Background

RNAi, or RNA interference, was discovered when puzzling results were obtained in experiments conducted by biologists Su Guo and Kenneth Kenpheus. They observed that sense and antisense RNA were equally effective in suppressing specific gene expression (Guo and Kempheus, 1995). In 1998, Fire and his colleagues resolved this paradox by finding that small amounts of dsRNA contaminate sense and antisense preparations. Even earlier, biologists had unknowingly observed RNA interference when performing experiments on Petunias. Rich Jorgensen et al., found that when they introduced a pigment-producing gene under the control of a promoter into flowers instead of the expected deep purple color flowers they obtained flowers that were variegated or white in color. Thus RNAi, a term coined after the groundbreaking discovery by Fire et al., refers to a phenomenon in which there is inhibition of expression of specific genes by double-stranded RNAs (dsRNAs). RNAi has been evolutionarily conserved among eukaryotes. It has been discovered in a wide variety of animals, including flies (Kennerdell and Carthew, 1998; Misquitta and Paterson, 1999), Trypanosoma brucei (Ngo et al., 1998), planaria, (Sanchez-Alvarado and Newmark, 1999), hydra (Lohmann et al., 1999), zebrafish (Wargelius et al., 1999), and mice (Wianny and Zernicka-Goetz, 2000). In plants this gene silencing phenomenon can be compared to cosuppression; Vaucheret et al.,1998; Waterhouse et al.,1998,1999; Baulcombe,1999). RNAi occurs posttranscriptionally and involves mRNA degradation (Montgomery et al., 1998; Ngo et al., 1998). In addition to playing a powerful role in creating loss-of-function mutants, it probably also plays an essential role in protecting the genome against instability caused by exogenous RNAs (eg. Viruses) (Kasschau et al., 1998) and accumulation of transposons and repetitive sequences (Ketting et al., 1999; Tabara et al., 1999; Hannnon GJ, 2002). Thus the vast nature of RNA interference-like processes may encompass not only gene silencing phenomena but also cellular programs for regulation of genes, inhibition of transposon mobilization, and anti-viral mechanism in plants.  RNAi occurs through a series of steps involving the generation of small interfering RNAs (siRNAs) in vivo through the action of a specific RNAaseIII endonuclease Dicer. The resulting siRNAs mediate the degradation of their complementary RNA by association of the siRNA with a nuclease complex to form what is called the RNA-induced silencing Complex (RISC). In the next step, an unwinding of the siRNA occurs which activates RISC. It is the activated RISC that binds to the target mRNA and finally leads to the loss of expression of the gene it coded (Zamore et al., 2000). For a while, the use of RNAi to study gene function had been restricted to plants, Caenorhabditis elegans and Drosophila where large dsRNA can efficiently cause gene-silencing to take place (Shi Y, 2003; Misquitta et al., 1999; Tuschl et al., 1999). The major hurdle to achieving RNAi in mammals was that dsRNAs longer than 30 nucleotides activate defense mechanisms that result in non-specific degradation of RNA transcripts and a general shutdown of host cell protein synthesis (Williams,BR 1997). This obstacle was overcome recently by using in vitro synthesized ~21 nucleotide siRNAs to mediate gene-specific suppression in mammalian cells. These siRNAs are long enough to cause gene suppression but not so long to cause interferon response to take place (Elbashir et al., 2001;Calpen et al., 2001). Once these molecules were identified, several DNA vector-based strategies were developed allowing use of RNAi into mammalian cells.  There are several plasmid vectors that express siRNA through RNA Polymerase II promoter. The most commonly used Pol III promoters are U6 and H1 that direct transcription of small repeats separated by a spacer region.

Advantages of Plasmid based vectors over synthetic siRNA:

Plasmid based vectors Synthetic siRNA Antibiotic resistance for selection Yes No Observe/ Monitor transfection efficiency of cells Yes No Cost per gene Moderate High Gene inhibition studies Long term Short term Stable cell line production Yes No Effective delivery of siRNA into cells that are hard to transfect Yes No

Applications

RNA interference technology has several potential applications not only in functional genomics analyses but also in therapeutics. This is owing to its unique feature of sequence specificity. A single base mismatch in the hairpin siRNA can dramatically reduce its RNAi effect (Brummelkamp et al., 2002). Such a high level of sequence specificity can help us use this technology to knockdown expression of genes that have either insertions, deletions or may be point mutations. This technology thus may potentially have several medical implications. For instance, RNAi may be used to inhibit cancer-related oncogenes that have been produced as a result of chromosomal translocations or point mutations. There are several hematopoietic cancers that are caused by dominantly acting oncoproteins that are encoded by fusion RNA transcripts resulting from chromosomal translocations. The chimeric RNA transcripts are therefore ideal targets for selective inhibition by siRNAs targeting the fusion sites, and this has been shown recently (Wilda et al., 2002).  RNAi technology has also been used successfully in inhibiting viral replication in cell culture (Jaque et al., 2002;Coburn et al., 2002). It has been used to suppress the cytotoxicity caused by overexpression of an androgen receptor, which is associated with the neurodegenerative disorder, spinobulbar muscular atrophy (SBMA) (Caplen et al., 2002). siRNAs also suppressed tumor production by inhibiting expression of K-ras v12 allele, but not that of the wild type allele (Brummelkamp et al.,2002). The discovery of RNAi has taken the scientific world to new realms where functional analysis of genes can be precisely carried out using these small RNA molecules. This novel technology will help in developing new reagents that can be used to target several human diseases. RNAi indeed is a breakthrough of the 21st century, which will revolutionize genetic, genomic and proteomic aspects of biology and signal a new era in the field of medicine.

References:

Guo S, and Kempheus KJ. (1995). Par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed. Cell 81: 611-620. Fire, A. et al. (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806–810 Kennerdell, J.R. & Carthew, R.W. (1998). Use of dsRNA-mediated genetic interference to demonstrate that frizzled and frizzled 2 act in the Wingless pathway. Cell 95, 1017-1026. Misquitta, L. & Paterson, B.M. (1999). Targeted disruption of gene function in Drosophila by RNA interference (RNA-i): A role for nautilus in embryonic somatic muscle formation. Proc. Natl. Acad. Sci. USA 96, 1451-1456. Ngo, H., Tschudi, C., Gull, K. & Ullu, E. (1998). Double-stranded RNA induces mRNA degradation in Trypanosoma brucei. Proc. Natl. Acad. Sci. USA 95, 14687-14692. Sánchez Alvarado, A. & Newmark, P.A. (1999). Double-stranded RNA specifically disrupts gene expression during planarian regeneration. Proc. Natl. Acad. Sci. USA 96, 5049-5054. Lohmann, J.U., Endl, I. & Bosch, T.C.G. (1999). Silencing of developmental genes in Hydra. Dev. Biol. 214, 211-214 Wargelius, A., Ellingsen, S. & Fjose, A. (1999). Double-stranded RNA induces specific developmental defects in zebrafish embryos. Biochem. Biophys. Res. Commun. 263, 156-161. Wianny, F. & Zernicka-Goetz, M. (2000). Specific interference with gene function by double-stranded RNA in early mouse development. Nature Cell Biology 2, 70-75. Waterhouse, P. M., Graham, M. W. and Wang, M.-B. (1998). Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA. Proc. Natl. Acad. Sci. USA 95, 13959-13964. Baulcombe, D.C. Fast forward genetics based on virus-induced gene silencing. Curr; Opin. Plant Biol. 2, 109-113 (1999). Vaucheret, H., Beclin, C., Elmayan, T., Feuerbach, F., Godon, C., Morel, J.B., Mourrain, P., Palauqui, J.C., and Vernhettes, S. 1998. Transgene-induced gene silencing in plants. Plant J. 16: 651-659. Montgomery, M. K., Xu, S., and Fire, A. (1998). RNA as a target of double-stranded RNA-mediated genetic interference in Caenorhabditis elegans. Proc Natl Acad Sci U S A 95, 15502-15507. Kasschau, K. D. and Carrington, J. C. (1998). A counterdefensive strategy of plant viruses: suppression of post-transcriptional gene silencing. Cell 95, 461-470. Ketting, R. F., Haverkamp, T. H., van Luenen, H. G. and Plasterk, R. H. (1999). Mut-7 of C. elegans, required for transposon silencing and RNA interference, is a homolog of Werner syndrome helicase and RNaseD. Cell 99, 133-141. Tabara, H., Sarkissian, M., Kelly, W. G., Fleenor, J., Grishok, A., Timmons, L., Fire, A. and Mello, C. C. (1999). The rde-1 gene, RNA interference, and transposon silencing in C. elegans. Cell 99, 123-132. Hannon, G.J. 2002. RNA Interference. Nature 418: 244-251. Zamore, P.D., Tuschl, T., Sharp, P.A. & Bartel, D.P. (2000). RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101, 25-33. Misquitta, L. & Paterson, B.M. (1999). Targeted disruption of gene function in Drosophila by RNA interference (RNA-i): A role for nautilus in embryonic somatic muscle formation. Proc. Natl. Acad. Sci. USA 96, 1451-1456. Tuschl, T., Zamore, P.D., Lehmann, R., Bartel, D.P. & Sharp, P.A. (1999). Targeted mRNA degradation by double-stranded RNA in vitro. Genes Dev. 13, 3191-3197. Williams,B.R. (1997)Role of the double-strandedRNA-activated protein kinase (PKR) in cell regulation. Biochem. Soc. Trans. 25, 509–513. Elbashir, S.M. et al. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498. Calpen, N.J. et al. (2001) Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems. Proc. Natl Acad. Sci. USA 98, 9742–9747. Brummelkamp, T.R. et al. (2002) A system for stable expression of short interfering RNAs in mammalian cells. Science 296, 550–553. Wilda, M. et al. (2002) Killing of leukemic cells with a BCR/ABL fusion gene by RNA interference (RNAi). Oncogene 21, 5716–5724. Jacque, J.M. et al. (2002) Modulation of HIV-1 replication by RNA interference. Nature 418, 435–438. Shi, Y (2003)  Mammalian RNAi for the masses. Trends in Genetics 19, 9-12. Coburn, G.A. and Cullen, B.R. (2002) Potent and specific inhibition of human immunodeficiency virus type 1 replication by RNA interference. J. Virol. 76, 9225–9231. Caplen, N.J. et al. (2002) Rescue of polyglutamine-mediated cytotoxicity by double-stranded RNA-mediated RNA interference. Hum. Mol. Genet. 11, 175–184. Brummelkamp, T.R. et al. (2002) Stable suppression of tumorigenicity by virus-mediated RNA interference. Cancer Cell 2, 243–247.