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. 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. The single strand of RNA acts as a template for RISC to recognize complementary messenger RNA (mRNA) 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 infections, as it is triggered by the presence of double-stranded RNA (dsRNA). 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. 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. The newly generated miRNA or siRNA act as single-stranded guide sequences for RISC to target mRNA for degradation.
- The strand with the less thermodynamically stable 5' end is selected by the protein Argonaute and integrated into RISC. 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.
thumb|Part of the RNA interference pathway with the different ways RISC can silence genes via their messenger RNA.
Gene regulation
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.
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. 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.
- 3'-to-5' degradation of the transcript is conducted by the exosome and Ski complex.
<u>Translation can be regulated at post-initiation steps by:</u>
- peptide degradation,
- 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, 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.
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.
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.
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.
DNA elimination
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.
{| class="wikitable"
|+Table 1: Complexes implicated in RISC assembly and function
|+Based on table by Sontheimer (2005) || 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 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 || eIF2C2 (ago2), Gemin3, 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>
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
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.
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 (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.
{| class="wikitable"
|+Table 2: Biochemically documented proteins associated with RISC
|+Based on the table by Sontheimer (2005) || D. melanogaster, T. brucei
|-
| eIF2C1 (Ago1)
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.
See also
- RNA-induced transcriptional silencing (RITS)
- RNA interference