mRNA Silencing by RNA Interference

mRNA silencing by RNA interference (RNAi) is a regulatory cellular mechanism which was recently elucidated. In 1990, two research groups observed that the insertion of several extra copies of a pigment gene into petunias did not result in more intensely colored plants but rather to a partial or total loss of pigment production. It was determined later that the reduced pigment synthesis was due to a posttranscriptional down-regulation of the mRNA transcribed from the pigment gene. This type of gene silencing was also observed in worms, fungi, fruit flies, and mammals. It is now clear that RNAi is a fundamental regulatory mechanism which evolved more than a billion years ago.

Following the initial observations of this genetic regulation, a period of intense research started. Due to this research, the basic principles of RNA interference are now well understood. These are outlined in the figure below. More comprehensive information can be found in numerous review articles, such as Hannon GJ, Nature 418:244-251 (2002), Agrawal N et al., Microbiol Mol Biol Rev 67:657-685 (2003), Milhavet O et al., Pharmacol Rev 55:629-648 (2003).

The action of RNA interference is based around double-stranded RNA (dsRNA). Since dsRNA is usually not part of a differentiated somatic cell, its presence indicates that something has gone wrong and a cellular defense mechanism is triggered. First, the dsRNA is degraded by a highly conserved cellular RNAse named Dicer into small oligoribonucleotides with a length of about 22 base pairs, including 2-nucleotide long 3´ overhangs. This type of small dsRNA is called small interfering RNA or siRNA. In a second step, siRNA binds to a multimolecular protein complex consisting of several proteins named RISC (RNA Induced Silencing Complex). The double-stranded siRNA molecule is unwound resulting in a ribonucleoprotein particle consisting of the RISC proteins and one siRNA strand. If an mRNA with a sequence complementary to the siRNA moiety is encountered by this complex, the mRNA is cleaved by an RNAse named Slicer and thereby rendered inactive. If the complementarity is not perfect, RISC may only bind to the mRNA which also blocks translation inhibiting expression.

The mechanisms that can give rise to the generation of dsRNA are quite diverse. Two non-physiological causes are: artificially high-copy numbers of a gene, for instance in transfected cells, or infection of cells with an RNA virus. However, RNA interference most likely is also part of normal gene regulation. In the last few years, micro-RNA (miRNA), which in some respects behaves like siRNA, has been detected in the cells of many organisms. miRNA is encoded by the cellular genome and seems to be important for gene regulation during development.

RNAi can be Used for Selective Gene Silencing

The fact that dsRNA can trigger cellular defense mechanisms is not new. For instance, it has been known for a long time that dsRNA is a strong inducer of the interferon response cascade. However, interferon response is induced by long dsRNA molecules and is completely non-specific, leading to the destruction of all mRNAs. On the contrary, RNA interference is induced by small dsRNAs which do not induce an interferon response and is highly specific since only mRNAs with sequences complementary to the interfering RNA are degraded or blocked. As is also shown in the figure, RNA interference is not only induced by endogenous dsRNA but also by exogenous siRNA, for instance after transfection of synthetic siRNA molecules or shRNA-encoding plasmids. This makes it possible to analyze the function of a gene by the selective elimination of its transcript (gene knockdown), a method that has opened new horizons in biomedical research. Since RNAi technology makes use of natural cellular mechanisms, it is much more efficient than the artificial antisense RNA approach that has failed in many experimental settings.

Although a rather new technique, gene knockdown by RNAi has already become an essential method in molecular biology where it is predominantly used for the analysis of gene function as well as for target identification and target validation. It is also realistic that it will be useful for the treatment of many diseases, such as dominant hereditary disorders, virus infections, and cancer.