Inhibition Of Cellular Gene Expression Rna Interference

The previous edition of this textbook (2004) mentioned RNA interference as a new technique with great potential for simple manipulation of gene expression. In September 2006, Andrew Z. Fire and Craig C. Mello were awarded a Nobel Prize for this versatile discovery. RNA interference (RNAi) is believed to be a biologically conserved function in a wide range of eukaryotic species. It may play a role in protection against double-stranded RNA viruses (Sijen et al. 2001) and genome-invading transposable elements (Provost et al. 2002; Volpe et al. 2002). Triggered by dsRNA, RNAi identifies and destroys the mRNA that shares homology with the dsRNA. Thus, the expression of a particular gene can be suppressed by introducing dsRNA whose antisense strand sequence matches the mRNA sequence. Fire et al. (1998) first described RNAi in the nematode Caenorhabditis elegans as sequence-specific gene silencing in response to double-stranded RNA. The mechanism of RNAi is partly understood, and key proteins involved in the pathway have been identified. In brief, the basic process of RNAi involves the following steps. In a first initiation step, Dicer, an enzyme of the RNase III family, initiates ATP-dependent fragmentation of long dsRNA into 21- to 25-nucleotide double-stranded fragments, termed small interfering RNAs (siRNAs). These siRNAs are specifically characterized by overhanging 3' ends of two nucleotides and phosphorylated 5' ends. The siRNA duplexes bind with Dicer, which facilitates the formation of an siRNA/multiprotein complex called RISC loading complex (RLC). The siRNA duplex in RLC then unwinds (which requires the protein Ago2) to form an active RNA-induced silencing complex (RISC) that contains a single-stranded RNA (called the guide strand). The RISC recognizes the target RNA through Watson-Crick base pairing with the guide strand and cleaves the target RNA. Finally, the RISC releases its cleaved product and goes on to catalyze a new cycle of target recognition and cleavage (Figure 2-6) (Tomari and Zamore 2005; Xia et al. 2005).

FIGURE 2-6. Schematic of the mechanism of RNA interference (RNAi) posttranscriptional knockdown of a gene product.

mRNA degradation

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The procedure starts with introduction (transfection, electroporation, or injection) of double-stranded RNA (dsRNA) or small interfering RNA (siRNA) into cells, or expression of small hairpin RNA (shRNA) in cells with vectors encoding shRNAs. The cellular ribonuclease (RNase) Dicer recognizes the long dsRNA molecules and shRNA. Subsequently the dsRNA is cleaved, resulting in 21-nt RNA duplexes, the siRNAs. These siRNA molecules are then incorporated into the RNA-induced silencing complex (RISC) multiprotein complex, where they are unwound by an adenosine triphosphate (ATP)-dependent process, transforming the complex into an active state. Activated RISC uses one strand of the RNA as a bait to bind homologous RNA molecules. The target RNA is cleaved and degraded, resulting in gene silencing.

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