Emergence of chemical biology approaches to the RNAi/miRNA pathway

Abstract

RNA interference (RNAi) is a well-conserved mechanism that uses small noncoding RNAs to silence gene expression posttranscriptionally. Gene regulation by RNAi is now recognized as one of the major regulatory pathways in eukaryotic cells. Although the main components of the RNAi/miRNA pathway have been identified, the molecular mechanisms regulating the activity of the RNAi/miRNA pathway have only begun to emerge within the last couple of years. Recently, high-throughput reporter assays to monitor the activity of the RNAi/miRNA pathway have been developed and used for proof-of-concept pilot screens. Both inhibitors and activators of the RNAi/miRNA pathway have been found. Although still in its infancy, a chemical biology approach using high-throughput chemical screens should open up a new avenue for dissecting the RNAi/miRNA pathway, as well as developing novel RNAi- or miRNA-based therapeutic interventions.

Figure 1. The RNAi/miRNA pathway

Genes encoding microRNAs are initially transcribed by RNA polymerase II or III to generate the primary miRNA transcripts (pri-miRNA) within the nucleus. The stem-loop structure of the pri-miRNA is recognized and cleaved on both strands by a Microprocessor complex, which consists of the nuclear RNase III enzyme Drosha and an RNA-binding protein, DGCR8, to yield a precursor miRNA (pre-miRNA) 60–70 nucleotides in length. The pre-miRNA is then exported from the nucleus through a nuclear pore by exportin-5 in a Ran-GTP-dependent manner and processed in the cytoplasm by the RNase III Dicer-TRBP. Sliced RNA strands are further unwound. One strand of the miRNA/miRNA* or siRNA duplex (the antisense, or guide strand) is then preferentially incorporated into the RNA-induced silencing complex (RISC) and will guide the RISC to a target mRNA in a sequence-specific manner. Once directed to a target mRNA, the RISC can mediate translational regulation by inhibiting the initiation or elongation step or through destabilization of the target mRNA. The illustrated number of base pairs does not reflect the actual length of either miRNA or siRNA.

Figure 2. Development of a reporter system to monitor the activity of the RNAi pathway

Human 293 cells stably expressing EGFP (293-EGFP) were infected with lentivirus producing shRNA against EGFP (shRNA-EGFP); the resulting RNAi-293-EGFP cells with reduced GFP expression were isolated. The RNAi-293-EGFP cells transfected with 2-O-methyl RNA against the GFP siRNA are shown on the right with the recovery of GFP expression. The RNAi-293-EGFP cells could be used for a chemical screen to identify both inhibitors and enhancers of the RNAi pathway.