14.11: RNA Interference

RNA interference (RNAi) is a process in which a small non-coding RNA molecule blocks the post-transcriptional expression of a gene by binding to its messenger RNA (mRNA) and preventing the protein from being translated.

This process occurs naturally in cells, often through the activity of genomically-encoded microRNAs. Researchers can take advantage of this mechanism by introducing synthetic RNAs to deactivate specific genes for research or therapeutic purposes. For example, RNAi could be used to suppress genes that are overactive in diseases such as cancer.

The Process

First, researchers synthesize double-stranded RNA with a sequence complementary to the targeted gene. Different types of double-stranded RNA can be used, including small interfering RNA (siRNA) and short hairpin RNA (shRNA). shRNA is one strand of RNA that is folded over—creating a double-stranded RNA with a hairpin loop on one side—and is a precursor of siRNA. The double-stranded RNA is then introduced into cells by methods such as injection or delivery by vectors, such as modified viruses. If shRNA is used, RNase enzymes in the cell, such as Dicer, cleave it down to the shorter siRNA, removing the hairpin loop.

The siRNA then binds to an enzyme Argonaute, which is part of a complex called the RNA-induced silencing complex (RISC). Here, the two strands of the siRNA separate. One floats away while the other—called the guide strand—remains attached to the RISC. It is known as the "guide strand" because it is the strand that binds the mRNA through complementary base pairing, bringing the RISC to the mRNA. This binding is very specific because the siRNA is usually designed to be completely complementary to the targeted mRNA. Argonaute then cleaves and degrades the mRNA, preventing it from being translated into protein— effectively silencing the gene.

In RNA interference, or RNAi, small non-coding RNAs bind to complementary messenger RNAs, mRNAs, to prevent their translation to proteins.

Three classes of small RNAs can accomplish RNAi— microRNAs or miRNAs are encoded by a cell's genome; small interfering or siRNAs are derived from exogenous viral double-stranded RNAs; and PIWI-interacting or piRNAs, are specific to the germline.

In general, the RNAi pathway begins when the enzyme Dicer cleaves double-stranded RNA to an approximately 20-25 nucleotide-long siRNA. siRNA then binds to a group of proteins called the RNA-induced silencing complex, RISC.

In RISC, the RNA guide strand remains in the complex while its complementary strand is removed.

The guide strand then binds to its complementary sequence in mRNA. Argonaute, an enzyme that is part of RISC, then cleaves the mRNA, thereby silencing the targeted gene.