Gene Silencing, Mechanism and Applications

Gene silencing is arguably the hottest topic in science at present and has been proclaimed to be the biggest breakthrough of the year 2002, as per the journal Science. Gene expression studies have always fascinated the scientists and are consequently studded with a number of nobel prizes. Gene silencing refers to general processes of interruption or suppression of transcription or translation of the mRNA of the target gene by mechanisms other than genetic modification. It is mediated by small RNA molecules known as siRNAs or miRNAs. These are produced by Dicer either from exogenously introduced dsRNAs or from endogenously transcribed MIR genes. These further activate RISC which inhibits transcription or translation of the target mRNA. Gene silencing maintains the genomic structure, differentiation and maintenance of stem cells and provides a promising treatment for diseases like AIDS and cancer, if the various limitations like problems of in vitro delivery and offtarget effects, associated with its applications as a curing tool, are overcome. Introduction RNA interference (RNAi) is arguably the hottest topic in science at present. The number of papers published on RNAi has exploded over the last few years and the journal Science proclaimed it the biggest scientific breakthrough of 2002. Why is RNAi causing such a fuss? Well, imagine you could identify the role of a gene in a disease by switching it off easily, in the space of just a day, and in almost any organism. Imagine you could then take this tool and treat certain diseases, such as cancer or AIDS, by switching off the causative genes. That's the promise that RNAi offers. Gene silencing is a general term describing epigenetic processes of gene regulation. It is used to describe the “switching off” of a gene by a mechanism other than genetic modification, i.e; a gene which would be expressed (turned on) under normal circumstances is switched off by machinery in the cell. The interruption or suppression of the expression of a gene at transcriptional or translational level is referred to as gene silencing. The silencing of a gene could be achieved by: i) Drugs: These bind to target protein and cause protein inhibition ii) RNase H–independent ODNs: These oligo deoxynucleotides iii) hybridize to target mRNA and cause inhibition of translation of target protein. iv) RNase H–dependent ODNs: These hybridize to target mRNA and (DHR-IJBLS), Vol. 3, Issue 1, 2012 (DHR-IJBLS) ISSN: 2278-8301 115 mediate its degradation by RNase H. v) Ribozymes and DNA enzymes: These catalyze cleavage of mRNA and hence cause its degradation. vi) SiRNA and miRNA: These hybridize to target mRNA by antisense strand and guide it into endoribonuclease enzyme complex, thereby causing its degradation or inhibition of translation. History and Discovery 1. Rich Jorgensen et al. in an attempt to alter flower colours in petunias, introduced additional copies of a gene encoding chalcone synthase, key enzyme for flower pigmentation, into flowers of normally pink or violet colour. Unexpectedly the flowers produced were less pigmented, fully or partially white. It was observed that both the transgene and endogenous gene were down regulated in white flowers. This phenomenon was called cosuppression of gene expression. 2. Quelling was observed in fungus, Neurospors crassa, in an attempt to boost production of orange pigment produced by the gene aL1 of the fungus. Attempts to enhance orange pigment in the fungus by introducing extra copies of carotenoid pigment genes failed when the orange pigment gene was suppressed in a third of the engineered mould. In some strains, the effect was passed on through multiple generations. This was later found to be similar to post – transcriptional silencing. 3. Plant virologists working on improving plant resistance to viral diseases observed a similar unexpected phenomenon. It was observed that plants carrying only short, non-coding regions of viral RNA sequences would show similar levels of protection as the plants expressing virus specific proteins. It was believed that viral RNA produced by transgenes could also inhibit viral replication. The reverse experiment in which short sequences of plant genes were introduced into viruses showed that targeted genes were suppressed in an infected plant. This phenomenon was labeled virus induced gene silencing (VIGS) and the set of such phenomenon was called as post transcriptional gene silencing. 4. Guo and Kempheus, attempted to use antisense RNA to shut down expression of the par1 gene in Caenorhabditis elegans in order to assess its function. As expected injection of antisense RNA disrupted expression of par-1 but injection of the sense strand control also did. The result remained a puzzle for three years and for this phenomenon they coined the term antisense mediated silencing. 5. Three years later Andrew Fire and Craig C.Mello studied phenotypic effect of singlestranded and doublestranded unc-22 RNA into gonads of C .elegans (1998). They observed that only the double stranded RNA consisting of both sense and antisense strand produced the typical twitcher in C. elegans while sense and antisense strands individually did not produced the twitcher. They concluded the results of their experiments as: i) Silencing was triggered by injecting dsRNA but weakly or not at all by ssRNA ii) Silencing was specific for an mRNA homologous to dsRNA iii) The dsRNA had to correspond to mature mRNA sequence, neither intron nor promoter sequence triggered the response. iv) Targeted mRNA was degraded. v) dsRNA are amplified in cell, as very few are required to produce the effect. vi) Effect of dsRNA spread between tissues and even to progeny. For their discovery of gene silencing by double stranded RNA they coined the term RNA (DHR-IJBLS), Vol. 3, Issue 1, 2012 (DHR-IJBLS) ISSN: 2278-8301 116 interference (RNAi) and were subsequently awarded the Nobel prize in physiology or medicine (2006). 6. RNAi was also observed subsequently in insects, frog and other animals including mice and in humans. 7. In 2001, Thomas Tuschl, discovered with his colleagues that RNAi could be prompted through the use of shorter pieces of RNA known as small interfering RNAs (siRNAs). Soon thereafter, they showed that duplexes of 21nucleotide siRNAs mediated RNAi in cultured mammalian cells and demonstrated that siRNAs could be designed to silence specific genes without activating the interferon response. In other words, scientists could potentially silence any gene of interest in a highly predictable, reproducible, and accurate fashion. 8. Gregory Hannon and his colleagues identified, described, and named the "Dicer" enzyme, which chops dsRNA into siRNAs, as well as the RNA-induced silencing complex (RISC), which mediates the silencing process by degrading the homologous mRNA. 9. In 2002, the use in mammalian cells of socalled short hairpin RNAs (shRNAs), which generate endogenous siRNAs within cells and thus provide stable, heritable gene silencing (in contrast, administered siRNAs are transient in their silencing effect). This effect was named "short hairpin-activated gene silencing" or SHAGging. This discovery allowed the development of cell lines and animal models with permanently silenced genes, a major step forward for basic science in general, and especially for functional genomics. Cellular components of Gene Silencing  MicroRNAs (miRNAs)  Small interference RNAs (siRNAs)