In eukaryotes, small non-coding RNAs regulate gene expression, helping to control cellular metabolism, growth and differentiation, to maintain genome integrity, and to combat viruses and mobile genetic elements. These pathways involve two specialized ribonucleases that control the production and function of small regulatory RNAs. The enzyme Dicer cleaves double-stranded RNA precursors, generating short interfering RNAs and microRNAs in the cytoplasm. These small RNAs are transferred to Argonaute proteins, which guide the sequence-specific silencing of messenger RNAs that contain complementary sequences by either enzymatically cleaving the mRNA or repressing its translation. The molecular structures of Dicer and the Argonaute proteins, free and bound to small RNAs, have offered exciting insights into the molecular mechanisms that are central to RNA silencing pathways.
This is a preview of subscription content, access via your institution
Open Access articles citing this article.
Cellular & Molecular Biology Letters Open Access 17 December 2022
Establishment of a miRNA profile in paediatric HIV-1 patients and its potential as a biomarker for effectiveness of the combined antiretroviral therapy
Scientific Reports Open Access 06 December 2021
Cancer Cell International Open Access 18 November 2021
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Get time limited or full article access on ReadCube.
All prices are NET prices.
Lewis, B. P., Burge, C. B. & Bartel, D. P. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 120, 15–20 (2005).
Stefani, G. & Slack, F. J. Small non-coding RNAs in animal development. Nature Rev. Mol. Cell Biol. 9, 219–230 (2008).
Bernstein, E., Caudy, A. A., Hammond, S. M. & Hannon, G. J. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409, 363–366 (2001).
Hutvagner, G. & Simard, M. J. Argonaute proteins: key players in RNA silencing. Nature Rev. Mol. Cell Biol. 9, 22–32 (2008).
Grewal, S. I. & Elgin, S. C. Transcription and RNA interference in the formation of heterochromatin. Nature 447, 399–406 (2007).
Klattenhoff, C. & Theurkauf, W. Biogenesis and germline functions of piRNAs. Development 135, 3–9 (2008).
Haasnoot, J., Westerhout, E. M. & Berkhout, B. RNA interference against viruses: strike and counterstrike. Nature Biotechnol. 25, 1435–1443 (2007).
Li, F. & Ding, S. W. Virus counterdefense: diverse strategies for evading the RNA-silencing immunity. Annu. Rev. Microbiol. 60, 503–531 (2006).
Lee, Y. et al. The nuclear RNase III Drosha initiates microRNA processing. Nature 425, 415–419 (2003).
Nykänen, A., Haley, B. & Zamore, P. D. ATP requirements and small interfering RNA structure in the RNA interference pathway. Cell 107, 309–321 (2001).
Elbashir, S. M., Lendeckel, W. & Tuschl, T. RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev. 15, 188–200 (2001).
Elbashir, S. M., Martinez, J., Patkaniowska, A., Lendeckel, W. & Tuschl, T. Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate. EMBO J. 20, 6877–6888 (2001).
Macrae, I. J. & Doudna, J. A. Ribonuclease revisited: structural insights into ribonuclease III family enzymes. Curr. Opin. Struct. Biol. 17, 138–145 (2007).
Blaszczyk, J. et al. Crystallographic and modeling studies of RNase III suggest a mechanism for double-stranded RNA cleavage. Structure 9, 1225–1236 (2001).
Zhang, H., Kolb, F. A., Jaskiewicz, L., Westhof, E. & Filipowicz, W. Single processing center models for human Dicer and bacterial RNase III. Cell 118, 57–68 (2004). This paper elegantly showed that Dicer contains a single processing centre and proposed that the two RNaseIII domains of Dicer function as an intramolecular dimer.
Gan, J. et al. Structural insight into the mechanism of double-stranded RNA processing by ribonuclease III. Cell 124, 355–366 (2006).
Lingel, A., Simon, B., Izaurralde, E. & Sattler, M. Structure and nucleic-acid binding of the Drosophila Argonaute 2 PAZ domain. Nature 426, 465–469 (2003).
Yan, K. S. et al. Structure and conserved RNA binding of the PAZ domain. Nature 426, 468–474 (2003).
Song, J. J. et al. The crystal structure of the Argonaute2 PAZ domain reveals an RNA binding motif in RNAi effector complexes. Nature Struct. Biol. 10, 1026–1032 (2003).
Macrae, I. J. et al. Structural basis for double-stranded RNA processing by Dicer. Science 311, 195–198 (2006). This paper described the three-dimensional architecture of Dicer, revealing how Dicer functions as a molecular ruler to determine the length of its dsRNA products.
Macrae, I. J., Zhou, K. & Doudna, J. A. Structural determinants of RNA recognition and cleavage by Dicer. Nature Struct. Mol. Biol. 14, 934–940 (2007).
Ma, J. B., Ye, K. & Patel, D. J. Structural basis for overhang-specific small interfering RNA recognition by the PAZ domain. Nature 429, 318–322 (2004). This paper revealed the mode of recognition of the 3′ end of siRNAs by the PAZ domain, identifying conserved residues that bind the 3′ nucleotide.
Lingel, A., Simon, B., Izaurralde, E. & Sattler, M. Nucleic acid 3′-end recognition by the Argonaute2 PAZ domain. Nature Struct. Mol. Biol. 11, 576–577 (2004). This paper showed that the PAZ domain provides a conserved hydrophobic binding pocket for the 3′ nucleotide of ssRNA.
Du, Z., Lee, J. K., Tjhen, R., Stroud, R. M. & James, T. L. Structural and biochemical insights into the dicing mechanism of mouse Dicer: a conserved lysine is critical for dsRNA cleavage. Proc. Natl Acad. Sci. USA 105, 2391–2396 (2008).
Ma, E., Macrae, I. J., Kirsch, J. F. & Doudna, J. A. Autoinhibition of human Dicer by its internal helicase domain. J. Mol. Biol. 380, 237–243 (2008).
Jankowsky, E. & Fairman, M. E. RNA helicases — one fold for many functions. Curr. Opin. Struct. Biol. 17, 316–324 (2007).
Provost, P. et al. Ribonuclease activity and RNA binding of recombinant human Dicer. EMBO J. 21, 5864–5874 (2002).
Zhang, H., Kolb, F. A., Brondani, V., Billy, E. & Filipowicz, W. Human Dicer preferentially cleaves dsRNAs at their termini without a requirement for ATP. EMBO J. 21, 5875–5885 (2002).
Han, J. et al. The Drosha–DGCR8 complex in primary microRNA processing. Genes Dev. 18, 3016–3027 (2004).
Gregory, R. I. et al. The Microprocessor complex mediates the genesis of microRNAs. Nature 432, 235–240 (2004).
Han, J. et al. Molecular basis for the recognition of primary microRNAs by the Drosha–DGCR8 complex. Cell 125, 887–901 (2006).
Sohn, S. Y. et al. Crystal structure of human DGCR8 core. Nature Struct. Mol. Biol. 14, 847–853 (2007).
Farazi, T. A., Juranek, S. A. & Tuschl, T. The growing catalog of small RNAs and their association with distinct Argonaute/Piwi family members. Development 135, 1201–1214 (2008).
Brennecke, J. et al. Discrete small RNA-generating loci as master regulators of transposon activity in Drosophila . Cell 128, 1089–1103 (2007).
Gunawardane, L. S. et al. A slicer-mediated mechanism for repeat-associated siRNA 5′ end formation in Drosophila . Science 315, 1587–1590 (2007).
Hutvágner, G. & Zamore, P. D. A microRNA in a multiple-turnover RNAi enzyme complex. Science 297, 2056–2060 (2002).
Doench, J. G., Petersen, C. P. & Sharp, P. A. siRNAs can function as miRNAs. Genes Dev. 17, 438–442 (2003).
Chiu, Y. L. & Rana, T. M. RNAi in human cells: basic structural and functional features of small interfering RNA. Mol. Cell 10, 549–561 (2002).
Jackson, R. J. & Standart, N. How do microRNAs regulate gene expression? Sci. STKE 2007, re1 (2007).
Cerutti, L., Mian, N. & Bateman, A. Domains in gene silencing and cell differentiation proteins: the novel PAZ domain and redefinition of the Piwi domain. Trends Biochem. Sci. 25, 481–482 (2000).
Song, J. J., Smith, S. K., Hannon, G. J. & Joshua-Tor, L. Crystal structure of Argonaute and its implications for RISC slicer activity. Science 305, 1434–1437 (2004). This paper revealed the molecular architecture of Argonaute proteins and showed that the PIWI domain resembles RNaseH, suggesting that Argonaute is the 'slicer'.
Nowotny, M., Gaidamakov, S. A., Crouch, R. J. & Yang, W. Crystal structures of RNase H bound to an RNA/DNA hybrid: substrate specificity and metal-dependent catalysis. Cell 121, 1005–1016 (2005).
Parker, J. S., Roe, S. M. & Barford, D. Crystal structure of a PIWI protein suggests mechanisms for siRNA recognition and slicer activity. EMBO J. 23, 4727–4237 (2004).
Yuan, Y. R. et al. Crystal structure of A. aeolicus argonaute, a site-specific DNA-guided endoribonuclease, provides insights into RISC-mediated mRNA cleavage. Mol. Cell 19, 405–419 (2005).
Ma, J. B. et al. Structural basis for 5′-end-specific recognition of guide RNA by the A. fulgidus Piwi protein. Nature 434, 666–670 (2005). This paper revealed that the 5′ phosphate group of the guide RNA strand binds to a pocket at the interface of the MID and PIWI domains in the A. fulgidus Piwi protein and that the first nucleotide of the guide strand does not base-pair with the target RNA.
Liu, J. et al. Argonaute2 is the catalytic engine of mammalian RNAi. Science 305, 1437–1441 (2004). This paper showed that human AGO2 has slicer activity and that mutations in its PIWI domain, based on the structure of archaeal Argonaute, abolish RISC activity in vivo.
Martinez, J. & Tuschl, T. RISC is a 5′ phosphomonoester-producing RNA endonuclease. Genes Dev. 18, 975–980 (2004).
Schwarz, D. S., Tomari, Y. & Zamore, P. D. The RNA-induced silencing complex is a Mg2+-dependent endonuclease. Curr. Biol. 14, 787–791 (2004).
Rivas, F. V. et al. Purified Argonaute2 and an siRNA form recombinant human RISC. Nature Struct. Mol. Biol. 12, 340–349 (2005).
Irvine, D. V. et al. Argonaute slicing is required for heterochromatic silencing and spreading. Science 313, 1134–1137 (2006).
Meister, G. et al. Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs. Mol. Cell 15, 185–197 (2004).
Saito, K. et al. Specific association of Piwi with rasiRNAs derived from retrotransposon and heterochromatic regions in the Drosophila genome. Genes Dev. 20, 2214–2222 (2006).
Förstemann, K., Horwich, M. D., Wee, L., Tomari, Y. & Zamore, P. D. Drosophila microRNAs are sorted into functionally distinct Argonaute complexes after production by Dicer-1. Cell 130, 287–297 (2007).
Elbashir, S. M. et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498 (2001).
Parker, J. S., Roe, S. M. & Barford, D. Structural insights into mRNA recognition from a PIWI domain–siRNA guide complex. Nature 434, 663–666 (2005). The paper revealed how the 5′ phosphate group of the guide RNA strand is recognized by Argonaute, and it highlights the importance of the seed region in mediating guide-target recognition.
Haley, B. & Zamore, P. D. Kinetic analysis of the RNAi enzyme complex. Nature Struct. Mol. Biol. 11, 599–606 (2004).
Mi, S. et al. Sorting of small RNAs into Arabidopsis argonaute complexes is directed by the 5′ terminal nucleotide. Cell 133, 116–127 (2008).
Montgomery, T. A. et al. Specificity of ARGONAUTE7–miR390 interaction and dual functionality in TAS3 trans-acting siRNA formation. Cell 133, 128–141 (2008).
Ohara, T. et al. The 3′ termini of mouse Piwi-interacting RNAs are 2′-O-methylated. Nature Struct. Mol. Biol. 14, 349–350 (2007).
Kirino, Y. & Mourelatos, Z. Mouse Piwi-interacting RNAs are 2′-O-methylated at their 3′ termini. Nature Struct. Mol. Biol. 14, 347–348 (2007).
Schwarz, D. S. et al. Asymmetry in the assembly of the RNAi enzyme complex. Cell 115, 199–208 (2003).
Miyoshi, K., Tsukumo, H., Nagami, T., Siomi, H. & Siomi, M. C. Slicer function of Drosophila Argonautes and its involvement in RISC formation. Genes Dev. 19, 2837–2848 (2005).
Matranga, C., Tomari, Y., Shin, C., Bartel, D. P. & Zamore, P. D. Passenger-strand cleavage facilitates assembly of siRNA into Ago2-containing RNAi enzyme complexes. Cell 123, 607–620 (2005).
Rand, T. A., Petersen, S., Du, F. & Wang, X. Argonaute2 cleaves the anti-guide strand of siRNA during RISC activation. Cell 123, 621–629 (2005).
Leuschner, P. J., Ameres, S. L., Kueng, S. & Martinez, J. Cleavage of the siRNA passenger strand during RISC assembly in human cells. EMBO Rep. 7, 314–320 (2006).
Gregory, R. I., Chendrimada, T. P., Cooch, N. & Shiekhattar, R. Human RISC couples microRNA biogenesis and posttranscriptional gene silencing. Cell 123, 631–640 (2005).
Macrae, I. J., Ma, E., Zhou, M., Robinson, C. V. & Doudna, J. A. In vitro reconstitution of the human RISC-loading complex. Proc. Natl Acad. Sci. USA 105, 512–517 (2008).
Maniataki, E. & Mourelatos, Z. A human, ATP-independent, RISC assembly machine fueled by pre-miRNA. Genes Dev. 19, 2979–2990 (2005).
Förstemann, K. et al. Normal microRNA maturation and germ-line stem cell maintenance requires Loquacious, a double-stranded RNA-binding domain protein. PLoS Biol. 3, e236 (2005).
Saito, K., Ishizuka, A., Siomi, H. & Siomi, M. C. Processing of pre-microRNAs by the Dicer-1–Loquacious complex in Drosophila cells. PLoS Biol. 3, e235 (2005).
Jiang, F. et al. Dicer-1 and R3D1-L catalyze microRNA maturation in Drosophila . Genes Dev. 19, 1674–1679 (2005).
Tomari, Y. et al. RISC assembly defects in the Drosophila RNAi mutant armitage . Cell 116, 831–841 (2004).
Liu, X., Jiang, F., Kalidas, S., Smith, D. & Liu, Q. Dicer-2 and R2D2 coordinately bind siRNA to promote assembly of the siRISC complexes. RNA 12, 1514–1520 (2006).
Liu, Q. et al. R2D2, a bridge between the initiation and effector steps of the Drosophila RNAi pathway. Science 301, 1921–1925 (2003).
Tomari, Y., Du, T. & Zamore, P. D. Sorting of Drosophila small silencing RNAs. Cell 130, 299–308 (2007).
Stark, A., Brennecke, J., Russell, R. B. & Cohen, S. M. Identification of Drosophila microRNA targets. PLoS Biol. 1, e60 (2003).
Lewis, B. P., Shih, I. H., Jones-Rhoades, M. W., Bartel, D. P. & Burge, C. B. Prediction of mammalian microRNA targets. Cell 115, 787–798 (2003).
Doench, J. G. & Sharp, P. A. Specificity of microRNA target selection in translational repression. Genes Dev. 18, 504–511 (2004).
Wang, Y., Sheng, G., Juranek, S. A., Tuschl, T. & Patel, D.J. Structure of the guide-strand-containing argonaute silencing complex. Nature 456, 209–213 (2008). This paper revealed that binding of the guide strand to Argonaute orders the seed region in a helical conformation, poised to initiate base-pairing with the target strand.
Wang, Y. et al. Structure of an argonaute silencing complex with a seed-containing guide DNA and target RNA duplex. Nature 456, 921–926 (2008).
Filipowicz, W. RNAi: the nuts and bolts of the RISC machine. Cell 122, 17–20 (2005).
Tomari, Y. & Zamore, P. D. Machines for RNAi. Genes Dev. 19, 517–529 (2005).
Pillai, R. S., Artus, C. G. & Filipowicz, W. Tethering of human Ago proteins to mRNA mimics the miRNA-mediated repression of protein synthesis. RNA 10, 1518–1525 (2004).
Mathonnet, G. et al. MicroRNA inhibition of translation initiation in vitro by targeting the cap-binding complex eIF4F. Science 317, 1764–1767 (2007).
Kiriakidou, M. et al. An mRNA m7G cap binding-like motif within human Ago2 represses translation. Cell 129, 1141–1151 (2007).
Jakymiw, A. et al. Disruption of GW bodies impairs mammalian RNA interference. Nature Cell Biol. 7, 1267–1274 (2005).
Behm-Ansmant, I. et al. mRNA degradation by miRNAs and GW182 requires both CCR4:NOT deadenylase and DCP1:DCP2 decapping complexes. Genes Dev. 20, 1885–1898 (2006).
Liu, J. et al. A role for the P-body component GW182 in microRNA function. Nature Cell Biol. 7, 1261–1266 (2005).
Eulalio, A., Huntzinger, E. & Izaurralde, E. GW182 interaction with Argonaute is essential for miRNA-mediated translational repression and mRNA decay. Nature Struct. Mol. Biol. 15, 346–353 (2008).
Till, S. et al. A conserved motif in Argonaute-interacting proteins mediates functional interactions through the Argonaute PIWI domain. Nature Struct. Mol. Biol. 14, 897–903 (2007).
Meister, G. et al. Identification of novel argonaute-associated proteins. Curr. Biol. 15, 2149–2155 (2005).
Höck, J. et al. Proteomic and functional analysis of Argonaute-containing mRNA–protein complexes in human cells. EMBO Rep. 8, 1052–1060 (2007).
We are grateful to D. Patel for communicating results in advance of publication. We also thank members of the Doudna laboratory for discussions and critical reading of the manuscript. Research in the Doudna laboratory is supported by the Howard Hughes Medical Institute and the National Institutes of Health. M.J. was supported by the European Molecular Biology Organization and is now a postdoctoral fellow of the Human Frontier Science Program.
The authors declare no competing financial interests.
Reprints and permissions information is available at http://www.nature.com/reprints.
Correspondence should be addressed to J.A.D. (email@example.com).
About this article
Cite this article
Jinek, M., Doudna, J. A three-dimensional view of the molecular machinery of RNA interference. Nature 457, 405–412 (2009). https://doi.org/10.1038/nature07755
This article is cited by
Cellular & Molecular Biology Letters (2022)
Design and Fabrication of a DNA-copper Nanocluster-based Biosensor for Multiple Detections of Circulating miRNAs in Early Screening of Breast Cancer
Journal of Fluorescence (2022)
Expression levels of plasma exosomal miR-124, miR-125b, miR-133b, miR-130a and miR-125b-1-3p in severe asthma patients and normal individuals with emphasis on inflammatory factors
Allergy, Asthma & Clinical Immunology (2021)
Cancer Cell International (2021)
Establishment of a miRNA profile in paediatric HIV-1 patients and its potential as a biomarker for effectiveness of the combined antiretroviral therapy
Scientific Reports (2021)