Review
RNAi: nature abhors a double-strand

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Abstract

In organisms as diverse as nematodes, trypanosomes, plants, and fungi, double-stranded RNA triggers the destruction of homologous mRNAs, a phenomenon known as RNA interference. RNA interference begins with the transformation of the double-stranded RNA into small RNAs that then guide a protein nuclease to destroy their mRNA targets.

Introduction

Confronted with double-stranded RNA (dsRNA), eukaryotic cells respond in a rather surprising way: they destroy their own mRNAs that share sequence with the double strand. This phenomenon, termed RNA interference (RNAi), has provided biologists with a remarkable tool for reverse genetics [1]. Thus, investigators studying Caenorhabditis elegans, Drosophila melanogaster, and a host of other invertebrates, plants such as Arabidopsis thaliana, fungi like Neurospora crassa (but not Saccharomyces cerevisiae), and mouse embryonic stem cells, oocytes, and early embryos can disrupt expression of virtually any gene by delivering dsRNA corresponding to that gene's sequence 2., 3., 4., 5., 6., 7., 8., 9., 10., 11., 12., 13., 14.. Recently, the use of RNAi has been extended to differentiated cultured mammalian cells 15., 16••..

Here, we first describe the two models recently proposed to explain the mechanism of RNAi. We then discuss our evolving understanding of the biological functions of the RNAi pathway.

Section snippets

It dices… it slices…?

Biochemical experiments conducted in Drosophila embryo lysates and cultured S2 cells support a four-step model for the RNAi pathway (Fig. 1). The model envisions that RNAi is initiated by the ATP-dependent, processive cleavage of long dsRNA into 21–25 nucleotide (nt) double-stranded fragments, termed small interfering RNAs (siRNAs) 16••., 17., 18•., 19•., 20••.. These siRNA duplexes are then incorporated into a protein complex that is not yet competent to mediate RNAi [21•]. ATP-dependent

Random degradative PCR?

Screens for genes required for gene silencing in plants, fungi, and worms have identified a family of proteins whose sequences suggest they are RNA-dependent RNA polymerases (RdRPs) (32., 33., 34., 35•.; Table 1). The discovery of such RdRP proteins in the RNAi and post-transcriptional gene-silencing pathways provides a possible explanation for the remarkable efficacy of dsRNA in gene silencing. It has been estimated that in Drosophila embryos, ∼35 molecules of dsRNA can silence a target mRNA

Dicer, development, and small temporal RNAs

Mutations in some genes required for RNAi and in orthologs of these genes have dramatic developmental defects, especially in the germline or in proliferative tissues, suggesting a link between the RNAi pathway and development 39., 40., 41., 42., 43., 44., 45., 46.. Mutations in the worm RdRP, ego-1, block RNAi in the germline and disrupt oogenesis. Deletion of the worm dcr-1 gene, the C. elegans homolog of Dicer, not only abrogates RNAi but also leads to misregulation of developmental timing 27.

miRNAs, your RNAs

Recently, >60 potential small regulatory RNAs (microRNAs or miRNAs) were identified in worms, fly embryos, and cultured human cells 53., 54., 55.. These RNAs are encoded in regions of the genome predicted to form ∼70nt stem-loop RNAs remarkably like stRNA precursors, and two in worms have been shown to require Dicer for their production [55]. Although many of the miRNAs are constitutively expressed, others are restricted in expression to specific times in development. Some appear to be

Conclusions and future challenges

RNAi has been a boon to biologists, bringing reverse genetics (‘functional genomics’) to organisms lacking established genetic tools, and quickening the pace of genetic analysis in traditional genetic models such as C. elegans and Drosophila. Large-scale RNAi analysis of all the genes in C. elegans is well underway 68., 69., 70., and the discovery that synthetic siRNAs trigger RNAi in mammalian cells will surely lead to similar screens for human genes. The outlines of the RNAi pathway are

Acknowledgements

The authors thank David Bartel, Craig Mello, and Antti Nykänen for comments on the manuscript, and members of the Zamore lab for many helpful discussions.

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:• of special interest•• of outstanding interest

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