RNAi

RNA interference (also called "RNA-mediated interference", abbreviated RNAi) is a mechanism for RNA-guided regulation of gene expression in which double-stranded ribonucleic acid inhibits the expression of genes with complementary nucleotide sequences. Conserved in most eukaryotic organisms, the RNAi pathway is thought to have evolved as a form of innate immunity against viruses and also plays a major role in regulating development and genome maintenance.
The RNAi pathway is initiated by the enzyme dicer, which cleaves double-stranded RNA (dsRNA) to short double-stranded fragments of 20–25 base pairs. One of the two strands of each fragment, known as the guide strand, is then incorporated into the RNA-induced silencing complex (RISC) and base-pairs with complementary sequences. The most well-studied outcome of this recognition event is a form of post-transcriptional gene silencing. This occurs when the guide strand base pairs with a messenger RNA (mRNA) molecule and induces degradation of the mRNA by argonaute, the catalytic component of the RISC complex. The short RNA fragments are known as small interfering RNA (siRNA) when they derive from exogenous sources and microRNA (miRNA) when they are produced from RNA-coding genes in the cell's own genome. The RNAi pathway has been particularly well-studied in certain model organisms such as the nematode worm Caenorhabditis elegans, the fruit fly Drosophila melanogaster, and the flowering plant Arabidopsis thaliana.
The selective and robust effect of RNAi on gene expression makes it a valuable research tool, both in cell culture and in living organisms; synthetic dsRNA introduced into cells can induce suppression of specific genes of interest. RNAi may also be used for large-scale screens that systematically shut down each gene in the cell, which can help identify the components necessary for a particular cellular process or an event such as cell division. Exploitation of the pathway is also a promising tool in biotechnology and medicine.
Historically, RNA interference was known by other names, including post transcriptional gene silencing, transgene silencing, and quelling. Only after these apparently-unrelated processes were fully understood did it become clear that they all described the RNAi phenomenon. RNAi has also been confused with antisense suppression of gene expression, which does not act catalytically to degrade mRNA but instead involves single-stranded RNA fragments physically binding to mRNA and blocking translation. In 2006, Andrew Fire and Craig C. Mello shared the Nobel Prize in Physiology or Medicine for their work on RNA interference in the nematode worm C. elegans,which they published in 1998.

Cellular mechanism

RNAi is an RNA-dependent gene silencing process that is mediated by the RNA-induced silencing complex (RISC) and is initiated by short double-stranded RNA molecules in the cytoplasm, where they interact with the catalytic RISC component argonaute. When the dsRNA is exogenous, coming from infection by a virus with an RNA genome or laboratory manipulations, the RNA is imported directly into the cytoplasm and cleaved to short fragments by the enzyme dicer. The initiating dsRNA can also be endogenous, as in pre-microRNAs expressed from RNA-coding genes in the genome. The primary transcripts from such genes are first processed to the characteristic stem-loop structure of pre-miRNA in the nucleus, then exported to the cytoplasm to be cleaved by dicer. Thus the two pathways for exogenous and endogenous dsRNA converge at the RISC complex, which mediates gene silencing effects.

dsRNA cleavage

Exogenous dsRNA initiates RNAi by activating the ribonuclease protein dicer,which binds and cleaves double-stranded RNAs (dsRNA)s to produce double-stranded fragments of 20–25 base pairs with a few unpaired overhang bases on each end.Bioinformatics studies on the genomes of multiple organisms suggest this length maximizes target-gene specificity and minimizes non-specific effects.These short double-stranded fragments are called small interfering RNAs (siRNAs). These siRNAs are then separated into single strands and integrated into an active RISC complex. After integration into the RISC, siRNAs base-pair to their target mRNA and induce cleavage of the mRNA, thereby preventing it from being used as a translation template.
Exogenous dsRNA is detected and bound by an effector protein known as RDE-4 in C. elegans and R2D2 in Drosophila that stimulates dicer activity.This protein only binds long dsRNAs, but the mechanism producing this length specificity is unknown.These RNA-binding proteins then facilitate transfer of cleaved siRNAs to the RISC complex.
This initiation pathway may be amplified by the cell through the synthesis of a population of 'secondary' siRNAs using the dicer-produced initiating or 'primary' siRNAs as templates.These siRNAs are structurally distinct from dicer-produced siRNAs and appear to be produced by an RNA-dependent RNA polymerase (RdRP).

microRNA
MicroRNAs (miRNAs) are genomically encoded non-coding RNAs that help regulate gene expression, particularly during development. The phenomenon of RNA interference, broadly defined, includes the endogenously induced gene silencing effects of miRNAs as well as silencing triggered by foreign dsRNA. Mature miRNAs are structurally similar to siRNAs produced from exogenous dsRNA, but miRNAs must first undergo extensive post-transcriptional modification. An miRNA is expressed from a much longer RNA-coding gene as a primary transcript known as a pri-miRNA, which is processed in the cell nucleus to a 70-nucleotide stem-loop structure called a pre-miRNA by the microprocessor complex. This complex consists of an RNase III enzyme called Drosha and a dsRNA-binding protein Pasha. The dsRNA portion of this pre-miRNA is bound and cleaved by dicer to produce the mature miRNA molecule that can be integrated into the RISC complex; thus, miRNA and siRNA share the same cellular machinery downstream of their initial processing.
The siRNAs derived from long dsRNA precursors differ from miRNAs in that miRNAs, especially those in animals, typically have incomplete base pairing to a target and inhibit the translation of many different mRNAs with similar sequences. In contrast, siRNAs typically base-pair perfectly and induce mRNA cleavage only in a single, specific target.In Drosophila and C. elegans, miRNA and siRNA are processed by distinct argonaute proteins and dicer enzymes.

RISC activation and catalysis
The catalytically-active components of the RISC complex are endonucleases called argonaute proteins, which cleave the target mRNA strand complementary to their bound siRNA. As the fragments produced by dicer are double-stranded, they could each in theory produce a functional siRNA. However, only one of the two strands, which is known as the guide strand, binds the argonaute protein and directs gene silencing. The other anti-guide strand or passenger strand is degraded during RISC activation.Although it was first believed that an ATP-dependent helicase separated these two strands,the process is actually ATP-independent and performed directly by the protein components of RISC.The strand selected as the guide tends to be that with a more stable 5' end, but strand selection is unaffected by the direction in which dicer cleaves the dsRNA before RISC incorporation.Instead, the R2D2 protein may serve as the differentiating factor by binding the less-stable 5' end of the passenger strand.The structural basis for binding of RNA to the argonaute protein was examined by X-ray crystallography of the binding domain of an RNA-bound argonaute protein. Here, the phosphorylated 5' end of the RNA strand enters a conserved basic surface pocket and makes contacts through a divalent cation such as magnesium and by aromatic stacking between the 5' nucleotide in the siRNA and a conserved tyrosine residue. This site is thought to form a nucleation site for the binding of the siRNA to its mRNA target.
It is not understood how the activated RISC complex locates complementary mRNAs within the cell. Although the cleavage process has been proposed to be linked to translation, translation of the mRNA target is not essential for RNAi-mediated degradation.Indeed, RNAi may be more effective against mRNA targets that are not translated.Argonaute proteins, the catalytic components of RISC, are localized to specific regions in the cytoplasm called P-bodies (also cytoplasmic bodies or GW bodies), which are regions with high rates of mRNA decay;miRNA activity is also clustered in P-bodies.Disruption of P bodies in cells decreases the efficiency of RNA interference, suggesting that they are the site of a critical step in the RNAi process.

Variation among organisms
Organisms vary in their ability to take up foreign dsRNA and use it in the RNAi pathway. The effects of RNA interference can be both systemic and heritable in plants and C. elegans, although not in Drosophila or mammals. In plants, RNAi is thought to propagate by the transfer of siRNAs between cells through plasmodesmata.A broad general distinction between plants and animals lies in the targeting of endogenously produced miRNAs; in plants, miRNAs are usually perfectly or nearly perfectly complementary to their target genes and induce direct mRNA cleavage by RISC, while animals' miRNAs tend to be more divergent in sequence and induce translational repression.This translational effect may be produced by inhibiting the interactions of translation initiation factors with the messenger RNA's polyadenine tail.Some eukaryotic protozoa such as Leishmania major and Trypanosoma cruzi lack the RNAi pathway entirely.Most or all of the components are also missing in some fungi, most notably the model organism Saccharomyces cerevisiae.Certain ascomycetes and basidiomycetes are also missing RNA interference pathways; this observation indicates that proteins required for RNA silencing have been lost independently from many fungal lineages, possibly due to the evolution of a novel pathway with similar function, or to the lack of selective advantage in certain niches.