By Bruce Nash, PhD
DNA Learning Center (DNALC), Cold Spring Harbor Laboratory

In my previous article, I introduced an experiment that allows students to turn off genes using RNA interference, or RNAi. In this article, I introduce an experiment that lets students investigate the RNAi mechanism in the roundworm C. elegans. Since discovery of RNAi in 1998, research has revealed its roles in protecting our genome from viruses and transposons, and in gene regulation. RNAi is also a cutting-edge tool to rapidly assay gene function.

How RNAi works

Unlike mutation, RNAi down-regulates gene function without changing the DNA sequence. The trigger for RNAi is double-stranded RNA (dsRNA), which when introduced into a cell is recognized and processed by the RNAi machinery. The key components of this machinery are 2 proteins: Dicer and Argonaute. Dicer is an RNase III protein that recognizes dsRNA and cleaves the RNA into short pieces, called siRNAs. After cleavage by Dicer, the siRNAs bind to Argonaute, which is the catalytic component of a larger complex called the RNA-induced silencing complex, or RISC. Once bound, 1 strand of the siRNA is unwound from Argonaute. The remaining strand acts as a guide, allowing RISC to scan for complementary mRNAs. Once base-paired to a target message, Argonaute cleaves the mRNA opposite the guide RNA, inactivating the gene.

The RNAi machinery also processes the products of endogenous genes, called microRNAs (miRNAs), a type of RNA-coding gene first discovered in C. elegans. The precursors to miRNAs contain sections that fold back on themselves and base pair. Dicer recognizes these double-stranded sections and cleaves them to give rise to short pieces equivalent to siRNAs. As with siRNAs, the miRNAs bind to Argonaute, which uses 1 strand of the small RNA as a guide to find target mRNAs. In animals, most miRNAs do not match completely with the target RNAs. Instead, the miRNA and target base pair, but there are mismatches that lead to bulges between the 2 RNAs. The bulges interfere with Argonaute cleaving activity. Instead of being cleaved, the translation of mRNAs is inhibited. Although the mechanism is different, this still inhibits the function of the gene.

Using single-worm PCR to study RNAi in C. elegans

In this experiment, the mechanism of RNAi is investigated in 2 C. elegans strains with an identical “dumpy” trait—1 induced by RNAi and 1 caused by a chromosomal deletion. The experiment begins with bacterial culture of 2 strains of E. coli. One is a control strain. The second strain targets the dpy-13 gene by RNAi. Through genetic modification, this strain produces dsRNA with sequence identical to part of the dpy-13 gene. Wild-type worms on plates with control bacteria are fed the RNAi strain. dpy-13 mutant worms are also grown on control bacteria. Once the worms have grown, genomic DNA is isolated from worms on each plate. “Single-worm” PCR is used to amplify DNA from the dpy-13 locus, and the DNA from wild-type worms is compared to DNA from RNAi-treated worms and chromosomal mutants. Gel electrophoresis identifies the deletion in the mutant and shows that the dumpy gene is intact in wild-type and RNAi-treated strains. This provides evidence that RNAi does not alter the genetic code, potentially challenging students’ prior knowledge that phenotype is solely the result of genotype. Bioinformatics exercises complement the experiment, predicting the results of PCR and allowing students to investigate dpy-13.

Covering key concepts and technologies

This experiment exposes students to many key concepts and technologies, including PCR, gel electrophoresis, model organisms, and genetic modification, while they investigate the nature of this fascinating and powerful mechanism.

Author’s note:

The RNAi experiment discussed can be done with your students using one of Carolina’s Examining the RNAi Mechanism kits.