Recently scientists working in different research fields observed a phenomenon they could not immediately understand. Plant biologists were attempting to boost the activity of the gene for chalcone synthase, an enzyme involved in the production of anthocyanin pigments, by introducing a powerful promotor sequence into their petunias. However, instead of a deep purple colour, many of the flowers grew variegated, or virgin white. The researchers concluded that the introduced chalcone synthase gene had somehow muted both itself and a normal petunia gene. Joergensen et al termed this phenomenon of gene silencing "cosuppression" (1).
Their discoveries were supported by another group studying plant RNA viruses. Baulcombe et al (2) were expressing genes from the potato virus X in tobacco plants. The researchers hoped that viral proteins produced by the plants would stimulate its defence allowing the plants to resist subsequent attack by the virus itself. To their surprise the plants with the strongest resistance were those in which the introduced gene was silent. The researchers concluded that the introduced gene was co-suppressing both itself and the same gene in the virus.
In fungi, gene silencing was observed during attempts to boost the production of an orange pigment by the mould Neurospora crassa. Macino and Cogoni introduced extra copies of a gene involved in making a carotenoid pigment. In their experiments a third of the engineered mould bleached out, rather than turning to a deeper orange. Something had suppressed the pigment gene. They termed the observed phenomenon of gene silencing "quelling" (3,4).
Other scientists working with Caenorhabditis elegans obtained strange results in their antisense experiments. The theory behind the antisense approach is to inject complementary RNA sequences into the target organism to block the targeted mRNA. The two sequences should then hybridize stopping the production of the encoded protein. To Guo's surprise even the injected sense strand was active (5). This was later explained as the sense strand used was contaminated with very small amounts of the corresponding antisense strand . In a classic antisense approach these small contaminations would have shown no effect at all.
In 1998 Fire et al., suggested a new mechanism for the phenomenon of gene silencing. In their experiments using Caenorhabditis elegans they showed that double stranded RNA (dsRNA) was even more effective in gene silencing than both sense or antisense strands alone (7). They found that only a few molecules of injected dsRNA were required per affected cell. Fire et al. described this mechanism as extremely gene specific and suggested that the dsRNA mediated silencing was part of a complex biological regulation system. Fire et al. named the phenomenon of gene silencing RNA interference (RNAi).
RNAi Mechanism and Short Interfering RNA (siRNA)
Consistent with gene silencing by dsRNA, Hamilton et al., described the existence of small (about 25nt) RNAs that correspond to the gene that has been silenced in plants .
While looking for a common principle Hammond et al., detected similar short RNAs in Drosophila. They suggested that these are incorporated into a RNA induced silencing complex (RISC) and then are used as a guide in the RNAi mechanism, which then leads to degradation of the corresponding mRNA (9).
Today the basic mechanism of RNA interference (as it has been shown for Drosophila) can be understood as a two step process (10).
First, the dsRNA is cleaved to yield short interfering RNAs (siRNAs) of about 21-23nt length (8, 9, 11-13) with 5' terminal phosphate and 3' short overhangs (~2nt) (12). Then the siRNAs target the corresponding mRNA sequence specific for destruction (Fig. 1) (7-9,11,13,14).
Fig. 1: After dsRNA (of >30nt) is transfered into the cellular system, Dicer (Drosophila; 13) or another RNase III-like enzyme breaks the dsRNA into shorter RNA sequences (about 21-23 nt). These short sequences are called short interfering RNA (siRNA). siRNAs direct target specific mRNA degradation in the RNA induced silencing complex (RISC) (9).
Hammond et al., concluded that the identical size of RNA fragments in plants and animals must be the result of a highly conserved mechanism in nature (9). This theory has been supported by many studies showing that dsRNA induced gene silencing can be found in a number of different species (7, 15-24).
Non-Specific Response of Mammalian Cells
Even though it has been shown that dsRNA can mediate gene-specific interference in early mouse embryos and in mouse oocytes (25, 26), the introduction of dsRNA into somatic mammalian cells is limited. Instead of triggering RNAi, the introduced dsRNA generates a general, non-specific decrease of mRNA often followed by cell death. One response to dsRNA in mammalian cells is mediated by the dsRNA-dependent protein kinase (PKR) which phophorylates and inactivates the translation factor eIF2a, leading to a generalized suppression of protein synthesis, and in some cases apoptosis (27).
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