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The RNA-induced silencing complex, or RISC, is a ribonucleoprotein complex involved in the RNA interference pathway. The pathway scans the cell for mRNA that are viral in nature or ones that have been transcribed from viral DNA and/or transposable elements in the genome. In doing so, the pathway interferes with the translation process of these mRNA molecules, thereby stopping the production of viral proteins that could be harmful to the cell. As part of this pathway, RISC processes and incorporates microRNA or small interfering RNA produced from viral DNA and/or transposable elements in the genome. By using the incorporated microRNA or siRNA as a template, the RISC complex searches the cell for mRNA that are complementary to the template. Based on the degree of complementarity between the mRNA and the template, the RISC interferes with the translation of protein from the mRNA. The end result is gene silencing, which is a defense mechanism against viral infections in many eukaryotes.

Discovery of RISC


RISC was first discovered in 2000 by Gregory Hannon and his colleagues at the Cold Spring Harbor Laboratory.

Hannon and his colleagues noted that, by introducing double-stranded RNAs into organisms such as C. elegans and Drosophila, gene expression could be inhibited in a sequence-specific manner via RNA interference. They sought out to determine the underlying mechanism behind RNA interference. By introducing double stranded RNAs, a 'loss-of-function' phenotype could appear in cultured Drosophila cells. This was correlated with a decrease in the levels of mRNA in the cells, implying that the absence of mRNA led to the loss of function. The Drosophila cells also contained nuclease activity that specifically cleaved mRNA transcripts that were homologous to the introduced double-stranded RNA. They found that this particular enzyme contained an essential RNA component. The source of this nuclease activity turned out to be a multiprotein complex, later called RISC.

Structure of RISC
The structure of RISC varies between different organisms and cell types. However, the three most common components are Argonaute, Dicer and TBPR, with Argonaute being present in most RISC. Together, the three components form the RISC-loading complex.

RISC-loading complex
The RISC-loading complex (RLC) is the essential structure required to load double stranded RNA fragments into RISC in order to target mRNA. The double-stranded RNA can either be microRNA or small interfering RNA. The RLC consists of dicer, the transactivating response RNA-binding protein (TRBP) and Argonaute 2. Dicer associates with TRBP and Argonaute 2 to facilitate the transfer of the dsRNA fragments generated by Dicer to Argonaute 2.

Dicer endonuclease
Dicer is an endonuclease that is encoded by the DICER1 gene in humans. Being part of the RNase III family, Dicer cleaves double-stranded RNA (dsRNA) and pre-microRNA (pre-miRNA) into short double-stranded RNA fragments called small interfering RNA and microRNA, respectively. These fragments are approximately 21 to 23 base pairs in length and are then delivered to Argonaute proteins straight after cleavage.

Argonaute proteins
A major component of RISC, Argonaute is a ubiquitous protein found in plants, animals and fungi. It holds the dsRNA template received from Dicer and also provides a region in which the dsRNA can interact with the target mRNA. Argonaute then cleaves the mRNA with its endonuclease activity, preventing translation and silencing the gene.

There are many families of Argonaute proteins. The prokaryotic ones are often functionally redundant and can replace one another. The eukaryotic ones differ by the means with which they bind to dsRNA. The largest family of eukaryotic Argonaute is known as Ago, with Ago2 being the major component of human RISC.

However, the structure of eukaryotic Argonaute is as of yet unclear. The structures of prokaryotic Argonaute has been better described. The protein has two lobes, each responsible for binding opposite end of the small interfering RNA. The N-terminal lobe contains a PAZ domain, which binds the 3'-end of the dsRNA. On the other hand, the C-terminal lobe contains the middle domain and the PIWI domain. The middle domain binds to the 5'-phosphate of the dsRNA whilst the PIWI domain acts as a RNAse to cleave the target mRNA in a process called 'slicing'. Furthermore, there is a flexible hinge region between the two lobes which provides structural flexibility. This allows the two lobes to pivot around one another, opening a region for the dsRNA template and mRNA template to bind.

TRBP
TRBP is a Dicer-binding protein that regulates Dicer activity. It works together with another Dicer-binding protein called PACT to regulate Dicer activity, though both may appear functionally redundant on first inspection. When TRBP binds to Dicer, the endonuclease activity of Dicer is activated and Dicer proceeds to cleave 70-base-pair dsRNA into siRNA or microRNA. On the other hand, when PACT binds to Dicer, the endonuclease activity is inhibited, leading to no yield of siRNA or microRNA.

Loading of template RNA
RISC activity is triggered by the appearance of double stranded RNA in the cytoplasm of a eukaryotic cell. The process starts in the nucleus with a precursor microRNA, which is an RNA transcript that can fold back on itself to form a double stranded RNA. The precursor microRNA is cleaved by the ribonuclease Drosha, trimming down to a double stranded molecule which is around 70 base pairs in length and contains hairpins. This RNA molecule is then exported out of the nucleus and into the cytoplasm, where it becomes substrate for the Dicer-containing pre-RISC complex. The Dicer endonuclease trims the 70-base-pair microRNA down to around 21 to 23 base pairs, which is then incorporated into the pre-RISC complex to form an active mature RISC. While incorporated, the double-stranded RNA splits into two single strands. The single strand with the higher free energy is thermodynamically favorable and therefore kept within RISC. This is known as the guide strand. The strand with lower free energy is known as the passenger strand; it detaches from RISC before being degraded.

mRNA binding
By this stage, RISC is a ribonucleotide particle containing a single-stranded RNA, known as the guide strand, which is bound to an Argonaute protein. While bound to Argonaute, the bases 2 to 6 of the guide strand protrude out from the structure to face the cytoplasm. This protruding region is known as the seed region and is the primary site for target recognition. This explains why RISC is so efficient in searching the cytoplasm for free-floating mRNA. Once a target is found, the seed region binds to the complementary region on the target. This binding causes a conformational change in the Argonaute which opens up a binding cleft to accommodate for both the guide strand and mRNA target. The level of complementarity between the guide strand and the mRNA will determine the pathway which RISC will take in order to silence the gene: either mRNA degradation or translational repression.

mRNA degradation
Also known as splicing, mRNA degradation is the simplest silencing pathway. If there is complete complementarity between the guide strand in the RISC and the mRNA, then the nuclease activity of Argonaute 2 is activated. The PIWI domain of Argonaute 2 catalyses the slicing of the target mRNA. The amino acids of the PIWI domain coordinate catalytic metal ions such that a water molecule can perform nucleophilic attack on the phosphodiester backbone of the target mRNA. After hydrolysis, the fragments of the target can still be translated, but the resulting protein product will contain frameshift and stop codons, rendering them dysfunctional and targeted for degradation.

Translational repression
Translational repression is the most prevalent pathway of gene silencing in mammals. If there is incomplete complementarity between the microRNA and target mRNA, the nuclease activity of Ago2 is not activated. Therefore, mRNA is not cleaved and its 3' end remains bound to RISC. This prevents the 3' end of the mRNA from joining with the 5' end. Without circularisation of the mRNA, the ribosome cannot initiate translation and viral protein is not produced.

However, there are two other mechanisms by which RISC can repress translation. Both are from Drosophila but involve different Argonaute proteins. The first pathway involves Ago2 blocking protein-protein interaction within the translation initiation complex. Without the complex, translation cannot occur. The second path involve Ago1 promoting the deadenylation and degradation of the target mRNA.

It is important to note that translational repression can only take place if the Argonaute-bound guide strand is a microRNA. The process cannot occur if the guide strand is a siRNA. In addition, with regards to incomplete complementarity, the only region on the microRNA that is required to be complementary to the mRNA is bases 2 to 7.

Heterochromatin formation
Instead of binding to mRNA like in the previous two pathways, some RISCs are able to directly target the genome by recruiting histone methyltransferases to form heterochromatin at the gene locus, 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. 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.

DNA elimination
The following RISC pathway is unique to Tetrahymena thermophila, which has two nuclei in its cell. One is a macronucleus, which is active during vegetative growth, whilst the other is a micronucleus, which is active during germ line. After sexual conjugation, a new macronucleus is formed from the mated micronucleus and the old macronucleus needs to be discarded. During the formation of the new macronucleus, parts of the DNA that were not present in the old macronucleus are eliminated by RISC. The proposed model is that the genetic content of the newly formed macronucleus is transcribed into siRNA, which is then loaded into RISCs as a template. RISCs then scan the old macronucleus, binding to any complementary region, and are discarded along with the old macronucleus. This leaves RISCs that did not bind to the old macronucleus and, therefore, only target unnecessary sequences in the new macronucleus, eliminating them so the new nucleus is identical to the old.