From Science News

Here's How RNA Interference Works. Issue: Oct, 2000

Using genetically altered strains of the roundworm C. elegans, scientists have revealed some of the genetic components responsible for a cellular process called RNA interference (RNAi) in which double-stranded RNA triggers the degradation of a homologous messenger RNA. Some of the same genes appear to be involved in both RNAi and nonsense-mediated decay, a protective mechanism that may be used by cells to proofread newly created messenger RNA (mRNA) and to prevent the production of defective protein molecules.

In an article published in the September 15, 2000, issue of Science, Howard Hughes Medical Institute investigator Brenda Bass, geneticist Susan Mango, and their colleagues at the University of Utah report that three of seven genes of the smg gene family are involved in both nonsense-mediated decay and RNAi.

Molecular biologists have used double-stranded RNAi as a tool to degrade mRNA in cells to shut down the effects of specific genes in C. elegans, Drosophila, and many other cell-types. Since nonsense-mediated decay also involves mRNA degradation, Bass and Mango decided to explore whether RNAi requires the smg genes, which were known to be involved in nonsense-mediated decay.

Bass and Mango injected smg-deficient strains of C. elegans and a wild-type strain with double-stranded RNA designed to interfere with production of myosin, a protein that is critical to muscle development. Thus, the scientists could quantify the effectiveness of RNAi by measuring paralysis in the worms using a crawl assay - a test of the worms' ability to move across a laboratory dish.

"In wild-type worms, we found that the progeny treated with RNAi were paralyzed from day one and remained paralyzed," says Bass. "So, the RNAi technique completely interfered with their muscle development."

The smg-deficient worms were likewise paralyzed on day one, but began recovering by day two. Nonsense-mediated decay is not necessarily required for RNAi because the worms are paralyzed on day one. However, the experiments showed that smg-2, smg-5, and smg-6 were required for the persistence of the RNAi effect.

The levels of mRNA for the myosin-related gene were monitored as the smg-mutant animals proceeded through the paralysis induced by RNAi and subsequent recovery. These levels were very low in the completely paralyzed worms. As the worms recovered, the mRNA levels rebounded.

"Nonsense-mediated decay is thought to involve some sort of sensing or scanning of the mRNA to make sure it's okay in terms of its sequence or perhaps its assembly into a three-dimensional RNA- protein complex," explains Bass. "Stop codons in the wrong place are a signal that the mRNA is not okay and should be degraded. It's intriguing to speculate that maybe the interaction of an mRNA with a complementary sequence can feed into this pathway and also provide a signal for degradation."