Schematic Overview of the RNAi Mechanism
The presence of double-stranded RNA (dsRNA) triggers the posttranscriptional silencing of the gene which is targeted by the dsRNA. Different RNA molecules can induce gene silencing:
- siRNAs, which are delivered into the cell or
- shRNAs, which are transcribed from a plasmid.
Although the origin and the structure of the RNA molecules differ, they share the subsequent mode of action. The common entry point for gene silencing is the formation of the RNA-induced silencing complex (RISC) from 5'-phosphorylated siRNAs and endogenous Argonaute protein. shRNAs require cleavage by the endogenous cytoplasmic nuclease Dicer to yield siRNAs which are 21 to 27 nucleotides long.
The Argonaute protein and one strand of the siRNA form the RNA-induced silencing complex (RISC). The incorporated RNA strand determines the sequence-specificity of the gene silencing. RISC binds to the mRNA which is targeted by the single RNA strand within the complex and cleaves the mRNA. The cleaved mRNA cannot be translated. RISC dissociates and can cleave other mRNAs. By that even a few numbers of the RNA-induced silencing complex can lead to high-level gene silencing.