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Killing the messenger: short RNAs that silence gene expression

Key Points

  • 21–23-nucleotide (nt) double-stranded (ds), short interfering (si)RNAs can silence gene expression by directing the sequence-specific cleavage of target messenger RNA in a process that is referred to as RNA interference (RNAi).

  • Although long dsRNAs have been used successfully for the silencing of gene expression in various organisms including Caenorhabditis elegans, plants, Drosophila and mouse oocytes, their use in mammalian systems has been limited primarily because the introduction of dsRNA longer than 30 nt induces a nonspecific interferon response.

  • The evidence that siRNAs could be introduced into mammalian cells and cause the sequence-specific degradation of mRNA without inducing an interferon response has revolutionized the way in which mammalian somatic-cell genetic studies are approached, laid the ground work for the development of short-RNA-based silencing technologies, and holds promise for the development of potential siRNA-based therapeutic strategies.

  • The use of synthetic siRNAs for gene silencing leads to a transient response. DNA-vector-based delivery systems have been developed, which prolonged the silencing effect indefinitely.

  • Most of these expression systems take advantage of the ability of Dicer to process short hairpin RNAs into siRNAs and thereby silence the target gene. These technologies have made it possible to carry out gene-silencing experiments in various cell types and cell lines, as well as to create transgenic animals that stably silence a target gene.

  • This article discusses the rapid advances in RNAi-based gene-silencing technologies and the impact these advances have on the study of gene function, including the development of transgenic animals, siRNA-mediated gene silencing in somatic tissues and functional-genomic studies.

Abstract

Short interfering RNAs can be used to silence gene expression in a sequence-specific manner in a process that is known as RNA interference. The application of RNA interference in mammals has the potential to allow the systematic analysis of gene expression and holds the possibility of therapeutic gene silencing. Much of the promise of RNA interference will depend on the recent advances in short-RNA-based silencing technologies.

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Figure 1: The RNA interference pathway.
Figure 2: Methods to generate short RNAs that silence gene expression.

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Acknowledgements

Owing to the large amount of work that has been done in this field, it was impossible to cover every paper in this review, and we apologize for any oversights. We thank Helen Cargill for preparing the figures and A. Grishok, J. Doench and C. Petersen for their critical reading of the manuscript. Work in our laboratory was supported by a United States Public Health Service MERIT Award from the National Institutes of Health (NIH), a grant from the National Cancer Institute to P.A.S., and partially by a Cancer Center Support core grant from the National Cancer Institute. C.D.N. was supported by the NIH.

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Correspondence to Phillip A. Sharp.

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DATABASES

Swiss-Prot

β-catenin

β-galactosidase

Bim1

CCR5

CD25

CDH1

Dicer

eIF2α

eIF2C2

Fas receptor

Gal4

GFP

IL-2

lamin A/C

p53

FURTHER INFORMATION

National Center for Biotechnology Information

Ambion's siRNA target finder and design tool

Biocomputing at Whitehead Institute

The Hannon lab's siRNA selection site

The Tuschl lab's siRNA user guide

Jack Lin's siRNA sequence finder

Glossary

RIBOZYME TECHNOLOGY

This method uses an RNA molecule that binds the target messenger RNA in a sequence-specific manner and catalyses the cleavage of the mRNA. This ribozyme thereby prevents translation of the target mRNA into protein.

ANTISENSE TECHNOLOGY

This method uses either DNA or RNA molecules that are complementary to sequences on the target messenger RNA and inhibits protein production.

RNASE III

A double-stranded (ds)RNA-specific endoribonuclease that cleaves long dsRNA into short fragments that have a characteristic 3′ overhang and a recessed 5′ phosphate on each strand.

PAZ

(PIWI, argonaute and zwille). A putative protein interaction domain named after the founding members that contain this domain.

PIWI DOMAIN PROTEINS

Proteins that have a conserved protein domain of unknown function. In Drosophila, this family has been implicated in translational control and silencing of numerous copies of the alcohol dehydrogenase gene.

PPD PROTEIN

A protein that has a PAZ/PIWI domain.

INTERFERON

A small and highly potent molecule that functions in an autocrine and paracrine manner, and that induces cells to resist viral replication.

2′–5′ OLIGOADENYLATE SYNTHASE

A component of the interferon-response pathway that, when activated by long double-stranded RNA, catalyses the conversion of ATP to 2′–5′ A oligonucleotides.

RNASE L

An enzyme that is activated by 2′–5′ A oligonucleotides, leading to the cleavage of several RNA species including ribosomal RNA, resulting in an inhibition of messenger RNA translation.

PKR

A protein kinase that, when activated by long double-stranded RNA, phosphorylates and inactivates the translation initiation factor eIF2α, resulting in an inhibition of messenger RNA translation initiation.

SYNTHETIC OLIGODEOXYRIBONUCLEOTIDE/ RNASEH METHOD

A method that is used for mapping endonuclease-sensitive sites and for inhibiting gene expression. Synthetic single-stranded oligodeoxy-ribonucleotide and a complementary sequence to a target messenger RNA are transfected into cells, leading to the formation of an RNA–DNA hybrid. Endogenous RNase H cleaves the RNA molecule of an RNA–DNA hybrid and prevents protein synthesis.

RNA POLYMERASE II

(pol II). The enzyme that transcribes messenger RNA and most of the small nuclear RNAs of eukaryotes, in conjunction with various transcription factors.

RNA POLYMERASE III

(pol III). The enzyme that transcribes stable RNA products that are not translated into proteins, particularly transfer RNAs. However, pol III also transcribes the 5S ribosomal RNA, 7SL RNA and U6 small nuclear RNA.

CIS-ACTING ELEMENT

An arrangement of sequences on a contiguous piece of DNA.

TANDEM PROMOTERS

Promoters that are arranged in the same orientation in close proximity on a contiguous piece of DNA.

LONG TERMINAL REPEAT

(LTR). A sequence that is repeated at both ends of a retroviral DNA that is required for retroviral insertion into its target genomic DNA.

REVERSE TRANSCRIPTASE

An enzyme that is used by retroviruses and retrotransposons to synthesize DNA.

RAG-DEFICIENT BLASTOCYSTS

Blastocysts derived from mice that lack the recombinase-activating gene. Mice that are RAG deficient are unable to produce mature B and T cells and are therefore immunocompromised.

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Dykxhoorn, D., Novina, C. & Sharp, P. Killing the messenger: short RNAs that silence gene expression. Nat Rev Mol Cell Biol 4, 457–467 (2003). https://doi.org/10.1038/nrm1129

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