Slicing & Dicing: How RNA Interference Selectively Inhibits Gene Expression
RNA interference (RNAi) is a eukaryotic post-transcriptional mechanism used to decide whether or not to degrade or otherwise inhibit translation of a given mRNA. This decision is based on the level of sequence similarity between a double stranded RNA (dsRNA) and the mRNA sequence to be translated. There are two pathways of RNAi that differ in the level of sequence similarity, and therefore, response.
The first pathway results in mRNA degradation. It evolved as a viral defense; single stranded RNA (ssRNA) viruses are temporarily at a dsRNA stage when replicating their genetic material in the cell. (The complementary nature of RNA and other nucleic acids makes this a logical copying mechanism for these viruses.) However, this dsRNA is not normal in the eukaryotic cytoplasm; the only use we have for RNA is in a single stranded (or stem loop, in the case of tRNA) form. The presence of dsRNA in the cell sets off alarm bells and an angry enzyme, aptly named Dicer, dices the dsRNA up into small interfering RNAs (siRNAs). These pieces are transported to the RNA-induced silencing complex (RISC), which, upon activation, cleaves (using the argonaute endonuclease) any mRNA that matches the exact sequence of the presented siRNAs, for fear of these sequences being integrated by viruses into the host chromosome. By this mechanism, the mRNA matching the host sequence is degraded, thus preventing viral expression of the sequences it may have integrated into the host genome.
The second pathway results in translational inhibition; however, it is not in response to a viral threat. Rather, the dsRNA sequences originate within the nucleus, and are produced by our own cellular machinery (or, in some cases, by a scientist). These sequences are called microRNAs (miRNAs), and they begin in the nucleus as single stranded sequences. An enzyme called Drosha processes these miRNAs within the nucleus, and they emerge into the cytoplasm as double stranded entities (they take advantage of their palindromic areas to form stem loops). In the cytoplasm, Dicer cleaves the ds miRNA into fragments which bind the RISC complex. Rather than degrade the mRNA, however, the RISC complex simply inhibits translation of the target mRNA by other means.
This second pathway can be used to selectively prevent gene expression by introducing ds miRNA sequences matching those of the genes to silence. These knockout experiments provide valuable information about gene functions, and help create negative control groups for certain experiments.
Here are some resources I found helpful:
https://youtu.be/WJ_0afWCmhk - this video is short and to the point.
https://youtu.be/xDg6pu7HWz4 - this video is more thorough and covers more background/applications of the process.
