Question

Mello and Fire conducted an experiment in which they tested sense RNA (single-stranded RNA with an identical sequence to the target mRNA sequence), anti-sense RNA (single-stranded RNA that is complementary to the target mRNA sequence) and double-stranded RNA (the sense and anti-sense RNA molecules base-paired together). They found that although anti-sense RNA molecules had a small effect, the double-stranded RNA molecules had the largest silencing effect.

1. What do these data suggest about the mechanism of RNAi-induced gene silencing?

2. The researchers also showed that just a few RNA molecules could lead to extensive silencing effects. What do these data suggest about the mechanism of RNAi-induced gene silencing?

Answer 1

1) RNA interference (RNAi) is a genetic regulatory system that functions to silence the activity of specific genes. RNAi occurs naturally, through the production of nuclear-encoded pre-microRNA (pre-miRNA), and can be induced experimentally, using short segments of synthetic double-stranded RNA (dsRNA). The synthetic dsRNA employed is typically either a small hairpin RNA (shRNA) or a short interfering RNA (siRNA). In both the natural and the experimental pathways, an enzyme known as DICER is necessary for the formation of miRNA from pre-miRNA or of siRNA from shRNA. The miRNA or siRNA then binds to an enzyme-containing molecule known as RNA-induced silencing complex (RISC). The miRNA-RISC or siRNA-RISC complex binds to target, or complementary, messengerRNA (mRNA) sequences, resulting in the enzymatic cleavage of the target mRNA. The cleaved mRNA is rendered nonfunctional and hence is “silenced.”

RNAi is an RNA-dependent gene silencing process that is controlled by the RNA-induced silencing complex (RISC) and is initiated by short double-stranded RNA molecules in a cell's cytoplasm, where they interact with the catalytic RISC component argonaute.When the dsRNA is exogenous (coming from infection by a virus with an RNA genome or laboratory manipulations), the RNA is imported directly into the cytoplasm and cleaved to short fragments by Dicer.

2) The advantages of RNAi include the high efficiency of the gene knockdown, the ability to easily target the gene of interest, as well as stable and long-term silencing by expressing shRNAs. This makes for a powerful tool that has been successfully applied to answer many questions in cell biology.

RNAi is widely used by researchers to silence genes in order to learn something about their function. siRNAs can be designed to match any gene, can be manufactured cheaply, and can be readily administered to cells.