What is RNA interference (RNAi)?

RNA interference (RNAi) is a mechanism of gene silencing occurring in a broad range of eukaryotic organisms. This process involves the digestion of cytoplasmic double-stranded RNA (dsRNA) into smaller fragments of a defined length.

Dicer, a cytoplasmic RNase present in eukaryotic cells, catalyzes this ATP-dependent reaction, generating 21-25 nt dsRNA molecules with phosphorylated 5’ ends, and 3’ dinucleotide overhangs carrying an intact hydroxyl group. Dicer chops dsRNA into two types of smaller RNA – microRNA (miRNA) and small interfering RNA (siRNA). The guide strand of these small RNA molecules couple to a protein complex called RISC (RNA-induced silencing complex), which then catalyzes the mRNA-specific gene silencing event (RNA interference or gene knock down).

This occurs via one of two mechanisms. RISC-siRNA complexes induce sequence-specific mRNA degradation. In contrast, RISC-miRNA complexes lead to the sequence-specific inhibition of mRNA translation. Endogenously expressed siRNA have not been found in mammals. However, they are a powerful functional genomics tool when delivered to mammalian cells experimentally via transfection or viral transduction (see the next FAQ). Upon being processed by Dicer and RISC, the antisense strands of these molecules guide RISC to mRNA of a complementary sequence, and target the mRNA for endonucleolytic destruction.

MicroRNA are naturally expressed as polycistronic transcripts that are converted into primary microRNA or pri-miRNA (approximately 70 nt in length) in the nucleus of mammalian cells. These species are processed into pre-miRNA, which are then exported from the nucleus to the cytoplasm where they are cleaved by Dicer to generate mature miRNA. The miRNA-RISC complex forms and binds to complementary sites in the targeted mRNA near the 3’ untranslated region (UTR), resulting in the repression of translation.