RNA silencing is a sequence specific RNA degradation process that is triggered by the formation of double stranded RNA that can be introduced by virus or transgenes. Duplexes 21- nucleotide (nt) RNAs with symmetric 2-nt 3'overhangs are introduced into the cell mediating the degradation of mRNA. According to central dogma of molecular biology, proteins are made in two steps. The first step, transcription, copies genes from double stranded deoxyribonucleic acid (ds DNA) molecules to mobile, single- stranded ribonucleic acid (RNA) molecules called mRNA. In the second step, translation, the mRNA is converted to its functional protein form. Since there are two steps to making a protein, there are two ways of preventing one from being made. Scientists have made exciting progress in blocking the protein synthesis through the second step, translation. One way they have accomplished this is by inserting synthetic molecules that triggers a cellular process called RNA interference.
Molecular biologists had the dream to knockout gene expression at the mRNA level for the last 15 years. Efforts were made to generate loss of cell functions or organisms various molecules that included from eg antisense sequences, ribozymes and chimeric oligonucleotides. Alternative methods for silencing specific genes have also provided potentially powerful approaches. Antisense methods, using either DNA or RNA, are relatively straightforward techniques for probing gene functions; however, these methodologies have suffered setbacks because of lack of specificity and incomplete efficiency. Moreover, the desired effects were difficult to predict and often only a weak suppression was achieved (Guru, 2000). PTGS in plants involves down-regulation of gene expression at the post transcriptional level, by targeting specific RNAs for degradation. Transgenes are subject to suppression by PTGS, as are other genes that share significant sequence homology with the silenced genes. Plant PTGS is similar to other eukaryotic evidence such as suppression of transgenes and transposons, and cellular responses to double-stranded RNAs.
First described in worms in 1998, RNAi operates in plants, fungi, flies and mammals. Long molecules of double stranded RNA (dsRNA) trigger the process. The dsRNA comes from virus and transposon activity in natural RNAi process, while it can be injected in the cells in experimental processes (Elbashir et al. 2001a). The strand of the dsRNA that is identical in sequence to a region in target mRNA molecule is called the sense strand, and the other strand which is complimentary is termed the antisense strand. An enzyme complex called DICER in D. melanogaster, thought to be similar to RNAase III then recognizes ds RNA, and cuts it into roughly 22- nucleotide long fragments. These fragments termed siRNAs for "small interfering RNAs" which remain in double stranded duplexes with very short 3' overhangs. Then act as templates for the RNAi inducing silencing complex to destroy the homologous message, thus specifically suppressing its expression. This form of RNAi is termed as PTGS, other forms are also thought to operate at the genomic or transcriptional level in some organisms.
The future impact of PTGS is immense that it has a role in viral defence and transposon silencing mechanism.
Salient features of RNAi
• Double stranded RNA rather than single-stranded antisense RNA is the interfering agent.
• High degree of specific gene silencing with less effort.
• Highly potent and effective (only a few double stranded RNA molecules per cell are required for effective interference).
• Silencing can be introduced in different developmental stages.
• Systemic silencing.
• Avoids problems with abnormalities caused by a knocked out gene in early stages (which could mask desired observations).
• Silencing effects passed through generations.
The use of RNAi as a method to alter gene expression has been attempted in a diverse group of organisms, employing different methods, with different rates of success. In C. elegans, Drosophila and plants, RNAi seems to be an effective, specific and valuable tool for reverse genetics. A second group including zebrafish, Xenopus and mouse show RNAi with some limitations. RNA interference employing short dsRNA oligonucleotides will permit to decipher the functions of genes being only partially sequenced. One of the first commercial products of RNA silencing was tomato in which the target was to reduce the expression of these genes in the silenced plants meant that the tomatoes were firm after ripening and were not damaged by handling. Virus induced gene silencing is potentially a powerful tool to silence the endogenous genes that are homologus to any sequences carried within the virus. This technology will enable to use plant virus induced gene silencing approach for plant genetic studies.
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