RISC

The **RNA-induced silencing complex**, or **RISC**, is a multiprotein complex, specifically a ribonucleoprotein, which incorporates one strand of a single-stranded RNA (ssRNA) fragment, such as microRNA (miRNA), or double-stranded small interfering RNA (siRNA). The single strand acts as a template for RISC to recognize 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 defense against viral infections.

## Discovery

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 protein 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.

### Gene regulation

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.

#### mRNA degradation

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.

mRNA degradation is localised in cytoplasmic bodies called P-bodies.

#### Translational repression

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:

* promoting premature termination of translation ribosomes, or,
* slowing elongation.

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 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.

#### DNA elimination

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.

## 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.

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

Main articles: 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. There are eight family members in human Argonautes of which only Argonaute 2 is exclusively involved in targeted RNA cleavage in RISC.

From Wikipedia:Argonaute:

For the diseases that are involved with selective or elevated expression of particular identified genes, such as pancreatic cancer, the high sequence specificity of RNA interference might make it suitable to be a suitable treatment, particularly appropriate for combating cancers associated with mutated endogenous gene sequences. It has been reported several tiny non-coding RNAs(microRNAs) are related with human cancers, like miR-15a and miR-16a are frequently deleted and/or down-regulated in patients. Even though the biological functions of miRNAs are not fully understood, the roles for miRNAs in the coordination of cell proliferation and cell death during development and metabolism have been uncovered. It is trusted that the miRNAs can direct negative or positive regulation at different levels, which depends on the specific miRNAs and target base pair interaction and the cofactors that recognize them.

Because it has been widely known that many viruses have RNA rather than DNA as their genetic material and go through at least one stage in their life cycle when they make double-stranded RNA, RNA interference has been considered to be a potentially evolutionarily ancient mechanism for protecting organisms from viruses. The small interfering RNAs produced by Dicer cause sequence specific, post-transcriptional gene silencing by guiding an endonuclease, the RNA-induced silencing complex (RISC), to mRNA. This process has been seen in a wide range of organisms, such as Neurospora fungus(in which it is known as quelling), plants(post-transcriptionl gene silencing) and mammalian cells(RNAi). If there is a complete or near complete sequence complementarity between the small RNA and the target, the Argonaute protein component of RISC mediates cleavage of the target transcript, the mechanism involves repression of translation predominantly.

### 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 (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.

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

### Other proteins

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

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.