РНҚ-индукцияланған тыныштыру кешені және гендік үнсіздендіру механизмдері

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. 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.

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.

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. +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
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 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.

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