How do siRNAs lead to RNA degradation? Although the molecular details are still being worked out, a model has emerged. Duplex siRNAs are unwound by an ATP-dependent helicase and one strand is incorporated into a large multiprotein nuclease complex known as the RNA-induced silencing complex (RISC). Incorporation of small RNA activates RISC. Guided by the antisense strand of siRNA, the Argonaute2 endonuclease in the activated RISC recognizes and cleaves the corresponding mRNA, with a resultant decrease in the steady-state levels of mRNA. Argonaute proteins contain a conserved protein-protein interaction domain called PAZ and another conserved domain (PIWI) of uncertain function . Four proteins have so far been identified as candidate components of the RISC complex in mammalian cells. These are the Argonaute proteins eIF2C1 and eIF2C2 and gemin 3 (also known as DDX20, DEAD/H box polypeptide 20) and gemin 4, but their roles are not understood.
The RNAi response is an intrinsic defense mechanism for combating viruses and protecting the genome from invasion by endogenously generated transposons. The notion of homology-directed gene silencing as an anti-viral response derives from a large body of data obtained in plants. Most plant viruses have double-stranded RNA genomes. RNAi is induced in response to replicative stage double-stranded RNA intermediates in the viral life cycle. Arabidopsis mutants that are defective in genes involved in RNAi are highly susceptible to cucumovirus and tobacco mosaic viral infection. The importance of RNAi in antiviral defense is also suggested by the fact that plant and insect viruses have developed counter defensive strategies, encoding proteins that suppress different steps in RNA silencing. However, an innate RNAi antiviral response has not yet been reported in mammals. This may be because evolution has endowed mammals with alternative antiviral mechanisms that override the RNAi response. Introduction of dsRNA larger than 30 nt into mammalian cells results in global repression of gene expression because of the activation of protein kinase PKR and 2', 5'-oligoadenylate synthetase that form part of the interferon (IFN) defense pathway against viruses. Nevertheless, the RNAi pathway is functionally intact in mammalian cells. Exogenously introduced siRNAs guide degradation of cognate mRNA and abrogate gene expression in an exquisitely sequence-specific manner. The important discovery that RNAi works in mammalian cells was soon followed by a flurry of research papers demonstrating the antiviral potential of this powerful new technology against a multitude of mammalian viruses including HIV-1 . RNAi is also being used in basic HIV research to silence host genes to understand better the interaction of HIV with host genes during the viral life cycle.
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