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RNA Interference - The Art of Gene Silencing

BY: Pournami Gouthaman | Category: DNA | Submitted: 2011-01-25 20:25:15
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Article Summary: "Small RNA molecules, combined to a RNA induced silencing complex, have the ability to attach to complementary strands of messenger RNA, resulting in the degradation of messenger RNA, blocking its translation and eventually leading to gene silencing. Its targeted gene silencing ability has made it a potential tool in gene therapy.."


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The Central Dogma of Molecular Biology explains the fundamental gene expression pathway in an organism. According to this, DNA (De-oxy ribonucleic acid) is first transcribed to RNA (ribonucleic acid) in the nucleus and the mature messenger RNA (mRNA) resulting from splicing is then translated to proteins in the cytoplasm. So, initially what scientists thought was that all the mRNAs formed are translated to proteins. This notion was proved wrong by the early findings of gene silencing in some plants and fungi, which were termed as cosuppression, transcriptional or post-transcriptional gene silencing and quelling. The stunning realization that RNA plays an active role in gene regulation came about from the discovery of the catalytic function of RNA (ribozyme). The ability of ribozymes to catalyse self-replication and synthesis of other RNA molecules even led to the conclusion that RNA was the first genetic material on earth.

RNA interference (RNAi) is the term given to the phenomenon inside the living cell, by which short RNA molecules control the gene expression. The phenotypic effect of RNA injected into the worm Caenorhabditis elegans was studied by Andrew Fire and Craig Mellow, which led to their discovery of RNA interference. The RNAi mechanism of gene silencing was originally evolved in order to deal with the attack of foreign genetic material (especially double stranded RNA) and jumping genetic material called transposon during infection of cells by viruses like HIV. Two types of RNA molecules play major role in RNAi. These are microRNA (miRNA) and short interfering RNA (siRNA). miRNAs are small RNA molecules about 22 nucleotides long, encoded by the genome. It acts as post-transcriptional regulators by binding to complementary sequences on target mRNAs usually resulting in the blocking of translation and gene silencing. siRNA also has the same properties as miRNA, the only difference being the origin. siRNAs are formed from the degradation of double stranded RNAs (dsRNAs) in the cytoplasm, carried out by the enzyme Dicer. Thus, we can say that miRNA is endogenous and siRNA are exogenous in nature.

RNAi is found in eukaryotes, including plants and animals. The exact mechanism of RNA interference can be explained as follows. When a long dsRNA is introduced into a cell, it is broken down to short interfering RNAs. These strands are separated to form two single stranded RNA (ssRNA) called the passenger strand and guide strand. The guide strand now attaches itself to the RNA induced silencing complex (RISC), whereas passenger strand is degraded. When the guide strand attaches to the complementary sequence of mRNA, the catalytic component of RISC complex called Argonaute cleaves the mRNA, thus preventing its translation. Even though the initial concentration of siRNA is small, the process of RNAi is said to spread throughout the organism in a systematic way and is observed to be hereditary also in some cases.

Because of its selective action, the process of RNA interference has wide applications in the field of genetics and applied biology, and can be studied both in cell cultures and organisms. The discovery of RNAi mechanism has great significance in understanding more about the general regulation of genes. In plants, worms and flies, RNAi acts as defense mechanism to fight against viral infections. It also helps to keep the activity of transposons in the genome under control, by inhibition at the transcriptional level. RNAi, with the help of miRNAs help to suppress protein synthesis and regulate the development of the organism. It was also found that RNAi like mechanisms keep the chromatin condensed and suppress transcription. The introduction of a dsRNA which is complementary to a specific mRNA can result in the silencing of a particular selected gene. This means that RNAi helps in targeted gene silencing, which is now very much researched upon, to develop its potential application in modern gene therapy.

RNAi is applied in functional genomics for carrying out genomic mapping in plants. It is also used for various applications in biotechnology, based on its special property of targeted gene silencing. Clinical trials in mammals are carried out by introducing siRNAs into cells, as the use of dsRNA becomes difficult due to the immune response of interferon. Several proposals have come up about the possible use of RNAi in treating neurodegenerative disorders, finding a cure for genetic diseases that are caused by defective genes and the prospective treatment of cancer, by controlling the gene for cell division or the gene that is over expressed in cancer cells. Before going to clinical trials in human, the possible adverse effects of RNAi should also be studied in detail. The possibility of off-targeting by the introduced dsRNA is seen as 10%, which can result in the silencing of other genes, the mRNA of which will be having similar nucleotide sequence as the targeted gene.

The current research that is advancing in this field is the development of a safe delivery method for siRNA into the cells. RNA interference has not only opened a new doorway to study the function of genes, but has also led to the proposal of its future application in medicine.

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I am a biotech engineer interested in researching and writing biotech articles.

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