Editing RNA-induced silencing complex

{{Short description|Multiprotein complex}}
The '''RNA-induced silencing complex''', or '''RISC''', is a [[multiprotein complex]], specifically a [[ribonucleoprotein]], which functions in [[gene silencing]] via a variety of pathways at the transcriptional and translational levels.<ref name="Pratt2009">{{Cite journal|name-list-style=amp|vauthors=Pratt AJ, MacRae IJ|year=2009|title=The RNA-induced silencing complex: A versatile gene-silencing machine|journal=[[Journal of Biological Chemistry]]|volume=284|issue=27|pages=17897–17901|doi=10.1074/jbc.R900012200|pmc=2709356|pmid=19342379|doi-access=free}}</ref> Using single-stranded [[RNA]] (ssRNA) fragments, such as [[microRNA]] (miRNA), or double-stranded [[small interfering RNA]] (siRNA), the complex functions as a key tool in gene regulation.<ref name="Filipowicz2008">{{Cite journal|vauthors=Filipowicz W, Bhattacharyya SN, Sonenber N | title= Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight? | journal=[[Nature Reviews Genetics]] | volume=9 | issue=2 | year=2008 | pages=102–114 | doi=10.1038/nrg2290 | pmid=18197166| s2cid= 11824239 | url= https://escholarship.mcgill.ca/concern/articles/cz30pz55f }}</ref> The single strand of RNA acts as a template for RISC to recognize [[complementary DNA|complementary]] [[messenger RNA]] (mRNA) [[transcription (genetics)|transcript]]. Once found, one of the proteins in RISC, [[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 defense against [[viral disease|viral infections]], as it is triggered by the presence of double-stranded RNA (dsRNA).<ref name="Fire1998">
{{Cite journal |vauthors=Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC | title=Potent and specific genetic interference by double-stranded RNA in ''Caenorhabditis elegans'' | journal=[[Nature (journal)|Nature]] | volume=391 | issue=6669 | year=1998 | pages=806–811 | doi=10.1038/35888 | pmid=9486653| bibcode=1998Natur.391..806F | s2cid=4355692 | url=http://www.dspace.cam.ac.uk/handle/1810/238264 }}
</ref><ref name="Watson2008">
{{cite book| last=Watson | first=James D. | title=Molecular Biology of the Gene | year=2008 | publisher=Cold Spring Harbor Laboratory Press | location=San Francisco, CA |isbn=978-0-8053-9592-1| pages=641–648 }}</ref><ref name="Pratt2009" />

==Discovery==
The [[biochemistry|biochemical]] identification of RISC was conducted by [[Gregory Hannon]] and his colleagues at the [[Cold Spring Harbor Laboratory]].<ref name="Hammond2000">
{{Cite journal |vauthors=Hammond SM, Bernstein E, Beach D, Hannon GJ | author-link2=Emily Bernstein|title=An RNA-directed nuclease mediates post-transcriptional gene silencing in ''Drosophila'' cells | journal=[[Nature (journal)|Nature]] | volume=404 | issue=6775 | year=2000 | pages=293–296 | doi=10.1038/35005107 | pmid=10749213| bibcode=2000Natur.404..293H | s2cid=9091863 }}</ref> 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]].<ref name="Fire1998"/>

[[File:Drosophila melanogaster - side (aka).jpg|200px|thumb|''Drosophila melanogaster'']]

Hannon and his colleagues attempted to identify the RNAi mechanisms involved in [[gene silencing]], by dsRNAs, in ''[[Drosophila]]'' cells. ''Drosophila'' [[Schneider 2 cells|S2 cells]] were [[transfection|transfected]] with a ''[[lac operon|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 (molecular biology)|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|G<sub>1</sub> 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 phase|G<sub>2</sub>]] and [[M phase|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.<ref name="Hammond2000"/> Later Devanand Sarkar and his colleagues Prasanna K. Santhekadur and Byoung Kwon Yoo at the Virginia Commonwealth University elucidated the RISC activity and its molecular mechanism in cancer cells and they identified another new component of the RISC, called AEG-1 [47].

==Function in RNA interference==
[[File:1ytu argonaute dsrna.png|thumb|The PIWI domain of an Argonaute protein in complex with double-stranded RNA.]]

=== Incorporation of siRNA/miRNA ===
The [[Ribonuclease III|RNase III]] [[Dicer]] is a critical member of RISC that initiates the RNA interference process by producing double-stranded siRNA or single-stranded miRNA. Enzymatic cleavage of dsRNA within the cell produces the short siRNA fragments of 21-23 [[nucleotide]]s in length with a two-nucleotide [[Directionality (molecular biology)|3']] overhang.<ref name="Zamore20002">{{Cite journal|vauthors=Zamore PD, Tuschl T, Sharp PA, Bartel DP|year=2000|title=RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals|journal=[[Cell (journal)|Cell]]|volume=101|issue=1|pages=25–33|doi=10.1016/S0092-8674(00)80620-0|pmid=10778853|doi-access=free}}</ref><ref name="Vermeulen20052">{{Cite journal|vauthors=Vermeulen A, Behlen L, Reynolds A, Wolfson A, Marshall W, Karpilow J, Khvorova A|year=2005|title=The contributions of dsRNA structure to Dicer specificity and efficiency|journal=[[RNA (journal)|RNA]]|volume=11|issue=5|pages=674–682|doi=10.1261/rna.7272305|pmc=1370754|pmid=15811921}}</ref> Dicer also processes pre-miRNA, which forms a hairpin loop structure to mimic dsRNA, in a similar fashion. dsRNA fragments are loaded into RISC with each strand having a different fate based on the asymmetry rule phenomenon, the selection of one strand as the guide strand over the other based on thermodynamic stability.<ref>{{Cite journal|last=Hutvagner|first=Gyorgy|date=2005|title=Small RNA asymmetry in RNAi: Function in RISC assembly and gene regulation|journal=FEBS Letters|language=en|volume=579|issue=26|pages=5850–5857|doi=10.1016/j.febslet.2005.08.071|pmid=16199039|issn=1873-3468|doi-access=free|bibcode=2005FEBSL.579.5850H |hdl=10453/15313|hdl-access=free}}</ref><ref name="Schwarz20032">{{Cite journal|vauthors=Schwarz DS, Hutvágner G, Du T, Xu Z, Aronin N, Zamore PD|year=2003|title=Asymmetry in the assembly of the RNAi enzyme complex|journal=[[Cell (journal)|Cell]]|volume=115|issue=2|pages=199–208|doi=10.1016/S0092-8674(03)00759-1|pmid=14567917|doi-access=free}}</ref><ref name="Khvorova20032">{{Cite journal|vauthors=Khvorova A, Reynolds A, Jayasena SD|year=2003|title=Functional siRNAs and miRNAs exhibit strand bias|journal=[[Cell (journal)|Cell]]|volume=115|issue=2|pages=209–216|doi=10.1016/S0092-8674(03)00801-8|pmid=14567918|s2cid=2500175|doi-access=free}}</ref><ref name="Siomi20092">{{Cite journal|name-list-style=amp|vauthors=Siomi H, Siomi MC|year=2009|title=On the road to reading the RNA-interference code|journal=[[Nature (journal)|Nature]]|volume=457|issue=7228|pages=396–404|doi=10.1038/nature07754|pmid=19158785|bibcode=2009Natur.457..396S|s2cid=205215974}}</ref> The newly generated miRNA or siRNA act as single-stranded guide sequences for RISC to target mRNA for degradation.<ref>{{Cite journal|date=2005-11-18|title=RNAi: RISC Gets Loaded|journal=Cell|language=en|volume=123|issue=4|pages=543–545|doi=10.1016/j.cell.2005.11.006|issn=0092-8674|last1=Preall|first1=Jonathan B.|last2=Sontheimer|first2=Erik J.|pmid=16286001|doi-access=free}}</ref><ref>{{Cite web|title=RNA interference overview {{!}} Abcam|url=https://www.abcam.com/pathways/rna-interference---a-comprehensive-overview|access-date=2021-03-07|website=www.abcam.com}}</ref>

* The strand with the less thermodynamically stable [[Directionality (molecular biology)|5' end]] is selected by the protein [[Argonaute]] and integrated into RISC.<ref name="Siomi20092" /><ref>{{Cite journal|last1=Preall|first1=Jonathan B.|last2=He|first2=Zhengying|last3=Gorra|first3=Jeffrey M.|last4=Sontheimer|first4=Erik J.|date=2006-03-07|title=Short Interfering RNA Strand Selection Is Independent of dsRNA Processing Polarity during RNAi in Drosophila|journal=Current Biology|language=English|volume=16|issue=5|pages=530–535|doi=10.1016/j.cub.2006.01.061|issn=0960-9822|pmid=16527750|doi-access=free|bibcode=2006CBio...16..530P }}</ref> This strand is known as the guide strand and targets mRNA for degradation.
* The other strand, known as the passenger strand, is degraded by RISC.<ref name="Gregory20052">{{Cite journal|vauthors=Gregory RI, Chendrimada TP, Cooch N, Shiekhattar R|year=2005|title=Human RISC couples microRNA biogenesis and posttranscriptional gene silencing|journal=[[Cell (journal)|Cell]]|volume=123|issue=4|pages=631–640|doi=10.1016/j.cell.2005.10.022|pmid=16271387|doi-access=free}}</ref>

[[File:Part of the RNA interference pathway focusing on RISC.png|thumb|Part of the RNA interference pathway with the different ways RISC can silence genes via their messenger RNA.]]

===Gene regulation===

[[File:MicroRNAs and Argonaute RNA binding.svg|thumb|right|AGO2 (grey) in complex with a microRNA (light blue) and its target mRNA (dark blue)]]

Major proteins of RISC, Ago2, SND1, and AEG-1, act as crucial contributors to the gene silencing function of the complex.<ref>{{Cite journal|date=2020-06-01|title=RISC assembly and post-transcriptional gene regulation in Hepatocellular Carcinoma|journal=Genes & Diseases|language=en|volume=7|issue=2|pages=199–204|doi=10.1016/j.gendis.2019.09.009|issn=2352-3042|doi-access=free|last1=Santhekadur|first1=Prasanna K.|last2=Kumar|first2=Divya P.|pmid=32215289|pmc=7083748}}</ref>

RISC uses the guide strand of miRNA or siRNA to target complementary [[Three prime untranslated region|3'-untranslated regions]] (3'UTR) of mRNA transcripts via [[Base pair|Watson-Crick base pairing]], allowing it to regulate gene expression of the mRNA transcript in a number of ways.<ref name="Wakiyama20072">{{Cite journal|vauthors=Wakiyama M, Takimoto K, Ohara O, Yokoyama S|year=2007|title=Let-7 microRNA-mediated mRNA deadenylation and translational repression in a mammalian cell-free system|journal=[[Genes & Development]]|volume=21|issue=15|pages=1857–1862|doi=10.1101/gad.1566707|pmc=1935024|pmid=17671087}}</ref><ref name="Pratt2009" />

====mRNA degradation====

The most understood function of RISC is degradation of target mRNA which reduces the levels of transcript available to be translated by [[ribosomes]]. The endonucleolytic cleavage of the mRNA complementary to the RISC's guide strand by Argonaute protein is the key to RNAi initiation.<ref name=":0">{{Cite journal|last1=ORBAN|first1=TAMAS I.|last2=IZAURRALDE|first2=ELISA|date=April 2005|title=Decay of mRNAs targeted by RISC requires XRN1, the Ski complex, and the exosome|journal=RNA|volume=11|issue=4|pages=459–469|doi=10.1261/rna.7231505|issn=1355-8382|pmc=1370735|pmid=15703439}}</ref> 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.<ref name="Pratt2009"/>

There are two major pathways of mRNA degradation once cleavage has occurred. Both are initiated through degradation of the mRNA's [[Polyadenylation|poly(A) tail]], resulting in removal of the mRNA's 5' cap.

* 5'-to-3' degradation of the transcript occurs by [[5'-3' exoribonuclease 1|XRN1 exonuclease]] in [[cytoplasm]]ic bodies called [[P-bodies]].<ref name="Sen20052">{{Cite journal|name-list-style=amp|vauthors=Sen GL, Blau HM|year=2005|title=Argonaute2/RISC resides in sites of mammalian mRNA decay known as cytoplasmic bodies|journal=[[Nature Cell Biology]]|volume=7|issue=6|pages=633–636|doi=10.1038/ncb1265|pmid=15908945|s2cid=6085169}}</ref>
* 3'-to-5' degradation of the transcript is conducted by the [[Exosome complex|exosome]] and [[Ski complex]].<ref name=":0" />

====Translational repression====
RISC can modulate the loading of ribosome and accessory factors in [[translation]] to [[Repressor|repress]] expression of the bound mRNA transcript. Translational repression only requires a partial sequence match between the guide strand and target mRNA.<ref name="Pratt2009" />

<u>Translation can be regulated at the initiation step by:</u>
*preventing the binding of the [[eukaryotic initiation factor|eukaryotic translation initiation factor]] (eIF) to the [[five-prime cap|5' cap]]. It has been noted RISC can [[deadenylation|deadenylate]] the 3' [[polyadenylation|poly(A) tail]] which might contribute to repression via the 5' cap.<ref name="Filipowicz2008"/><ref name="Wakiyama20072" />
* preventing the binding of the [[eukaryotic large ribosomal subunit (60S)|60S ribosomal subunit]] binding to the mRNA can repress translation.<ref name="Chendrimada2007">{{Cite journal |vauthors=Chendrimada TP, Finn KJ, Ji X, Baillat D, Gregory RI, Liebhaber SA, Pasquinelli AE, Shiekhattar R | title=MicroRNA silencing through RISC recruitment of eIF6 | journal=[[Nature (journal)|Nature]] | volume=447 | issue=7146 | year=2007 | pages=823–828 | doi=10.1038/nature05841 | pmid=17507929 | bibcode=2007Natur.447..823C | s2cid=4413327 }}</ref>

<u>Translation can be regulated at post-initiation steps by:</u>
* peptide degradation,
*promoting premature termination of translation ribosomes,<ref name="Petersen2006">{{Cite journal|vauthors=Petersen CP, Bordeleau ME, Pelletier J, Sharp PA | title=Short RNAs repress translation after initiation in mammalian cells | journal=[[Molecular Cell]] | volume=21 | issue=4 | year=2006 | pages=533–542 | doi=10.1016/j.molcel.2006.01.031 | pmid=16483934| doi-access=free }}</ref> or,
* slowing elongation.<ref name="Maroney2006">{{Cite journal |vauthors=Maroney PA, Yu Y, Fisher J, Nilsen TW | title=Evidence that microRNAs are associated with translating messenger RNAs in human cells | journal=[[Nature Structural & Molecular Biology]] | volume=13 | issue=12 | year=2006 | pages=1102–1107 | doi=10.1038/nsmb1174 | pmid=17128271 | s2cid=19106463 }}</ref>

There is still speculation on whether translational repression via initiation and post-initiation is mutually exclusive.

====Heterochromatin formation====
Some RISCs are able to directly target the [[genome]] by recruiting [[histone methyltransferase]]s to form [[heterochromatin]] at the gene [[Locus (genetics)|locus]], 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.<ref name="Pratt2009" /><ref name="Verdel20042">{{Cite journal|vauthors=Verdel A, Jia S, Gerber S, Sugiyama T, Gygi S, Grewal SI, Moazed D|year=2004|title=RNAi-mediated targeting of heterchromatin by the RITS complex|journal=[[Science (journal)|Science]]|volume=303|issue=5658|pages=672–676|doi=10.1126/science.1093686|pmc=3244756|pmid=14704433|bibcode=2004Sci...303..672V}}</ref><ref name="VerdelA20042">{{Cite journal|vauthors=Verdel A, Jia S, Gerber S, Sugiyama T, Gygi S, Grewal SI, Moazed D|year=2004|title=RITS acts in ''cis'' to promote RNA interference-mediated transcription and post-transcriptional silencing|journal=[[Nature Genetics]]|volume=36|issue=11|pages=1174–1180|doi=10.1038/ng1452|pmid=15475954|doi-access=free}}</ref>

RITS has been shown to direct heterochromatin formation at centromeres through recognition of centromeric repeats. Through base-pairing of siRNA (guide strand) to target chromatin sequences, histone-modifying enzymes can be recruited.<ref>{{Cite journal|last1=Shimada|first1=Yukiko|last2=Mohn|first2=Fabio|last3=Bühler|first3=Marc|date=2016-12-01|title=The RNA-induced transcriptional silencing complex targets chromatin exclusively via interacting with nascent transcripts|journal=Genes & Development|volume=30|issue=23|pages=2571–2580|doi=10.1101/gad.292599.116|issn=0890-9369|pmc=5204350|pmid=27941123}}</ref>

The mechanism is not well understood; however, 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.<ref name="Sugiyama20052">{{Cite journal|vauthors=Sugiyama T, Cam H, Verdel A, Moazed D, Grewal SI|year=2005|title=RNA-dependent RNA polymerase is an essential component of a self-enforcing loop coupling heterochromatin assembly to siRNA production|journal=[[Proceedings of the National Academy of Sciences of the United States of America]]|volume=102|issue=1|pages=152–157|doi=10.1073/pnas.0407641102|pmc=544066|pmid=15615848|doi-access=free|bibcode=2005PNAS..102..152S }}</ref>

In ''[[Schizosaccharomyces pombe]]'' and ''[[Arabidopsis]]'', the processing of dsRNA targets into siRNA by Dicer RNases can initiate a gene silencing pathway by heterochromatin formation. An Argonaute protein known as [[AGO4]] interacts with the small RNAs that define heterochromatic sequences. A [[Histone methyltransferase|histone methyl transferase]] (HMT), [[H3K9]], methylates histone H3 and recruits chromodomain proteins to the methylation sites. DNA methylation maintains the silencing of genes as the heterochromatin sequences can be established or spread.<ref name="Mochizuki20042">{{Cite journal|name-list-style=amp|vauthors=Mochizuki K, Gorovsky MA|year=2004|title=Small RNAs in genome arrangement in ''Tetrahymena''|journal=[[Current Opinion in Genetics & Development]]|volume=14|issue=2|pages=181–187|doi=10.1016/j.gde.2004.01.004|pmid=15196465}}</ref>

====DNA elimination====
The siRNA generated by RISCs seem to have a role in degrading DNA during [[Somatic (biology)|somatic]] [[macronucleus]] development in [[ciliates]] of the genus ''[[Tetrahymena]]''. It is similar to the epigenetic control of heterochromatin formation and is implied as a defense against invading genetic elements.<ref name="Mochizuki20042" />

Similar to heterochromatin formation in ''S. pombe'' and ''Arabidopsis'', a ''Tetrahymena''  protein related to the Argonaute family, Twi1p, catalyzes DNA elimination of target sequences known as internal elimination sequences (IESs). Using methyltransferases and chromodomain proteins, IESs are heterochromatized and eliminated from the DNA.<ref name="Mochizuki20042" />

==RISC-associated proteins==
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.<ref name="Sontheimer2005">{{Cite journal | author=Sontheimer EJ | title=Assembly and function of RNA silencing complexes | journal=[[Nature Reviews Molecular Cell Biology]] | volume=6 | issue=2 | year=2005 | pages=127–138 | doi=10.1038/nrm1568 | pmid=15654322 | s2cid=27294007 }}</ref>

{| class="wikitable"
|+'''Table 1: Complexes implicated in RISC assembly and function''' 
|+''Based on table by Sontheimer (2005)''<ref name="Sontheimer2005" /> 
|-
! Complex !! Source !! Known/apparent components !! Estimated size !! Apparent function in RNAi pathway 
|-
| Dcr2-R2D2<ref name="Liu2003">{{Cite journal |vauthors=Liu Q, Rand TA, Kalidas S, Du F, Kim HE, Smith DP, Wang X |title=R2D2, a bridge between the initiation and effector steps of the ''Drosophila'' RNAi pathway | journal=[[Science (journal)|Science]] | volume=301 | issue=5641 | year=2003 | pages=1921–1925 | doi=10.1126/science.1088710 | pmid=14512631|bibcode=2003Sci...301.1921L |s2cid=41436233 }}</ref> || ''D. melanogaster'' S2 cells || [[Dicer|Dcr2]], [[R2D2 (protein)|R2D2]] || ~250 kDa|| dsRNA processing, siRNA binding
|-
| RLC (A)<ref name="Pham2004">{{Cite journal |vauthors=Pham JW, Pellio JL, Lee YS, Carthew RW, Sontheimer EJ | title=A Dicer-2-dependent 80S complex cleaves targeted mRNAs during RNAi in ''Drosophila'' | journal=[[Cell (journal)|Cell]] | volume=117 | issue=1 | year=2004 | pages=83–94 | doi=10.1016/S0092-8674(04)00258-2 | pmid=15066284 | doi-access=free }}</ref><ref name="Tomari2004">{{Cite journal |vauthors=Tomari Y, Du T, Haley B, Schwarz DS, Bennett R, Cook HA, Koppetsch BS, Theurkauf WE, Zamore PD | title=RISC assembly defects in the ''Drosophila'' RNAi mutant ''armitage'' | journal=[[Cell (journal)|Cell]] | volume=116 | issue=6 | year=2004 | pages=831–841 | doi=10.1016/S0092-8674(04)00218-1 | pmid=15035985 | doi-access=free }}</ref> || ''D. melanogaster'' embryos || Dcr2, R2D2 || NR || dsRNA processing, siRNA binding, precursor to RISC 
|-
| Holo-RISC<ref name="Pham2004"/><ref name="Tomari2004"/> || ''D. melanogaster'' embryos || [[EIF2C2|Ago 2]], Dcr1, Dcr2, [[FMR1|Fmr1]]/[[FXR1|Fxr]], R2D2, [[Tudor domain|Tsn]], [[Vasa intronic gene|Vig]] || ~80S || Target-RNA binding and cleavage 
|-
| RISC<ref name="Hammond2000"/><ref name="Hammond2001">{{Cite journal |vauthors=Hammond SM, Boettcher S, Caudy AA, Kobayashi R, Hannon GJ | title=Argonaute2, a link between genetic and biochemical analyses of RNAi | journal=[[Science (journal)|Science]] | volume=293 | issue=5532 | pages=1146–1150 | doi=10.1126/science.1064023 | pmid=11498593 | year=2001| s2cid=5271290 }}</ref><ref name="Caudy2002">{{Cite journal |vauthors=Caudy AA, Myers M, Hannon GJ, Hammond SM | title=Fragile X-related protein and VIG associate with the RNA interference machinery | journal=[[Genes & Development]] | volume=16 | issue=19 | pages=2491–2496 | doi=10.1101/gad.1025202 | pmc=187452 | pmid=12368260 | year=2002}}</ref><ref name="Caudy2003">{{Cite journal |vauthors=Caudy AA, Ketting RF, Hammond SM, Denli AM, Bathoorn AM, Tops BB, Silva JM, Myers MM, Hannon GJ, Plasterk RH | title=A micrococcal nuclease homologue in RNAi effector complexes | journal=[[Nature (journal)|Nature]] | volume=425 | issue=6956 | year=2003 | pages=411–414 | doi=10.1038/nature01956 | pmid=14508492| bibcode=2003Natur.425..411C | s2cid=4410688 }}</ref> || ''D. melanogaster'' S2 cells || Ago2, Fmr1/Fxr, Tsn, Vig || ~500 kDa || Target-RNA binding and cleavage 
|-
| RISC<ref name="Rand2004">{{Cite journal |vauthors=Rand TA, Ginalski K, Grishin NV, Wang X | title=Biochemical identification of Argonaute 2 as the sole protein required for RNA-induced silencing complex activity | journal=[[Proceedings of the National Academy of Sciences of the United States of America]] | volume=101 | issue=40 | year=2004 | pages=14385–14389 | doi=10.1073/pnas.0405913101 | pmid=15452342 | pmc=521941| bibcode=2004PNAS..10114385R | doi-access=free }}</ref> || ''D. melanogaster'' S2 cells || Ago2 || ~140 kDa || Target-RNA binding and cleavage 
|-
| Fmr1-associated complex<ref name="Ishizuka2002">{{Cite journal |vauthors=Ishizuka A, Siomi MC, Siomi H | title=A ''Drosophila'' fragile X protein interacts with components of RNAi and ribosomal proteins | journal=[[Genes & Development]] | volume=16 | issue=19 | year=2002 | pages=2497–2508 | doi=10.1101/gad.1022002 | pmc=187455 | pmid=12368261}}</ref> || ''D. melanogaster'' S2 cells || [[Ribosomal protein L5|L5]], [[RPL11|L11]], [[5S ribosomal RNA|5S rRNA]], Fmr1/Fxr, Ago2, [[DDX5|Dmp68]] || NR || Possible target-RNA binding and cleavage
|-
| Minimal RISC<ref name="Martinez2002">{{Cite journal |vauthors=Martinez J, Patkaniowska A, Urlaub H, Luhrmann R, Tuschl T | title=Single-stranded antisense siRNAs guide target RNA cleavage in RNAi | journal=[[Cell (journal)|Cell]] | volume=110 | issue=5 | year=2002 | pages=563–574 | doi=10.1016/S0092-8674(02)00908-X | pmid=12230974| hdl=11858/00-001M-0000-0012-F2FD-2 | s2cid=10616773 | hdl-access=free }}</ref><ref name="Liu2004">{{Cite journal |vauthors=Liu J, Carmell MA, Rivas FV, Marsden CG, Thomson JM, Song JJ, Hammond SM, Joshua-Tor L, Hannon GJ | title=Argonaute2 is the catalytic engine of mammalian RNAi | journal=[[Science (journal)|Science]] | volume=305 | issue=5689 | year=2004 | pages=1437–1441 | doi=10.1126/science.1102513 | pmid=15284456| bibcode=2004Sci...305.1437L | s2cid=2778088 | doi-access=free }}</ref><ref name="Martinez2004">{{Cite journal |vauthors=Martinez J, Tuschl T|name-list-style=amp | title=RISC is a 5′ phosphomonoester-producing RNA endonuclease | journal=[[Genes & Development]] | volume=18 | issue=9 | year=2004 | pages=975–980 | doi=10.1101/gad.1187904 | pmc=406288 | pmid=15105377}}</ref><ref name="Meister2004"/>  || [[HeLa]] cells || [[EIF2C1|eIF2C1 (Ago1)]] or eIF2C2 (Ago2) || ~160 kDa || Target-RNA binding and cleavage 
|-
| miRNP<ref name="Mourelatos2002">{{Cite journal |vauthors=Mourelatos Z, Dostie J, Paushkin S, Sharma A, Charroux B, Abel L, [[Juri Rappsilber|Rappsilber J]], Mann M, Dreyfuss G | title=miRNPs: a novel class of ribonucleoproteins containing numerous microRNAs | journal=[[Genes & Development]] | volume=16 | issue=6 | year=2002 | pages=720–728 | doi=10.1101/gad.974702 | pmid=11914277 | pmc=155365}}</ref><ref name="Hutvagner2002">{{Cite journal |vauthors=Hutvágner G, Zamore PD|name-list-style=amp | title=A microRNA in a multiple-turnover RNAi enzyme complex | journal=[[Science (journal)|Science]] | volume=297 | issue=5589 | year=2002 | pages=2056–2060 | doi=10.1126/science.1073827 | pmid=12154197|bibcode=2002Sci...297.2056H |s2cid=16969059 }}</ref> || HeLa cells || eIF2C2 (ago2), [[DDX20|Gemin3]], [[GEMIN4|Gemin4]] || ~550 kDa || miRNA association, target-RNA binding and cleavage 
|}
<small>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.</small>

[[File:1u04-argonaute.png|thumb|A full-length argonaute protein from the archaea species ''Pyrococcus furiosus''.]]

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===
{{Main|Argonaute}}

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.<ref name="Hall2005">{{Cite journal | author=Hall TM | title=Structure and function of Argonaute proteins | journal=[[Cell (journal)|Cell]] | volume=13 | issue=10 | year=2005 | pages=1403–1408 | doi=10.1016/j.str.2005.08.005 | pmid=16216572 | doi-access=free }}</ref> There are eight family members in human Argonautes of which only Argonaute 2 is exclusively involved in targeted RNA cleavage in RISC.<ref name="Meister2004">{{Cite journal |vauthors=Meister G, Landthaler M, Patkaniowska A, Dorsett Y, Teng G, Tuschl T | title=Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs | journal=[[Molecular Cell]] | volume=15 | issue=2 | year=2004 | pages=1403–1408 | doi=10.1016/j.molcel.2004.07.007 | pmid=15260970| doi-access=free }}</ref>

[[File:RISC-loading complex.png|thumb|The RISC-loading complex allows the loading of dsRNA fragments (generated by Dicer) to be loaded onto Argonaute 2 (with the help of TRBP) as part of the RNA interference pathway.]]

===RISC-loading complex===
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 transactivating response RNA-binding protein ([[TARBP2|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 [[Protein domain|domains]]. 
* '''Argonaute 2''' is an RNase and is the catalytic centre of RISC.

Dicer associates with TRBP and Argonaute 2 to facilitate the transfer of the dsRNA fragments generated by Dicer to Argonaute 2.<ref name="Chendrimada2005">{{Cite journal |vauthors=Chendrimada TP, Gregory RI, Kumaraswamy E, Norman J, Cooch N, Nishikura K, Shiekhatter R | title=TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing | journal=[[Nature (journal)|Nature]] | volume=436 | issue=7051 | year=2005 | pages=740–744| doi=10.1038/nature03868 | pmid=15973356 | pmc=2944926| bibcode=2005Natur.436..740C }}</ref><ref name="Wang2009">{{Cite journal |vauthors=Wang HW, Noland C, Siridechadilok B, Taylor DW, Ma E, Felderer K, Doudna JA, Nogales E | title=Structural insights into RNA processing by the human RISC-loading complex | journal=[[Nature Structural & Molecular Biology]] | volume=16 | issue=11 | year=2009 | pages=1148–1153 | doi=10.1038/nsmb.1673| pmc=2845538 | pmid=19820710 }}</ref>

More recent research has shown the human [[RNA helicase A]] could help facilitate the RLC.<ref name="Fu2013">{{Cite journal |vauthors=Fu Q, Yuan YA|name-list-style=amp | title=Structural insights into RISC assembly facilitated by dsRNA-binding domains of human RNA helices A (DHX9) | journal=[[Nucleic Acids Research]] | volume=41 | issue=5 | year=2013 | pages=3457–3470 | doi=10.1093/nar/gkt042 | pmid=23361462 | pmc=3597700}}</ref>

===Other proteins===
Recently identified members of RISC are [[SND1]] and [[MTDH]].<ref name="Yoo2011">{{Cite journal|vauthors=Yoo BK, Santhekadur PK, Gredler R, Chen D, Emdad L, Bhutia S, Pannell L, Fisher PB, Sarkar D | title=Increased RNA-induced silencing complex (RISC) activity contributes to hepatocellular carcinoma | journal=Hepatology | volume=53 | issue=5 | year=2011 | pages=1538–1548 | doi=10.1002/hep.24216 | pmid=21520169 | pmc=3081619}}</ref> SND1 and MTDH are oncogenes and regulate various gene expression.<ref name="YooBK2011">{{Cite journal |vauthors=Yoo BK, Emdad L, Lee SG, Su Z, Santhekadur P, Chen D, Gredler R, Fisher PB, Sarkar D | title=Astrocyte elevated gene (AEG-1): a multifunctional regulator of normal and abnormal physiology | journal=[[Pharmacology & Therapeutics]] | volume=130 | issue=1 | year=2011 | pages=1–8 | doi=10.1016/j.pharmthera.2011.01.008 | pmid=21256156 | pmc=3043119}}</ref>

{| class="wikitable"
|+Table 2: Biochemically documented proteins associated with RISC
|+''Based on the table by Sontheimer (2005)''<ref name="Sontheimer2005"/>
|-
! Protein !! Species the protein is found
|-
| Dcr1<ref name="Pham2004"/> || ''D. melanogaster''
|-
| Dcr2<ref name="Liu2003"/><ref name="Pham2004"/><ref name="Tomari2004"/> || ''D. melanogaster''
|-
| R2D2<ref name="Pham2004"/><ref name="Tomari2004"/> || ''D. melanogaster''
|-
| Ago2<ref name="Pham2004"/><ref name="Hammond2001"/><ref name="Rand2004"/><ref name="Ishizuka2002"/> || ''D. melanogaster''
|-
| Dmp68<ref name="Ishizuka2002"/> || ''D. melanogaster''
|-
| Fmr1/Fxr<ref name="Pham2004"/><ref name="Caudy2002"/><ref name="Ishizuka2002"/> || ''D. melanogaster''
|-
| Tsn<ref name="Pham2004"/><ref name="Caudy2003"/> || ''D. melanogaster''
|-
| Vig<ref name="Pham2004"/><ref name="Caudy2002"/> || ''D. melanogaster''
|-
| [[Polysome|Polyribosomes]], ribosome components<ref name="Hammond2000"/><ref name="Pham2004"/><ref name="Hammond2001"/><ref name="Ishizuka2002"/><ref name="Djikeng2003">{{Cite journal |vauthors=Djikeng A, Shi H, Tschudi C, Shen S, Ullu E | title=An siRNA ribonucleoprotein is found associated with polyribosomes in ''Trypanosoma brucei'' | journal=[[RNA (journal)|RNA]] | volume=9 | issue=7 | year=2003 | pages=802–808 | doi=10.1261/rna.5270203 | pmc=1370447 | pmid=12810914}}</ref> || ''D. melanogaster'', ''[[Trypanosoma brucei|T. brucei]]''
|-
| eIF2C1 (Ago1)<ref name="Martinez2002"/> || ''[[Homo sapiens|H. sapiens]]''
|-
| eIF2C2 (Ago2)<ref name="Martinez2002"/><ref name="Liu2004"/><ref name="Meister2004"/><ref name="Hutvagner2002"/>  || ''H. sapiens''
|-
| Gemin3<ref name="Mourelatos2002"/><ref name="Hutvagner2002"/> || ''H. sapiens''
|-
| Gemin4<ref name="Mourelatos2002"/><ref name="Hutvagner2002"/> || ''H. sapiens''
|}

<small>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.</small>

==Binding of mRNA==
[[File:MiRNA.svg|thumb|350px|Diagram of RISC activity with miRNAs]]
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.<ref name="SenGL2005">{{Cite journal |vauthors=Sen GL, Wehrman TS, Blau HM | title=mRNA translation is not a prerequisite for small interfering RNA-mediated mRNA cleavage | journal=[[Differentiation (journal)|Differentiation]] | volume=73 | issue=6 | year=2005 | pages=287–293 | doi= 10.1111/j.1432-0436.2005.00029.x | pmid=16138829}}</ref>

Endogenously expressed miRNA in [[animal|metazoans]] is usually not perfectly complementary to a large number of genes and thus, they modulate expression via translational repression.<ref name="Saumet2006">{{Cite journal |vauthors=Saumet A, Lecellier CH|name-list-style=amp | title=Anti-viral RNA silencing: do we look like plants? | journal=[[Retrovirology (journal)|Retrovirology]] | volume=3 | year=2006 | pages=3 | doi=10.1186/1742-4690-3-3 | pmid=16409629 | pmc=1363733 |doi-access=free }}</ref><ref name="Bartel2009">{{Cite journal | author=Bartel DP | title=MicroRNAs: target recognition and regulatory functions | journal=[[Cell (journal)|Cell]] | volume=136 | issue=2 | year=2009 | pages=215–233 | doi=10.1016/j.cell.2009.01.002 | pmid=19167326 | pmc=3794896}}</ref> However, in [[plant]]s, 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.<ref name="Jones2006">{{Cite journal |vauthors=Jones-Rhoades MW, Bartel DP, Bartel B | title=MicroRNAs and their regulator roles in plants | journal=[[Annual Review of Plant Biology]] | volume=57 | year=2006 | pages=19–53 | doi=10.1146/annurev.arplant.57.032905.105218 | pmid=16669754}}</ref>

==See also==
*[[RNA-induced transcriptional silencing]] (RITS)
*[[RNA interference]]

==References==
{{Reflist|30em}}

==Further reading==
{{refbegin}}
*{{Cite journal | doi = 10.1038/nrm1568| title = Assembly and function of RNA silencing complexes| journal = [[Nature Reviews Molecular Cell Biology]]| volume = 6| issue = 2| pages = 127–138| year = 2005| last1 = Sontheimer | first1 = EJ| pmid = 15654322| s2cid = 27294007}}
* {{cite journal | vauthors = Fu Q, Yuan YA | title = Structural insights into RISC assembly facilitated by dsRNA-binding domains of human RNA helicase A (DHX9) | journal = Nucleic Acids Research | volume = 41 | issue = 5 | pages = 3457–70 | date = March 2013 | pmid = 23361462 | pmc = 3597700 | doi = 10.1093/nar/gkt042 }}
* {{cite journal |vauthors=Schwarz DS, Tomari Y, Zamore PD | title=The RNA-induced silencing complex is a Mg<sup>2+</sup>-dependent endonuclease | journal= [[Current Biology]] | volume=14 | issue=9 | pages=787–91 | year=2004 | pmid=15120070 |doi=10.1016/j.cub.2004.03.008| doi-access=free | bibcode=2004CBio...14..787S }}
{{refend}}

==External links==
* {{MeshName|RNA-Induced+Silencing+Complex}}

{{Nucleases}}

[[Category:RNA]]