RNA-induced silencing complex

The biochemical identification of RISC was conducted by Gregory Hannon and his colleagues at the Cold Spring Harbor Laboratory.

{{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 | volume=404 | issue=6775 | year=2000 | pages=293–296 | doi=10.1038/35005107 | pmid=10749213| bibcode=2000Natur.404..293H | s2cid=9091863 }} 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.

File:Drosophila melanogaster - side (aka).jpg

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. 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].

File:1ytu argonaute dsrna.png

The 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 nucleotides in length with a two-nucleotide 3' overhang.{{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|volume=101|issue=1|pages=25–33|doi=10.1016/S0092-8674(00)80620-0|pmid=10778853|doi-access=free}}{{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|volume=11|issue=5|pages=674–682|doi=10.1261/rna.7272305|pmc=1370754|pmid=15811921}} 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.{{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}}{{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|volume=115|issue=2|pages=199–208|doi=10.1016/S0092-8674(03)00759-1|pmid=14567917|doi-access=free}}{{Cite journal|vauthors=Khvorova A, Reynolds A, Jayasena SD|year=2003|title=Functional siRNAs and miRNAs exhibit strand bias|journal=Cell|volume=115|issue=2|pages=209–216|doi=10.1016/S0092-8674(03)00801-8|pmid=14567918|s2cid=2500175|doi-access=free}}{{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|volume=457|issue=7228|pages=396–404|doi=10.1038/nature07754|pmid=19158785|bibcode=2009Natur.457..396S|s2cid=205215974}} The newly generated miRNA or siRNA act as single-stranded guide sequences for RISC to target mRNA for degradation.{{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}}{{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}}

• The strand with the less thermodynamically stable 5' end is selected by the protein Argonaute and integrated into RISC.{{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 }} 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.{{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|volume=123|issue=4|pages=631–640|doi=10.1016/j.cell.2005.10.022|pmid=16271387|doi-access=free}}

File:Part of the RNA interference pathway focusing on RISC.png

File:MicroRNAs and Argonaute RNA binding.svg

Major proteins of RISC, Ago2, SND1, and AEG-1, act as crucial contributors to the gene silencing function of the complex.{{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}}

RISC uses the guide strand of miRNA or siRNA to target complementary 3'-untranslated regions (3'UTR) of mRNA transcripts via Watson-Crick base pairing, allowing it to regulate gene expression of the mRNA transcript in a number of ways.{{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}}

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.{{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}} 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.

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

• 5'-to-3' degradation of the transcript occurs by XRN1 exonuclease in cytoplasmic bodies called P-bodies.{{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}}
• 3'-to-5' degradation of the transcript is conducted by the exosome and Ski complex.

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.{{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 | volume=447 | issue=7146 | year=2007 | pages=823–828 | doi=10.1038/nature05841 | pmid=17507929 | bibcode=2007Natur.447..823C | s2cid=4413327 }}

Translation can be regulated at post-initiation steps by:

• peptide degradation,
• promoting premature termination of translation ribosomes,{{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 }} or,
• slowing elongation.{{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 }}

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, 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.{{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|volume=303|issue=5658|pages=672–676|doi=10.1126/science.1093686|pmc=3244756|pmid=14704433|bibcode=2004Sci...303..672V}}{{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}}

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.{{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}}

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.{{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 }}

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 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.{{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}}

The siRNA generated by RISCs seem to have a role in degrading DNA during 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.

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.

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.{{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 }}

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.

File:1u04-argonaute.png

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.

{{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.{{Cite journal | author=Hall TM | title=Structure and function of Argonaute proteins | journal=Cell | volume=13 | issue=10 | year=2005 | pages=1403–1408 | doi=10.1016/j.str.2005.08.005 | pmid=16216572 | doi-access=free }} There are eight family members in human Argonautes of which only Argonaute 2 is exclusively involved in targeted RNA cleavage in RISC.{{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 }}

File:RISC-loading complex.png

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 (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.

Dicer associates with TRBP and Argonaute 2 to facilitate the transfer of the dsRNA fragments generated by Dicer to Argonaute 2.{{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 | volume=436 | issue=7051 | year=2005 | pages=740–744| doi=10.1038/nature03868 | pmid=15973356 | pmc=2944926| bibcode=2005Natur.436..740C }}{{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 }}

More recent research has shown the human RNA helicase A could help facilitate the RLC.{{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}}

Recently identified members of RISC are SND1 and MTDH.{{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}} SND1 and MTDH are oncogenes and regulate various gene expression.{{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}}

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.

File:MiRNA.svg

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.{{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 | volume=73 | issue=6 | year=2005 | pages=287–293 | doi= 10.1111/j.1432-0436.2005.00029.x | pmid=16138829}}

Endogenously expressed miRNA in metazoans is usually not perfectly complementary to a large number of genes and thus, they modulate expression via translational repression.{{Cite journal |vauthors=Saumet A, Lecellier CH|name-list-style=amp | title=Anti-viral RNA silencing: do we look like plants? | journal=Retrovirology | volume=3 | year=2006 | pages=3 | doi=10.1186/1742-4690-3-3 | pmid=16409629 | pmc=1363733 |doi-access=free }}{{Cite journal | author=Bartel DP | title=MicroRNAs: target recognition and regulatory functions | journal=Cell | volume=136 | issue=2 | year=2009 | pages=215–233 | doi=10.1016/j.cell.2009.01.002 | pmid=19167326 | pmc=3794896}} 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.{{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}}

• RNA-induced transcriptional silencing (RITS)
• RNA interference

{{Reflist|30em}}

{{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 Mg²⁺-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}}

• {{MeshName|RNA-Induced+Silencing+Complex}}

{{Nucleases}}

Category:RNA