution past, present, and future.