RNA-induced silencing complex
The RNA-induced silencing complex, or RISC, is a multiprotein complex, specifically a ribonucleoprotein, which incorporates one strand of a double-stranded RNA (dsRNA) fragment, such as small interfering RNA (siRNA) or microRNA (miRNA). The single strand acts as a template for RISC to recognise complementary messenger RNA (mRNA) transcript. Once found, one of the proteins in RISC, called Argonaute, activates and cleaves the mRNA. This process is called RNA interference (RNAi) and it is found in many eukaryotes; it is a key process in gene silencing and defence against viral infections.
- 1 Discovery
- 2 Function in RNA interference
- 3 RISC-associated proteins
- 4 Binding of mRNA
- 5 See also
- 6 Further reading
- 7 External links
- 8 References
The biochemical identification of RISC was conducted by Gregory Hannon and his colleagues at the Cold Spring Harbor Laboratory. This was only a couple of years after the discovery of RNA interference in 1998 by Andrew Fire and Craig Mello, who shared the 2006 Nobel Prize in Physiology or Medicine.
Hannon and his colleagues attempted to identify the RNAi mechanisms involved in gene silencing, by dsRNAs, in Drosophila cells. Drosophila S2 cells were transfected with a lacZ expression vector to quantify gene expression with β-galactosidase activity. Their results showed co-transfection with lacZ dsRNA significantly reduced β-galactosidase activity compared to control dsRNA. Therefore, dsRNAs control gene expression via sequence complementarity.
S2 cells were then transfected with Drosophila cyclin E dsRNA. Cycline E is an essential gene for cell cycle progression into the S phase. Cyclin E dsRNA arrested the cell cycle at the G1 phase (before the S phase). Therefore, RNAi can target endogenous genes.
In addition, cyclin E dsRNA only diminished cyclin E RNA — a similar result was also shown using dsRNA corresponding to cyclin A which acts in S, G2 and M phases of the cell cycle. This shows the characteristic hallmark of RNAi: the reduced levels of mRNAs correspond to the levels of dsRNA added.
To test whether their observation of decreased mRNA levels was a result of mRNA being targeted directly (as suggested by data from other systems), Drosophila S2 cells were transfected with either Drosophila cyclin E dsRNAs or lacZ dsRNAs and then incubated with synthetic mRNAs for cyclin E or lacZ.
Cells transfected with cyclin E dsRNAs only showed degradation in cyclin E transcripts — the lacZ transcripts were stable. Conversely, cells transfected with lacZ dsRNAs only showed degradation in lacZ transcripts and not cyclin E transcripts. Their results led Hannon and his colleagues to suggest RNAi degrades target mRNA through a 'sequence-specific nuclease activity'. They termed the nuclease enzyme RISC.
Function in RNA interference
Loading of dsRNA
The RNase III Dicer aids RISC in RNA interference by cleaving dsRNA into 21-23 nucleotide long fragments with a two-nucleotide 3' overhang. These dsRNA fragments are loaded into RISC and each strand has a different fate based on the asymmetry rule phenomenon.
- The strand with the less stable 5' end is selected by the RNase Argonaute and integrated into RISC. This strand is known as the guide strand.
- The other strand, known as the passenger strand, is degraded by RISC.
RISC uses the bound guide strand to target complementary 3'-untranslated regions (3'UTR) of mRNA transcripts via Watson-Crick base pairing. RISC can now regulate gene expression of the mRNA transcript in a number of ways.
The most understood function of RISC is degrading target mRNA which reduces the levels of transcript available to be translated by ribosomes. There are two main requirements for mRNA degradation to take place:
- a near-perfect complementary match between the guide strand and target mRNA sequence, and,
- a catalytically active Argonaute protein, called a 'slicer', to cleave the target mRNA.
RISC can modulate the loading of ribosome and accessory factors in translation to repress expression of the bound mRNA transcript. Translational repression only requires a partial sequence match between the guide strand and target mRNA.
Translation can be regulated at the initiation step by:
- preventing the binding of the eukaryotic translation initiation factor (eIF) to the 5' cap. It has been noted RISC can deadenylate the 3' poly(A) tail which might contribute to repression via the 5' cap.
- preventing the binding of the 60S ribosomal subunit binding to the mRNA can repress translation.
Translation can be regulated at post-initiation steps by:
There is still speculation on whether translational repression via initiation and post-initiation is mutually exclusive.
Some RISCs are able to directly target the genome by recruiting histone methyltransferases to form heterochromatin at the gene locus and thereby, silencing the gene. These RISCs take the form of a RNA-induced transcriptional silencing complex (RITS). The best studied example is with the yeast RITS.
The mechanism is not well understood but RITS degrade nascent mRNA transcripts. It has been suggested this mechanism acts as a 'self-reinforcing feedback loop' as the degraded nascent transcripts are used by RNA-dependent RNA polymerase (RdRp) to generate more siRNAs.
RISCs seem to have a role in degrading DNA during somatic macronucleus development in protozoa Tetrahymena. It is similar to heterochromatin formation and is implied as a defence against invading genetic elements.
The complete structure of RISC is still unsolved. Many studies have reported a range of sizes and components for RISC but it is not entirely sure whether this is due to there being a number of RISC complexes or due to the different sources that different studies use.
|Complex||Source||Known/apparent components||Estimated size||Apparent function in RNAi pathway|
|Dcr2-R2D2||D. melanogaster S2 cells||Dcr2, R2D2||~250 kDa||dsRNA processing, siRNA binding|
|RLC (A)||D. melanogaster embryos||Dcr2, R2D2||NR||dsRNA processing, siRNA binding, precursor to RISC|
|Holo-RISC||D. melanogaster embryos||Ago 2, Dcr1, Dcr2, Fmr1/Fxr, R2D2, Tsn, Vig||~80S||Target-RNA binding and cleavage|
|RISC||D. melanogaster S2 cells||Ago2, Fmr1/Fxr, Tsn, Vig||~500 kDa||Target-RNA binding and cleavage|
|RISC||D. melanogaster S2 cells||Ago2||~140 kDa||Target-RNA binding and cleavage|
|Fmr1-associated complex||D. melanogaster S2 cells||L5, L11, 5S rRNA, Fmr1/Fxr, Ago2, Dmp68||NR||Possible target-RNA binding and cleavage|
|Minimal RISC||HeLa cells||eIF2C1 (Ago1) or eIF2C2 (Ago2)||~160 kDa||Target-RNA binding and cleavage|
|miRNP||HeLa cells||eIF2C2 (ago2), Gemin3, Gemin4||~550 kDa||miRNA association, target-RNA binding and cleavage|
Ago, Argonaute; Dcr, Dicer; Dmp68, D. melanogaster orthologue of mammalian p68 RNA unwindase; eIF2C1, eukaryotic translation initiation factor 2C1; eIF2C2, eukaryotic translation initiation factor 2C2; Fmr1/Fxr, D. melanogaster orthologue of the fragile-X mental retardation protein; miRNP, miRNA-protein complex; NR, not reported; Tsn, tudor-staphylococcal nuclease; Vig, vasa intronic gene.
Regardless, it is apparent that Argonaute proteins are present and are essential for function. Furthermore, there are insights into some of the key proteins (in addition to Argonaute) within the complex, which allow RISC to carry out its function.
Argonaute proteins are a family of proteins found in prokaryotes and eukaryotes. Their function in prokaryotes is unknown but in eukaryotes they are responsible for RNAi. There are eight family members in human Argonautes of which only Argonaute 2 is exclusively involved in targeted RNA cleavage in RISC.
The RISC-loading complex (RLC) is the essential structure required to load dsRNA fragments into RISC in order to target mRNA. The RLC consists of dicer, the human immunodeficiency virus transactivating response RNA-binding protein (TRBP) and Argonaute 2.
- Dicer is an RNase III endonuclease which generates the dsRNA fragments to be loaded that direct RNAi.
- TRBP is a protein with three double-stranded RNA-binding domains.
- Argonaute 2 is an RNase and is the catalytic centre of RISC.
|Protein||Species the protein is found|
|Polyribosomes, ribosome components||D. melanogaster, T. brucei|
|eIF2C1 (Ago1)||H. sapiens|
|eIF2C2 (Ago2)||H. sapiens|
Ago, Argonaute; Dcr, Dicer; Dmp68, D. melanogaster orthologue of mammalian p68 RNA unwindase; eIF2C1, eukaryotic translation initiation factor 2C1; eIF2C2, eukaryotic translation initiation factor 2C2; Fmr1/Fxr, D. melanogaster orthologue of the fragile-X mental retardation protein; Tsn, tudor-staphylococcal nuclease; Vig, vasa intronic gene.
Binding of mRNA
It is as yet unclear how the activated RISC complex locates the mRNA targets in the cell, though it has been shown that the process can occur in situations outside of ongoing protein translation from mRNA.
Endogenously expressed miRNA in metazoans is usually not perfectly complementary to a large number of genes and thus, they modulate expression via translational repression. However, in plants, the process has a much greater specificity to target mRNA and usually each miRNA only binds to one mRNA. A greater specificity means mRNA degradation is more likely to occur.
- Sontheimer, EJ (2005). "Assembly and function of RNA silencing complexes". Nature Reviews Molecular Cell Biology 6 (2): 127. doi:10.1038/nrm1568.
- Fu, Q.; Yuan, Y. A. (2013). "Structural insights into RISC assembly facilitated by dsRNA-binding domains of human RNA helicase A (DHX9)". Nucleic Acids Research 41 (5): 3457. doi:10.1093/nar/gkt042.
- Schwarz D S, Tomari Y, Zamore P D (2004). "The RNA-induced silencing complex is a Mg2+-dependent endonuclease". Current Biology 14 (9): 787–91. doi:10.1016/j.cub.2004.03.008. PMID 15120070.
- Dcr-1 and Dcr-2 have distinct but overlapping roles in the siRNA and miRNA silencing pathways
- RNA-Induced Silencing Complex at the US National Library of Medicine Medical Subject Headings (MeSH)
- RNAiAtlas - database of siRNA libraries and their target analysis results
- Filipowicz W, Bhattacharyya SN and Sonenber N (2008). "Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight?". Nature Reviews Genetics 9 (2): 102–114. doi:10.1038/nrg2290.
- Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE and Mello CC (1998). "Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans". Nature 391 (6669): 806–811. doi:10.1038/35888.
- Watson, James D. (2008). Molecular Biology of the Gene. San Francisco, CA: Cold Spring Harbor Laboratory Press. pp. 641–648. ISBN 978-0-8053-9592-1.
- Hammond SM, Bernstein E, Beach D and Hannon GJ (2000). "An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells". Nature 404 (6775): 293–296. doi:10.1038/35005107.
- Zamore PD, Tuschl T, Sharp PA and Bartel DP (2000). "RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals". Cell 101 (1): 25–33. doi:10.1016/S0092-8674(00)80620-0.
- Vermeulen A, Behlen L, Reynolds A, Wolfson A, Marshall W, Karpilow J and Khvorova A (2005). "The contributions of dsRNA structure to Dicer specificity and efficiency". RNA 11 (5): 674–682. doi:10.1261/rna.7272305.
- Schwarz DS, Hutvágner G, Du T, Xu Z, Aronin N and Zamore PD (2003). "Asymmetry in the assembly of the RNAi enzyme complex". Cell 115 (2): 199–208. doi:10.1016/S0092-8674(03)00759-1.
- Khvorova A, Reynolds A and Jayasena SD (2003). "Functional siRNAs and miRNAs exhibit strand bias". Cell 115 (2): 209–216. doi:10.1016/S0092-8674(03)00801-8.
- Siomi H and Siomi MC (2009). "On the road to reading the RNA-interference code". Nature 457 (7228): 396–404. doi:10.1038/nature07754.
- Preall JB, He Z, Gorra JM and Sontheimer EJ (2006). "Short interfering RNA strand selection is independent of dsRNA processing polarity during RNAi in Drosophila". Current Biology 16 (5): 530–535. doi:10.1016/j.cub.2006.01.061.
- Gregory RI, Chendrimada TP, Cooch N and Shiekhattar R (2005). "Human RISC couples microRNA biogenesis and posttranscriptional gene silencing". Cell 123 (4): 631–640. doi:10.1016/j.cell.2005.10.022.
- Wakiyama M, Takimoto K, Ohara O and Yokoyama S (2007). "Let-7 microRNA-mediated mRNA deadenylation and translational repression in a mammalian cell-free system". Genes & Development 21 (15): 1857–1862. doi:10.1101/gad.1566707.
- Pratt AJ and MacRae IJ (2009). "The RNA-induced silencing complex: A versatile gene-silencing machine". Journal of Biological Chemistry 284 (27): 17897–17901. doi:10.1074/jbc.R900012200.
- Sen GL and Blau HM (2005). "Argonaute2/RISC resides in sites of mammalian mRNA decay known as cytoplasmic bodies". Nature Cell Biology 7 (6): 633–636. doi:10.1038/ncb1265.
- Chendrimada TP, Finn KJ, Ji X, Baillat D, Gregory RI, Liebhaber SA, Pasquinelli AE and Shiekhattar R (2007). "MicroRNA silencing through RISC recruitment of eIF6". Nature 447 (7146): 823–828. doi:10.1038/nature05841.
- Petersen CP, Bordeleau M-E, Pelletier J and Sharp PA (2006). "Short RNAs repress translation after initiation in mammalian cells". Molecular Cell 21 (4): 533–542. doi:10.1016/j.molcel.2006.01.031.
- Maroney PA, Yu Y, Fisher J and Nilsen TW (2006). "Evidence that microRNAs are associated with translating messenger RNAs in human cells". Nature Structural & Molecular Biology 13 (12): 1102–1107. doi:10.1038/nsmb1174.
- Verdel A, Jia S, Gerber S, Sugiyama T, Gygi S, Grewal SI and Moazed D (2004). "RNAi-mediated targeting of heterchromatin by the RITS complex". Science 303 (5658): 672–676. doi:10.1126/science.1093686.
- Verdel A, Jia S, Gerber S, Sugiyama T, Gygi S, Grewal SI and Moazed D (2004). "RITS acts in cis to promote RNA interference-mediated transcription and post-transcriptional silencing". Nature Genetics 36 (11): 1174–1180. doi:10.1038/ng1452.
- Sugiyama T, Cam H, Verdel A, Moazed D and Grewal SI (2005). "RNA-dependent RNA polymerase is an essential component of a self-enforcing loop coupling heterochromatin assembly to siRNA production". Proceedings of the National Academy of Sciences of the United States of America 102 (1): 152–157. doi:10.1073/pnas.0407641102.
- Mochizuki K and Gorovsky MA (2004). "Small RNAs in genome arrangement in Tetrahymena". Current Opinion in Genetics & Development 14 (2): 181–187. doi:10.1016/j.gde.2004.01.004.
- Sontheimer EJ (2005). "Assembly and function of RNA silencing complexes". Nature Reviews Molecular Cell Biology 6 (2): 127–138. doi:10.1038/nrm1568.
- Liu Q, Rand TA, Kalidas S, Du F, Kim HE, Smith DP and Wang X (2003). "R2D2, a bridge between the initiation and effector steps of the Drosophila RNAi pathway". Science 301 (5641): 1921–1925. doi:10.1126/science.1088710.
- Pham JW, Pellio JL, Lee YS, Carthew RW and Sontheimer EJ (2004). "A Dicer-2-dependent 80S complex cleaves targeted mRNAs during RNAi in Drosophila". Cell 117 (1): 83–94. doi:10.1016/S0092-8674(04)00258-2.
- Tomari Y, Du T, Haley B, Schwarz DS, Bennett R, Cook HA, Koppetsch BS, Theurkauf WE and Zamore PD (2004). "RISC assembly defects in the Drosophila RNAi mutant armitage". Cell 116 (6): 831–841. doi:10.1016/S0092-8674(04)00218-1.
- Hammond SM, Boettcher S, Caudy AA, Kobayashi R and Hannon GJ. "Argonaute2, a link between genetic and biochemical analyses of RNAi". Science 293 (5532): 1146–1150. doi:10.1126/science.1064023.
- Caudy AA, Myers M, Hannon GJ and Hammond SM. "Fragile X-related protein and VIG associate with the RNA interference machinery". Genes & Development 16 (19): 2491–2496. doi:10.1101/gad.1025202.
- Caudy AA, Ketting RF, Hammond SM, Denli AM, Bathoorn AM, Tops BB, Silva JM, Myers MM, Hannon GJ and Plasterk RH (2003). "A micrococcal nuclease homologue in RNAi effector complexes". Nature 425 (6956): 411–414. doi:10.1038/nature01956.
- Rand TA, Ginalski K, Grishin NV and Wang X (2004). "Biochemical identification of Argonaute 2 as the sole protein required for RNA-induced silencing complex activity". Proceedings of the National Academy of Sciences of the United States of America 101 (40): 14385=14389. doi:10.1073/pnas.0405913101.
- Ishizuka A, Siomi MC and Siomi H (2002). "A Drosophila fragile X protein interacts with components of RNAi and ribosomal proteins". Genes & Development 16 (19): 2497–2508. doi:10.1101/gad.1022002.
- Martinez J, Patkaniowska A, Urlaub H, Luhrmann R and Tuschl T (2002). "Single-stranded antisense siRNAs guide target RNA cleavage in RNAi". Cell 110 (5): 563–574. doi:10.1016/S0092-8674(02)00908-X.
- Liu J, Carmell MA, Rivas FV, Marsden CG, Thomson JM, Song JJ, Hammond SM, Joshua-Tor L and Hannon GJ (2004). "Argonaute2 is the catalytic engine of mammalian RNAi". Science 305 (5689): 1437–1441. doi:10.1126/science.1102513.
- Martinez J and Tuschl T (2004). "RISC is a 5′ phosphomonoester-producing RNA endonuclease". Genes & Development 18 (9): 975–980. doi:10.1101/gad.1187904.
- Meister G, Landthaler M, Patkaniowska A, Dorsett Y, Teng G and Tuschl T (2004). "Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs". Molecular Cell 15 (2): 1403–1408. doi:10.1016/j.molcel.2004.07.007.
- Mourelatos Z, Dostie J, Paushkin S, Sharma A, Charroux B, Abel L, Rappsilber J, Mann M and Dreyfuss G (2002). "miRNPs: a novel class of ribonucleoproteins containing numerous microRNAs". Genes & Development 16 (6): 720–728. doi:10.1101/gad.974702.
- Hutvágner G and Zamore PD (2002). "A microRNA in a multiple-turnover RNAi enzyme complex". Science 297 (5589): 2056–2060. doi:10.1126/science.1073827.
- Hall TM (2005). "Structure and function of Argonaute proteins". Cell 13 (10): 1403–1408. doi:10.1016/j.str.2005.08.005.
- Chendrimada TP, Gregory RI, Kumaraswamy E, Norman J, Cooch N, Nishikura K and Shiekhatter R (2005). "TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing". Nature 436 (7051): 740–744. doi:10.1038/nature03868.
- Wang HW, Noland C, Siridechadilok B, Taylor DW, Ma E, Felderer K, Doudna JA and Nogales E (2009). "Structural insights into RNA processing by the human RISC-loading complex". Nature Structural & Molecular Biology 16 (11): 1148–1153. doi:10.1038/nsm.
- Fu Q and Yuan YA (2013). "Structural insights into RISC assembly facilitated by dsRNA-binding domains of human RNA helices A (DHX9)". Nucleic Acids Research 41 (5): 3457–3470. doi:10.1093/nar/gkt042.
- Yoo BK, Santhekadur PK, Gredler R, Chen D, Emdad L, Bhutia S, Pannell L, Fisher PB and Sarkar D (2011). "Increased RNA-induced silencing complex (RISC) activity contributes to hepatocellular carcinoma". Hepatology 53 (5): 1538–1548. doi:10.1002/hep.24216.
- Yoo BK, Emdad L, Lee SG, Su Z, Santhekadur P, Chen D, Gredler R, Fisher PB and Sarkar D (2011). "Astrocyte elevated gene (AEG-1): a multifunctional regulator of normal and abnormal physiology". Pharmacology & Therapeutics 130 (1): 1–8. doi:10.1016/j.pharmthera.2011.01.008.
- Djikeng A, Shi H, Tschudi C, Shen S and Ullu E (2003). "An siRNA ribonucleoprotein is found associated with polyribosomes in Trypanosoma brucei". RNA 9 (7): 802–808. doi:10.1261/rna.5270203.
- Sen GL, Wehrman TS and Blau HM (2005). "mRNA translation is not a prerequisite for small interfering RNA-mediated mRNA cleavage". Differentiation 73 (6): 287–293. doi:10.1111/j.1432-0436.2005.00029.
- Saumet A and Lecellier CH (2006). "Anti-viral RNA silencing: do we look like plants?". Retrovirology 3: 3. doi:10.1186/1742-4690-3-3.
- Bartel DP (2009). "MicroRNAs: target recognition and regulatory functions". Cell 136 (2): 215–233. doi:10.1016/j.cell.2009.01.002.
- Jones-Rhoades MW, Bartel DP and Bartel B (2006). "MicroRNAs and their regulator roles in plants". Annual Review of Plant Biology 57: 19–53. doi:10.1146/annurev.arplant.57.032905.105218.