Figure 1: Diagram of the mechanism of RNAi. Naturally occurring microRNA (miRNA) and short hairpin RNA (shRNA) are processed by Dicer in the cytoplasm into short interfering RNAs (siRNA). These are recognized by EIF2C and other proteins of the RNA-induced silencing complex (RISC). The siRNA is unwound and used as a template for identifying a homologous mRNA, which is cleaved by an RNase H-like enzyme in RISC. The siRNA/RISC complex is then free for further mRNA cleavage.
Post-transcriptional gene silencing (PTGS) was first thought to be a novel mechanism limited to plants, but has been shown to be a highly conserved mechanism found also in worms, flies, and mammals. It appears to be an early defense mechanism against viruses and transposons, effectively silencing foreign and unwanted genes. The discovery of RNA interference (RNAi) in C. elegans using injected double strand RNA (dsRNA) launched the current wave of RNAi discoveries in mammals. Initially, these experiments failed due to the activation of the interferon response pathway by long dsRNA, which caused global inhibition of protein synthesis. This was overcome by Tom Tuschl's lab, which discovered that small RNAs of 19-23 bases, called short interfering RNA (siRNA), avoided this response. These siRNAs can be synthesized and introduced directly into cells (Figure 1). Unlike the extended silencing effect seen in worms and flies, the effect of siRNA in mammals is transitory, usually lasting up to 72 hours in cultured cells. However, the use of plasmids to express short hairpin RNA (shRNA), which mimics the recently discovered endogenous microRNA (miRNA), allows for extended gene silencing (figure 2).
RNAi is a potent tool for specific gene silencing that only requires the target gene sequence. Because of the sequence specificity, even different alleles of the same gene can be silenced. For a thorough review of RNAi, see Nature Insight, Vol. 431 pp. 338-378, 16 September, 2004.
We are using RNAi to inhibit HIF-1 and COX-II in vivo to determine the effects on glioma and meningioma tumor growth. We are also investigating delivery methods that will facilitate moving this potential therapy into a clinical setting.
Figure 2: Diagram of RNAi mediated by shRNA production. First, a long dsDNA oligonucleotide is synthesized with the correct sequence (see methods for siRNA sequence selection) and cohesive ends, then inserted into a plasmid directly downstream of a RNA Pol III promoter (U6 or H1 are commonly used). The plasmid is amplified in bacteria, isolated and sequenced, then transfected into Your Favorite Cell line and stable clones are selected by antibiotic resistance (i.e., hygromycin). The U6 promoter constitutively expresses the shRNA insert, which folds into a hairpin structure and is exported from the nucleus. In the cytoplasm, Dicer recognizes the hairpin structure and processes it into a functional siRNA.