RNAi

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RNAi

RNAi or RNA interference is a mechanism of post transcriptional Gene regulation. This involves a complicated mechanism by which non coding RNAs degrade mRNA strands thus interefering in that particular genes expression. These non coding transcripts are thought to have an effect in regulating up to a third of Protein coding genes in humans by this method. The non coding RNAs that have an effect in RNAi are called micro or miRNAs. The miRNAs are synthesised the same way as all RNA transcripts are; through RNA polymerase II [1]. They are hairpin in structure due to palindromic sequences in their strand and tend to be around 75 Nucleotides long [2].

Mechanism of RNAi

The double stranded miRNA leaves the Nucleus in its hairpin structure. In the Cytosol it is modified and one of its strands is associated with RISC (RNA induced silencing complex), which is a large multi protein complex. The miRNA strand associated with RISC is complementary to gene coding mRNAs in the cytosol, it is held in the RISC complex by the protein Argonaute. Argonaute orientates the miRNA so that its 5" end is positioned to have maximal interaction with complementary mRNA transcripts. The contact between the two RNAs is usually 7 base pairs. Upon binding Argonaute can either cleave the mRNA or destabilise its poly-adenylate tail so its is degraded faster in the cytosol, this depends on the extent of the Watson-Crick base pairs between the two strands. The RISC and associated miRNA then dissociates from the mRNA further affects other transcripts of that gene so that the effect is amplified [3]. This is how RNAi causes gene silencing or knockdown.

The above process of gene silencing also aids in viral infection. Retroviruses (a Virus with an RNA genomes) inject dsRNA into the cell to manipulate the cells translational machinery into producing more retroviruses. The dsRNA is recognised by the Enzyme Dicer which cleaves it into siRNAs (small interfering RNAs). These are 22 nucleotides long and defined by a 2 nucleotide 3" overhang. The siRNA is "unzipped" by a helicase enzyme and the one of the strands, called the guide strand associates itself with RISC [4]. RISC then attaches itself to the retroviral dsRNA and allows Argonaute to cleave it. This leads to the dsRNA being degraded completely [5].

Medical and Experimental Applications of RNAi

RNAi has been shown in cultured cells to have an 80% efficiency in silencing oncogenes (Cancer producing genes). This could have wide applications and potentially revolutionise cancer treatments, however RNAi is still being studied and could have disastrous side effects in human cells [6].

As we have many organisms genomes sequenced we can use RNAi to knockdown genes with unknown functions. This technique is a reversal of classical genetics; we are silencing the known Genotype and waiting for the unknown Phenotype to appear, this allows functions to be assigned to genes that were previously unidentified. RNAi knockdown has been utilised in Drosophila and C. elegans [7]. This has worked well due to parent cells passing on siRNAs to the daughter cells so that RNAi can continue against the same gene that was silenced in the parent [8].

References

  1. Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P,2008, Molecular Biology of the Cell,5th Edition, New York, Garland Science pg 493-4
  2. Eliiot W, Elliot D, 2009, Biochemistry and Molecular Biology 4th Edition, New York, Oxford University Press pg 407
  3. Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P,2008, Molecular Biology of the Cell,5th Edition, New York, Garland Science pg 494
  4. Nature Publishing Group,2005, RNA interference, Available at; http://www.nature.com/focus/rnai/animations/index.html, 18/11/11
  5. Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P,2008, Molecular Biology of the Cell,5th Edition, New York, Garland Science pg 495
  6. Eliiot W, Elliot D, 2009, Biochemistry and Molecular Biology 4th Edition, New York, Oxford University Press pg 408
  7. Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P,2008, Molecular Biology of the Cell,5th Edition, New York, Garland Science pg 570
  8. Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P,2008, Molecular Biology of the Cell,5th Edition, New York, Garland Science pg 495
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