RNAi mechanisms and applications

Abstract

Within the past two decades we have become increasingly aware of the roles that RNAs play in regulation of gene expression. The RNA world was given a booster shot with the discovery of RNA interference (RNAi), a compendium of mechanisms involving small RNAs (less than 30 bases long) that regulate the expression of genes in a variety of eukaryotic organisms. Rapid progress in our understanding of RNAi-based mechanisms has led to applications of this powerful process in studies of gene function as well as in therapeutic applications for the treatment of disease. RNAi-based therapies involve two-dimensional drug designs using only identification of good Watson-Crick base pairing between the RNAi guide strand and the target, thereby resulting in rapid design and testing of RNAi triggers. To date there are several clinical trials using RNAi, and we should expect the list of new applications to grow at a phenomenal rate. This article summarizes our current knowledge about the mechanisms and applications of RNAi.

Figure 1. Cellular pathways of gene silencing by RNA interference

RNAi is multifaceted, and there are various pathways in which small double-stranded RNA (dsRNAs) regulate gene expression. The endogenous micro-RNA (miRNA) pathway begins with Pol II-transcribed primary miRNAs that are processed in the nucleus to pre-miRNAs, exported to the cytoplasm, and processed again into functional miRNAs. The primary function of miRNAs is to inhibit translation via incomplete Watson-Crick base pairing to the 3′ untranslated regions of targeted mRNAs. Alternatively, perfectly duplexed small interfering RNAs (siRNAs) can be produced intracellularly or supplied exogenously to cells. The guide strand is incorporated into the RNA-induced silencing complex (RISC), where it guides sequence-specific degradation of the target transcript, irrespective of where the base pairing occurs. The miRNA and siRNA pathways are interchangeable, and the important determinants are the positions within the message and the extent of base pairing with the targeted transcripts. siRNAs can also trigger transcriptional gene silencing via interactions with chromatin, wherein they guide histone and DNA methylation leading to inactive chromatin.