RNA interference, or RNAi, is a biological process that regulates gene expression through gene silencing. This system functions as a precise control within cells, determining which genes are active by intercepting their instructions to prevent a specific protein from being made. This mechanism does not destroy the genes themselves. By doing so, RNAi fine-tunes the vast array of activities occurring within a cell.
Initiation by Double-Stranded RNA
The RNAi process is set in motion by the appearance of double-stranded RNA (dsRNA) within the cell’s cytoplasm. This molecule acts as a trigger, signaling that a specific gene needs to be silenced. The presence of dsRNA is unusual in mammalian cells, so it serves as a distinct flag for the cellular machinery to take action.
This trigger molecule can originate from two sources. An external, or exogenous, source includes a virus with an RNA-based genome or synthetic dsRNA introduced by scientists. The other source is internal, or endogenous, where the cell produces its own regulatory molecules known as microRNAs (miRNAs). These are transcribed as a longer single strand that folds back on itself to create a hairpin shape that mimics a dsRNA structure.
Processing by the Dicer Enzyme
Once long dsRNA is present in the cytoplasm, it is recognized by a specialized enzyme called Dicer. This protein functions like molecular scissors, precisely measuring and cutting the dsRNA into much shorter, uniform segments. These resulting fragments are typically between 21 and 25 nucleotides in length.
The short dsRNA pieces produced by Dicer are the active molecules that will carry out the silencing. If the original trigger was external, these small fragments are called small interfering RNAs (siRNAs). If the trigger was an internal hairpin RNA, the fragments are called microRNAs (miRNAs). Dicer’s role is to convert the initial dsRNA signal into these specific instruction molecules, which are now prepared to be loaded into the next part of the machinery.
Formation of the RNA-Induced Silencing Complex
After Dicer generates the small siRNA or miRNA duplexes, the next step involves assembling the RNA-induced silencing complex, or RISC. The small dsRNA fragment is loaded into this complex. Within the RISC, the two strands of the small RNA duplex are separated.
One strand, known as the “guide strand,” is selected and remains bound to the complex based on the thermodynamic properties at its ends. The other strand, called the “passenger strand,” is ejected and subsequently degraded by the cell. The central component of RISC is a protein from the Argonaute family, which holds the guide strand. The final product is an activated RISC, which now carries a single-stranded RNA guide ready to patrol the cell.
Target Gene Silencing
The activated RISC, armed with its guide strand, scans the cell’s messenger RNA (mRNA) molecules. These mRNA transcripts are temporary copies of genes that carry instructions for building proteins. The RISC complex searches for a sequence that is complementary to its guide RNA.
When the RISC complex finds a matching mRNA, the method of silencing depends on the degree of complementarity. For siRNAs, the match is perfect. In this case, the Argonaute protein within RISC precisely cuts the target mRNA in two. This cleavage destabilizes the mRNA, leading to its rapid degradation and preventing the protein from being made.
In contrast, when the guide strand is an miRNA, the match with the target mRNA is often imperfect. This incomplete pairing leads to a different outcome. Instead of slicing the mRNA, the RISC complex remains bound to it, physically obstructing the ribosome from moving along the mRNA. This process, known as translational repression, blocks the protein-building machinery without immediately destroying the mRNA transcript.