RNA Interference Overview

References

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Definitions

Argonaute
Argonaute proteins are found in all RISC and miRNA-containing ribonucleoprotein complexes and have been shown to be the catalytic site of mRNA cleavage in RISC [47]. Argonautes are ~100 kDa proteins characterized by conserved domains called PAZ and PIWI. Argonautes can be organized by sequence into two subfamilies, Ago and Piwi, based on a higher degree of homology to either Arabidopsis AGO1 or to Drosophila Piwi. The Ago group functions in RNAi and miRNA silencing pathways.

Cosuppression
Silencing of an endogenous gene caused by the introduction of a trans-gene or infection by a virus. This term, which can refer to silencing at the post-transcriptional (PTGS; post-transcriptional gene silencing) or transcriptional (TGS; transcriptional gene silencing) level, is used primarily by researchers working with plants. In Neurospora crassa, this phenomenon is known as quelling.

Dicer
A ~200 kDa multidomain, RNase III family enzyme that functions in processing dsRNA to siRNA and assembly of the guide strand into RISC. Dicer progressively cleaves dsRNA at 21–25 bp intervals to generate siRNAs with 2-nt 3' overhangs and phosphorylated 5' ends. The predicted structure of Dicer includes an ATPase/RNA helicase domain, a conserved PAZ domain that is shared with Argonaute, two catalytic RNase III domains, and a C-terminal dsRNA binding domain (dsRBD).

dsRNA
Double-stranded RNA

Guide Strand
The strand of the siRNA that is assembled with RISC and provides sequence specificity for target mRNA cleavage. The guide strand is in the anti-sense orientation with respect to the mRNA.

miRNA
MicroRNA, a large class of evolutionarily conserved, noncoding, RNA originating from longer transcripts characterized by imperfect hairpin structures. miRNAs are 19–23 nt RNAs processed from pre-miRNA precursors by Dicer, the same enzyme that processes siRNAs.

Passenger Strand
The strand of the siRNA that dissociates from the siRNA during assembly with RISC.

PAZ Domain
Conserved domain found in both Dicer and Argonaute. It is thought that the 3' end of the guide strand of an siRNA is in contact with the PAZ in RISC.

PIWI Domain
Conserved domain found in Argonaute and thought to be the catalytic site for mRNA cleavage. It is thought that the 5' end of the guide strand of the siRNA contacts the PIWI domain in RISC.

Post-transcriptional Gene Silencing (PTGS)
Silencing of an endogenous gene caused by the introduction of a homologous dsRNA, trans-gene or virus. In PTGS, the transcript of the silenced gene is synthesized but does not accumulate because it is rapidly degraded. This is a more general term than RNAi, since it can be triggered by several different means.

R2D2
A small, dsRNA binding protein that works with Dicer in assembly of siRNA with RISC.

rasiRNA
Repeat-associated siRNA, an endogenous form of siRNA that originates from repetitive elements within the genome.

RdRPs
RNA-dependent RNA Polymerases. RdRPs may play a role in amplifying RNA triggers for silencing. Although they are not found in insects and mammals, they are present in other eukaryotes that have RNA silencing pathways.

RISC
RNA-induced silencing complex. A nuclease complex, composed of proteins and siRNA, that targets and cleaves endogenous mRNAs complementary to the siRNA within the complex.

RITS
RNA-induced transcriptional silencing complex. A complex of proteins and rasiRNA that inhibits transcription of target DNA through heterochromatin formation. RITS contains at least one protein in common with RISC, Argonaute [49].

RNAi, RNA Interference
A process of mRNA cleavage and degradation that is induced by double-stranded RNA in a sequence-specific manner.

RNA Silencing
The collective term for the pathways that use small RNAs as guides to specifically modify expression of targeted genes or genomic regions.

RNase III Family
A group of double-stranded RNA-specific endonucleases characterized by production of dsRNA fragments with 2–3-nt 3' overhangs and 5' phosphorylated ends, similar to those produced by Dicer.

Small RNA
A collective term for siRNA, rasiRNA, and miRNA

siRNA
Short interfering RNA, siRNAs are 21–25 bp dsRNA with dinucleotide 3' overhangs that are processed from longer dsRNA by Dicer in the RNA interference pathway. Introduction of synthetic siRNAs can induce RNA interference in mammalian cells. siRNAs can also originate from endogenous dsRNA precursors.

shRNA
Short hairpin RNA. shRNAs are used in plasmid- or vector-based approaches for supplying siRNAs to cells to produce stable gene silencing. A strong promoter is used to drive transcription of a target sequence designed to form hairpins and loops of variable length, which are then processed to siRNAs by the cellular RNAi machinery.

siRISC
A term for RISC assembled with the guide strand of an siRNA.