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.

RNA interference (RNAi) is a potent method using only a few double stranded RNA (dsRNA) molecule per cell to silence the expression which has made it one of the hottest topics in molecular biology in last two years. 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. 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.

Salient features of RNAi

Mechanism of RNAi

Intensive research efforts to understand this intriguing process elucidates the exact molecular mechanism.

In the initiation step the "trigger" ds RNA molecule, usually several hundred base pair long, is cleaved to form 21-23 bp double stranded fragments known as short interfering RNAs (siRNAs) or guide RNAs. siRNAs are produced when the enzyme Dicer a member of the RNAase III family of dsRNS- specific ribonucleases, processively cleaves dsRNA in ATP dependent, processive manner.

In the effector step the duplex siRNA are then unwound by a helicase activity associated with a distinct multiprotein complex known as the RNA-induced silencing complex or RISC. An ATP dependent unwinding of siRNA duplex is required for activation of RISC.

The siRNA strand that is complementary to the targeted mRNA is then used as primer by an RNA-dependent RNA polymerase (RdRP) to convert the cognate mRNA into dsRNA itself. This dsRNA form of mRNA then becomes a substrate for Dicer cleavage activity, which leads to the destruction of the mRNA and formation of new siRNAs. Effectively, this step amplifies the RNAi response and creates a self- perpetuating cycle of "degradative polymerase chain reaction" that will persist until no target mRNAs remain. This basic 'core' pathway defines the RNAi response as one of the most elegant and efficient biochemical mechanisms in nature.

Antisense methods , using either DNA or RNA, are straight forward techniques for probing gene functions, however the discrepancy lies in the fact that this process suffers from specificity and incomplete efficacy. RNA silencing is induced in plants at varying efficacies by transgenes designed to produce either sense or antisense transcripts. Furthermore, transgenes engineered to produce self complementary transcripts (dsRNA) are potent and consistent inducers of RNA silencing.

In context with the current status of knowledge about RNAi, it is a revolution in the field of plant molecular genetics that it has enormous potential for engineering control of gene expression , as well as for the use of a tool in functional genomics. The ability to manipulate RNA silencing has a wide variety of practical applications of biotechnology ranging from molecular biology to gene therapy in animals. This process can be induced experimentally with high efficiency and targeted to a single specific gene or a multigene family.

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. RNAi is important for inhibition of gene expression at the post transcriptional level in eukaryotic cells. Worms can be engineered for the generation of stable phenotypic null mutants. In this context RNAi is a straight forward tool to rapidly assess gene function and reveal null phenotypes. Development of RNAi technology for the use in post implantation embryos. Drosophila embryology studies reveal to the production of null phenotypes by injecting early stage embryos. RNAi technology can be applied as genetic tools in vertebrates to induce sequence specific silencing in early mouse embryos. The predominant economic significance of RNA interference is established by its application as a therapeutic principle. As so, RNAi may yield RNA based drugs to treat human diseases. siRNA is effective against parasites, so perhaps it can be used to silence parasitic genes or used against other pathogens to benefit host organisms like humans. In most mammalian cells , 400-500 base pair long dsRNA invokes a more general translational suppression through a pathway involving interferon, ultimately leading to cell death by apoptosis. dsRNA can silence the expression of exogenous genes in Chinese hamster ovary cells.

Hence, siRNA brings the possibility of specific gene silencing through mRNA degradation, something its precursor, dsRNA, cannot do, while possibly being more versatile than less stable single stranded antisense oligonucleotides.