RNA-induced silencing complex

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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. [1] 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. [2] 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). [3] [4] [1]

Contents

Discovery

The biochemical identification of RISC was conducted by Gregory Hannon and his colleagues at the Cold Spring Harbor Laboratory. [5] 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. [3]

Drosophila melanogaster Drosophila melanogaster - side (aka).jpg
Drosophila melanogaster

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. [5] 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].

Function in RNA interference

The PIWI domain of an Argonaute protein in complex with double-stranded RNA. 1ytu argonaute dsrna.png
The PIWI domain of an Argonaute protein in complex with double-stranded RNA.

Incorporation of siRNA/miRNA

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. [6] [7] 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. [8] [9] [10] [11] The newly generated miRNA or siRNA act as single-stranded guide sequences for RISC to target mRNA for degradation. [12] [13]

Part of the RNA interference pathway with the different ways RISC can silence genes via their messenger RNA. Part of the RNA interference pathway focusing on RISC.png
Part of the RNA interference pathway with the different ways RISC can silence genes via their messenger RNA.

Gene regulation

AGO2 (grey) in complex with a microRNA (light blue) and its target mRNA (dark blue) MicroRNAs and Argonaute RNA binding.svg
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. [16]

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. [17] [1]

mRNA degradation

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. [18] 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. [1]

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.

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. [1]

Translation can be regulated at the initiation step by:

Translation can be regulated at post-initiation steps by:

  • peptide degradation,
  • promoting premature termination of translation ribosomes, [21] or,
  • slowing elongation. [22]

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. [1] [23] [24]

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. [25]

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. [26]

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. [27]

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. [27]

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. [27]

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. [28]

Table 1: Complexes implicated in RISC assembly and functionBased on table by Sontheimer (2005) [28]
ComplexSourceKnown/apparent componentsEstimated sizeApparent function in RNAi pathway
Dcr2-R2D2 [29] D. melanogaster S2 cells Dcr2, R2D2 ~250 kDadsRNA processing, siRNA binding
RLC (A) [30] [31] D. melanogaster embryosDcr2, R2D2NRdsRNA processing, siRNA binding, precursor to RISC
Holo-RISC [30] [31] D. melanogaster embryos Ago 2, Dcr1, Dcr2, Fmr1/Fxr, R2D2, Tsn, Vig ~80STarget-RNA binding and cleavage
RISC [5] [32] [33] [34] D. melanogaster S2 cellsAgo2, Fmr1/Fxr, Tsn, Vig~500 kDaTarget-RNA binding and cleavage
RISC [35] D. melanogaster S2 cellsAgo2~140 kDaTarget-RNA binding and cleavage
Fmr1-associated complex [36] D. melanogaster S2 cells L5, L11, 5S rRNA, Fmr1/Fxr, Ago2, Dmp68 NRPossible target-RNA binding and cleavage
Minimal RISC [37] [38] [39] [40] HeLa cells eIF2C1 (Ago1) or eIF2C2 (Ago2)~160 kDaTarget-RNA binding and cleavage
miRNP [41] [42] HeLa cellseIF2C2 (ago2), Gemin3, Gemin4 ~550 kDamiRNA 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.

A full-length argonaute protein from the archaea species Pyrococcus furiosus. 1u04-argonaute.png
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. [43] There are eight family members in human Argonautes of which only Argonaute 2 is exclusively involved in targeted RNA cleavage in RISC. [40]

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.png
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 associates with TRBP and Argonaute 2 to facilitate the transfer of the dsRNA fragments generated by Dicer to Argonaute 2. [44] [45]

More recent research has shown the human RNA helicase A could help facilitate the RLC. [46]

Other proteins

Recently identified members of RISC are SND1 and MTDH. [47] SND1 and MTDH are oncogenes and regulate various gene expression. [48]

Table 2: Biochemically documented proteins associated with RISCBased on the table by Sontheimer (2005) [28]
ProteinSpecies the protein is found
Dcr1 [30] D. melanogaster
Dcr2 [29] [30] [31] D. melanogaster
R2D2 [30] [31] D. melanogaster
Ago2 [30] [32] [35] [36] D. melanogaster
Dmp68 [36] D. melanogaster
Fmr1/Fxr [30] [33] [36] D. melanogaster
Tsn [30] [34] D. melanogaster
Vig [30] [33] D. melanogaster
Polyribosomes, ribosome components [5] [30] [32] [36] [49] D. melanogaster, T. brucei
eIF2C1 (Ago1) [37] H. sapiens
eIF2C2 (Ago2) [37] [38] [40] [42] H. sapiens
Gemin3 [41] [42] H. sapiens
Gemin4 [41] [42] H. sapiens

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

Diagram of RISC activity with miRNAs MiRNA.svg
Diagram of RISC activity with miRNAs

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. [50]

Endogenously expressed miRNA in metazoans is usually not perfectly complementary to a large number of genes and thus, they modulate expression via translational repression. [51] [52] 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. [53]

See also

Related Research Articles

microRNA Small non-coding ribonucleic acid molecule

MicroRNA (miRNA) are small, single-stranded, non-coding RNA molecules containing 21 to 23 nucleotides. Found in plants, animals and some viruses, miRNAs are involved in RNA silencing and post-transcriptional regulation of gene expression. miRNAs base-pair to complementary sequences in mRNA molecules, then silence said mRNA molecules by one or more of the following processes:

  1. Cleavage of the mRNA strand into two pieces,
  2. Destabilization of the mRNA by shortening its poly(A) tail, or
  3. Reducing translation of the mRNA into proteins.

Gene silencing is the regulation of gene expression in a cell to prevent the expression of a certain gene. Gene silencing can occur during either transcription or translation and is often used in research. In particular, methods used to silence genes are being increasingly used to produce therapeutics to combat cancer and other diseases, such as infectious diseases and neurodegenerative disorders.

Gene knockdown is an experimental technique by which the expression of one or more of an organism's genes is reduced. The reduction can occur either through genetic modification or by treatment with a reagent such as a short DNA or RNA oligonucleotide that has a sequence complementary to either gene or an mRNA transcript.

<span class="mw-page-title-main">Small interfering RNA</span> Biomolecule

Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, is a class of double-stranded RNA at first non-coding RNA molecules, typically 20–24 base pairs in length, similar to miRNA, and operating within the RNA interference (RNAi) pathway. It interferes with the expression of specific genes with complementary nucleotide sequences by degrading mRNA after transcription, preventing translation. It was discovered in 1998, by Andrew Fire at Carnegie Institution for Science in Washington DC and Craig Mello at University of Massachusetts in Worcester.

<span class="mw-page-title-main">Dicer</span> Enzyme that cleaves double-stranded RNA (dsRNA) into short dsRNA fragments

Dicer, also known as endoribonuclease Dicer or helicase with RNase motif, is an enzyme that in humans is encoded by the DICER1 gene. 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 20–25 base pairs long with a two-base overhang on the 3′-end. Dicer facilitates the activation of the RNA-induced silencing complex (RISC), which is essential for RNA interference. RISC has a catalytic component Argonaute, which is an endonuclease capable of degrading messenger RNA (mRNA).

<span class="mw-page-title-main">Short hairpin RNA</span> Type of RNA

A short hairpin RNA or small hairpin RNA is an artificial RNA molecule with a tight hairpin turn that can be used to silence target gene expression via RNA interference (RNAi). Expression of shRNA in cells is typically accomplished by delivery of plasmids or through viral or bacterial vectors. shRNA is an advantageous mediator of RNAi in that it has a relatively low rate of degradation and turnover. However, it requires use of an expression vector, which has the potential to cause side effects in medicinal applications.

<span class="mw-page-title-main">Ribonuclease III</span> Class of enzymes

Ribonuclease III (RNase III or RNase C)(BRENDA 3.1.26.3) is a type of ribonuclease that recognizes dsRNA and cleaves it at specific targeted locations to transform them into mature RNAs. These enzymes are a group of endoribonucleases that are characterized by their ribonuclease domain, which is labelled the RNase III domain. They are ubiquitous compounds in the cell and play a major role in pathways such as RNA precursor synthesis, RNA Silencing, and the pnp autoregulatory mechanism.

<span class="mw-page-title-main">Argonaute</span> Protein that plays a role in RNA silencing process

The Argonaute protein family, first discovered for its evolutionarily conserved stem cell function, plays a central role in RNA silencing processes as essential components of the RNA-induced silencing complex (RISC). RISC is responsible for the gene silencing phenomenon known as RNA interference (RNAi). Argonaute proteins bind different classes of small non-coding RNAs, including microRNAs (miRNAs), small interfering RNAs (siRNAs) and Piwi-interacting RNAs (piRNAs). Small RNAs guide Argonaute proteins to their specific targets through sequence complementarity, which then leads to mRNA cleavage, translation inhibition, and/or the initiation of mRNA decay.

Piwi-interacting RNA (piRNA) is the largest class of small non-coding RNA molecules expressed in animal cells. piRNAs form RNA-protein complexes through interactions with piwi-subfamily Argonaute proteins. These piRNA complexes are mostly involved in the epigenetic and post-transcriptional silencing of transposable elements and other spurious or repeat-derived transcripts, but can also be involved in the regulation of other genetic elements in germ line cells.

<span class="mw-page-title-main">Piwi</span> Genes and regulatory proteins

Piwi genes were identified as regulatory proteins responsible for stem cell and germ cell differentiation. Piwi is an abbreviation of P-elementInduced WImpy testis in Drosophila. Piwi proteins are highly conserved RNA-binding proteins and are present in both plants and animals. Piwi proteins belong to the Argonaute/Piwi family and have been classified as nuclear proteins. Studies on Drosophila have also indicated that Piwi proteins have no slicer activity conferred by the presence of the Piwi domain. In addition, Piwi associates with heterochromatin protein 1, an epigenetic modifier, and piRNA-complementary sequences. These are indications of the role Piwi plays in epigenetic regulation. Piwi proteins are also thought to control the biogenesis of piRNA as many Piwi-like proteins contain slicer activity which would allow Piwi proteins to process precursor piRNA into mature piRNA.

RNA-induced transcriptional silencing (RITS) is a form of RNA interference by which short RNA molecules – such as small interfering RNA (siRNA) – trigger the downregulation of transcription of a particular gene or genomic region. This is usually accomplished by posttranslational modification of histone tails which target the genomic region for heterochromatin formation. The protein complex that binds to siRNAs and interacts with the methylated lysine 9 residue of histones H3 (H3K9me2) is the RITS complex.

RNA silencing or RNA interference refers to a family of gene silencing effects by which gene expression is negatively regulated by non-coding RNAs such as microRNAs. RNA silencing may also be defined as sequence-specific regulation of gene expression triggered by double-stranded RNA (dsRNA). RNA silencing mechanisms are conserved among most eukaryotes. The most common and well-studied example is RNA interference (RNAi), in which endogenously expressed microRNA (miRNA) or exogenously derived small interfering RNA (siRNA) induces the degradation of complementary messenger RNA. Other classes of small RNA have been identified, including piwi-interacting RNA (piRNA) and its subspecies repeat associated small interfering RNA (rasiRNA).

<span class="mw-page-title-main">EIF2C2</span> Protein-coding gene in the species Homo sapiens

Protein argonaute-2 is a protein that in humans is encoded by the EIF2C2 gene.

<span class="mw-page-title-main">EIF2C1</span> Protein-coding gene in the species Homo sapiens

Protein argonaute-1 is a protein that in humans is encoded by the EIF2C1 gene.

Trans-acting siRNA are a class of small interfering RNA (siRNA) that repress gene expression through post-transcriptional gene silencing in land plants. Precursor transcripts from TAS loci are polyadenylated and converted to double-stranded RNA, and are then processed into 21-nucleotide-long RNA duplexes with overhangs. These segments are incorporated into an RNA-induced silencing complex (RISC) and direct the sequence-specific cleavage of target mRNA. Ta-siRNAs are classified as siRNA because they arise from double-stranded RNA (dsRNA).

<span class="mw-page-title-main">Stable nucleic acid lipid particle</span>

Stable nucleic acid lipid particles (SNALPs) are microscopic particles approximately 120 nanometers in diameter, smaller than the wavelengths of visible light. They have been used to deliver siRNAs therapeutically to mammals in vivo. In SNALPs, the siRNA is surrounded by a lipid bilayer containing a mixture of cationic and fusogenic lipids, coated with diffusible polyethylene glycol.

<span class="mw-page-title-main">RevCen</span>

RevCen is a family of non-coding RNA found in Schizosaccharomyces. It is a megastructure containing several siRNA which use the RNAi pathway to regulate heterochromatin formation. The long RNA transcript forms a secondary structure with several stem-loops which are processed by dicer into siRNA. This siRNA then initiate the formation of heterochromatin at the centromeres of fission yeast. Northern blot analysis confirmed the siRNAs were produced from the large RNA structure RevCen in vivo. As with all siRNAs, the enzyme dicer is responsible for dissecting dsRNA into the 21nt stretch of double-stranded RNA. Human recombinant dicer enzyme processed the RevCen structure in vitro, though the same activity by yeast Dcr1 has not been confirmed.

<span class="mw-page-title-main">RNA interference</span> Biological process of gene regulation

RNA interference (RNAi) is a biological process in which RNA molecules are involved in sequence-specific suppression of gene expression by double-stranded RNA, through translational or transcriptional repression. Historically, RNAi was known by other names, including co-suppression, post-transcriptional gene silencing (PTGS), and quelling. The detailed study of each of these seemingly different processes elucidated that the identity of these phenomena were all actually RNAi. Andrew Fire and Craig C. Mello shared the 2006 Nobel Prize in Physiology or Medicine for their work on RNAi in the nematode worm Caenorhabditis elegans, which they published in 1998. Since the discovery of RNAi and its regulatory potentials, it has become evident that RNAi has immense potential in suppression of desired genes. RNAi is now known as precise, efficient, stable and better than antisense therapy for gene suppression. Antisense RNA produced intracellularly by an expression vector may be developed and find utility as novel therapeutic agents.

RDE-1 (RNAi-DEfective 1) is a primary Argonaute protein required for RNA-mediated interference (RNAi) in Caenorhabditis elegans. The rde-1 gene locus was first characterized in C. elegans mutants resistant to RNAi, and is a member of a highly conserved Piwi gene family that includes plant, Drosophila, and vertebrate homologs.

<span class="mw-page-title-main">DCL2</span> Dicer-like gene in plants

DCL2 is a gene in plants that codes for the DCL2 protein, a ribonuclease III enzyme involved in processing exogenous double-stranded RNA (dsRNA) into 22 nucleotide small interference RNAs (siRNAs).

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