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Assembly and function of RNA silencing complexes

Nature Reviews Molecular Cell Biology volume 6, pages 127–138 (2005)Cite this article

Key Points

  • RNA interference (RNAi) is a eukaryotic gene-regulatory pathway that silences the expression of specific genes in response to homologous double-stranded (ds)RNA. The RNA-induced silencing complex (RISC) assembles on short interfering (si)RNA fragments and cleaves target mRNAs that hybridize with the siRNA.

  • In Drosophila melanogaster, the RNase-III enzyme Dicer2 (Dcr2) — which cleaves dsRNAs into siRNAs — is important for RISC assembly, even when pre-cleaved siRNAs are used to trigger silencing. Dcr2 and the associated dsRNA-binding protein R2D2 are components of a RISC-loading complex (RLC) that binds siRNAs and escorts them into RISC.

  • RISC forms through an ATP-dependent assembly pathway that includes an siRNA-unwinding step. The relative thermodynamic stabilities of the ends of the siRNA duplex help to specify the strand that will ultimately assemble into RISC.

  • RISC is a multiple-turnover, divalent-metal-ion-dependent enzyme that hydrolyses the target phosphodiester linkage, leaving 3′-hydroxyl and 5′-phosphate termini.

  • All RISCs that have been characterized so far contain a member of the Argonaute (Ago) family of proteins, as defined by the presence of PAZ and PIWI domains. The PIWI domain has recently been shown to have structural homology to ribonuclease-H enzymes, which implicates it as the endonuclease that cleaves the target mRNA.

Abstract

In the RNA-interference pathway, double-stranded RNA induces sequence-specific mRNA degradation through the action of the RNA-induced silencing complex (RISC). Recent work has provided our first glimpses of the RISC-assembly pathway and uncovered the biochemical roles of critical RISC components. These advances have taken our mechanistic understanding of RNA interference to a new level and promise to improve our ability to exploit this biological process for use in experimental biology and medicine.

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Figure 1: The general pathway of RNAi in vitro.
Figure 2: RISC assembly in Drosophila melanogaster.
Figure 3: siRNA asymmetry, dsRNA processing and the implications for RISC assembly.
Figure 4: Chemical mechanism of RISC-catalysed target-mRNA cleavage.
Figure 5: The PIWI domains of Ago proteins harbour the endonuclease activity of RISC.

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Acknowledgements

I would like to thank R. W. Carthew, P. Bellare, J. Gorra, Z. He, J. L. Pellino, J. W. Pham and J. Preall for helpful discussions and comments on the manuscript. E.J.S. is supported by grants from the National Institutes of Health, National Science Foundation, March of Dimes and American Cancer Society.

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  1. Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, 2205 Tech Drive, Evanston, 60208-3500, Illinois, USA

    Erik J. Sontheimer

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DATABASES

Entrez

AGO1

AGO2

Flybase

Ago2

Armitage

Aubergine

Dcr1

Dcr2

Dmp68

Fmr1

R2D2

Spindle-E

Tsn

Vig

Swiss-Prot

AGO3

AGO4

DCR

HIWI

Wormbase

DCR-1

RDE-1

RDE-4

FURTHER INFORMATION

Author's laboratory

Glossary

TRANSPOSON

A mobile genetic element that can relocate within a host genome. An autonomous transposon encodes a transposase protein that catalyses its excision and reintegration in the genome, and the transposon can therefore direct its own transposition.

SHORT INTERFERING RNA

(siRNA). A non-coding RNA (∼22-nt long) that is processed from longer dsRNA during RNA interference. Such non-coding RNAs hybridize with mRNA targets, and confer target specificity to the silencing complexes in which they reside.

RIBONUCLEASE III

A family of nucleases that cleave dsRNAs, generally leaving 2-nt 3′-overhangs.

ARGONAUTE (Ago) FAMILY

A family of proteins that are characterized by the presence of two homology domains, PAZ and PIWI. These proteins are essential for diverse RNA silencing pathways.

PAZ DOMAIN

A conserved nucleic-acid-binding structure that is found in members of the Dcr and Ago protein families.

PIWI DOMAIN

A conserved structure that is found in members of the Ago protein family. It is structurally similar to ribonuclease-H domains and, in at least some cases, has endoribonuclease activity.

PARALOGUE

A sequence, or gene, that originates from a common ancestral sequence, or gene, by a duplication event.

RNA UNWINDASE

An enzyme that catalyses the dissociation of RNA base pairs.

MICRORNA

(miRNA). An ∼21–22-nt RNA silencing trigger that is processed from short stem–loop precursors that are encoded in the genomes of metazoans and certain viruses.

DEXD/H FAMILY

A family of enzymes that uses the energy from ATP hydrolysis to drive RNA unwinding.

OFF-TARGET EFFECT

In the context of RNA silencing, this refers to the decreased expression of genes other than the intended target gene.

HETEROCHROMATIN

A highly condensed and transcriptionally less active form of chromatin that occurs at defined sites, such as centromeres, silencer DNA elements or telomeres.

TRANSCRIPTIONAL GENE SILENCING

A form of gene silencing that inhibits RNA synthesis in response to dsRNA.

CHROMODOMAIN

A conserved protein structure that is common to some chromosomal proteins. It interacts with chromatin by binding to methylated lysine residues in histone proteins.

POLYSOME

(or polyribosome). Two or more ribosomes that are bound to different sites on the same mRNA.

A-FORM RNA

A helical structural form that is commonly adopted by dsRNA and RNA–DNA hybrid duplexes.

RIBONUCLEASE H

An enzyme that cleaves the RNA strand of a DNA–RNA hybrid duplex.

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Sontheimer, E. Assembly and function of RNA silencing complexes. Nat Rev Mol Cell Biol 6, 127–138 (2005). https://doi.org/10.1038/nrm1568

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