RNA Interference (RNAi)

Introduction

RNA interference (RNAi) or Post-Transcriptional Gene Silencing (PTGS) is a conserved biological response to double-stranded RNA that mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein-coding genes. This natural mechanism for sequence-specific gene silencing promises to revolutionize experimental biology and may have important practical applications in functional genomics, therapeutic intervention, agriculture and other areas.

Endogenous triggers of RNAi pathway

RNAi triggers

Endogenous triggers of RNAi pathway include foreign DNA or double-stranded RNA (dsRNA) of viral origin, aberrant transcripts from repetitive sequences in the genome such as transposons, and pre-microRNA (miRNA). In plants, RNAi forms the basis of virus-induced gene silencing (VIGS), suggesting an important role in pathogen resistance. A possible mechanism underlying the regulation of endogenous genes by the RNAi machinery was suggested from studies of C. elegans. In mammalian cells long (>30nt) double-stranded RNAs usually cause Interferon response.

A simplified model for the RNAi pathway

RNAi pathways

A simplified model for the RNAi pathway is based on two steps, each involving ribonuclease enzyme. In the first step, the trigger RNA (either dsRNA or miRNA primary transcript) is processed into an short, interfering RNA (siRNA) by the RNase II enzymes Dicer and Drosha. In the second step, siRNAs are loaded into the effector complex RNA-induced silencing complex (RISC). The siRNA is unwound during RISC assembly and the single-stranded RNA hybridizes with mRNA target. Gene silencing is a result of nucleolytic degradation of the targeted mRNA by the RNase H enzyme Argonaute (Slicer). If the siRNA/mRNA duplex contains mismatches the mRNA is not cleaved. Rather, gene silencing is a result of translational inhibition.

RNAi in experiments and therapeutics: how it works

RNAi pathways

RNAi can be triggered experimentally by exogenous introduction of dsRNA or constructs which express shRNAs.

The high degrees of efficiency and specificity are the main advantages of RNAi.

Consequently, RNAi is used in functional genomics (systematic analysis of loss-of-function phenotypes induced by RNAi triggers) and developing therapies for the treatment of viral infection, dominant disorders, neurological disorders, and many types of cancers (in vivo inactivation of gene products linked to human disease progression and pathology).