ReviewRNAi: nature abhors a double-strand
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
References (70)
- et al.
Use of dsRNA-mediated genetic interference to demonstrate that frizzled and frizzled 2 act in the wingless pathway
Cell
(1998) - et al.
Silencing of developmental genes in hydra
Dev Biol
(1999) - et al.
RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals
Cell
(2000) - et al.
ATP requirements and small interfering RNA structure in the RNA interference pathway
Cell
(2001) - et al.
Short 5′-phosphorylated double-stranded RNAs induce RNA interference in Drosophila
Curr Biol
(2001) - et al.
Functional anatomy of a dsRNA trigger. Differential requirement for the two trigger strands in RNA interference
Mol Cell
(2000) - et al.
Evidence that processed small dsRNAs may mediate sequence-specific mRNA degradation during RNAi in Drosophila embryos
Curr Biol
(2000) - et al.
RNAi as random degradative PCR. siRNA primers convert mRNA into dsRNAs that are degraded to generate new siRNAs
Cell
(2001) - et al.
Arabidopsis SGS2 and SGS3 genes are required for posttranscriptional gene silencing and natural virus resistance
Cell
(2000) - et al.
An RNA-dependent RNA polymerase gene in Arabidopsis is required for posttranscriptional gene silencing mediated by a transgene but not by a virus
Cell
(2000)
On the role of RNA amplification in dsRNA-triggered gene silencing
Cell
RNA-based silencing strategies in plants
Curr Opin Genet Dev
Listening to the silent genes: transgene silencing, gene regulation and pathogen control
Trends Plant Sci
Domains in gene silencing and cell differentiation proteins: the novel PAZ domain and redefinition of the Piwi domain
Trends Biochem Sci
EGO-1 is related to RNA-directed RNA polymerase and functions in germ-line development and RNA interference in C. elegans
Curr Biol
Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing
Cell
Maternal effects of the short integument mutation on embryo development in Arabidopsis
Dev Biol
The rde-1 gene, RNA interference, and transposon silencing in C. elegans
Cell
Mut-7 of C. elegans, required for transposon silencing and RNA interference, is a homolog of Werner syndrome helicase and RNaseD
Cell
A counterdefensive strategy of plant viruses: suppression of posttranscriptional gene silencing
Cell
Viral suppressors of RNA silencing
Curr Opin Biotechnol
Double-stranded RNA-mediated silencing of genomic tandem repeats and transposable elements in the D. melanogaster germline
Curr Biol
Large-scale analysis of gene function in Caenorhabditis elegans by high-throughput RNAi
Curr Biol
Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans
Nature
Suppression of gene expression by homologous transgenes
Antonie Van Leeuwenhoek Int J
RNA as a target and an initiator of post-transcriptional gene silencing in transgenic plants
Plant Mol Biol
Double-stranded RNA induces mRNA degradation in Trypanosoma brucei
Proc Natl Acad Sci USA
Specific interference by ingested dsRNA
Nature
Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA
Proc Natl Acad Sci USA
Double-stranded RNA specifically disrupts gene expression during planarian regeneration
Proc Natl Acad Sci USA
Total silencing by intron-spliced hairpin RNAs
Nature
Specific interference with gene function by double-stranded RNA in early mouse development
Nat Cell Biol
Specific and heritable genetic interference by double-stranded RNA in Arabidopsis thaliana
Proc Natl Acad Sci USA
Specific double-stranded RNA interference in undifferentiated mouse embryonic stem cells
Mol Cell Biol
Selective reduction of dormant maternal mRNAs in mouse oocytes by RNA interference
Development
Cited by (428)
The initiation of RNA interference (RNAi) in plants
2021, Current Opinion in Plant BiologyArgonaute-CLIP delineates versatile, functional RNAi networks in Aedes aegypti, a major vector of human viruses
2021, Cell Host and MicrobeCitation Excerpt :RNA interference (RNAi) is an essential biological process that regulates gene expression and silences target RNAs. RNAi includes both microRNA (miRNA) and short interfering RNA (siRNA) pathways (Bartel, 2004; Hutvágner and Zamore, 2002; Meister, 2013). In both pathways, double-stranded RNAs (dsRNAs) are processed into small, single-stranded RNAs, which are then loaded onto Argonaute (AGO) subfamily proteins to form RNA-induced silencing complexes (RISCs).
Targeting micro-ribonucleic acid (miRNA) in cancer using advanced drug delivery systems
2021, Advanced Drug Delivery Systems in the Management of CancerSins of fathers through a scientific lens: Transgenerational effects
2021, Genome Stability: From Virus to Human ApplicationmiR-155 Overexpression in OT-1 CD8<sup>+</sup> T Cells Improves Anti-Tumor Activity against Low-Affinity Tumor Antigen
2020, Molecular Therapy OncolyticsA CRISPR/Cas9 based polymeric nanoparticles to treat/inhibit microbial infections
2019, Seminars in Cell and Developmental Biology