The discovery of RNA interference (RNAi), an endogenous cellular gene-silencing
mechanism, has already provided a powerful tool for basic science researchers
to study gene function. The subsequent finding that RNAi also operates in
mammalian cells has generated excitement regarding potential therapeutic applications.
In this article we discuss the basic mechanism of RNAi and the therapeutic
opportunities and obstacles for harnessing RNAi for therapy of human disease.
Gene silencing by RNA interference can be initiated by introducing synthetic
small interfering RNAs (siRNAs) or their precursors into cells where they
are taken up by the endogenous microRNA processing machinery. The siRNA precursors
are cleaved by the Dicer enzyme into siRNAs, which are 19- to 21-nucleotide
double-stranded RNAs with a 2- to 3-nucleotide overhang and characteristic
5′-phosphate and 3′-hydroxyl groups at each end. As Dicer, an
RNAse III–like enzyme, hands off the siRNA to the RISC (the cellular
complex ultimately responsible for “slicing” messenger RNA [mRNA]),
the double-stranded RNA unwinds, the RISC becomes activated, and 1 strand
(the guide strand) remains within the activated complex. If the guide strand
is homologous to an mRNA sequence, Argonaute 2, an enzyme within the RISC
complex, cleaves the mRNA in the center of the region of homology. The cleaved
mRNA is rapidly degraded and the protein for which it encodes is not produced.
The guide strand small RNA is protected from degradation by the RISC and can
direct the cleavage of many mRNAs. Alternately, plasmids or viral vectors
encoding small stem-loop RNAs (called short hairpin RNAs [shRNAs]) can be
introduced into cells where they are incorporated into the cell’s genome,
resulting in the expression of shRNAs that are processed in the nucleus by
the same mechanism as endogenous microRNA precursors and exported to the cytoplasm
where they are processed similarly to the process described above.
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