For half a century, RNA has gotten second billing to DNA as biologists have probed ever deeper into the functioning of cells. RNA — which like DNA consists of chains of subunits known as nucleotides — is a vital intermediary in many cellular processes. But DNA has always seemed to run the show, both as a repository of genetic information and as a source of evolutionary innovations.
In recent years, RNA’s star has been burning brighter. Researchers have discovered that cells have previously unsuspected layers of genetic regulation orchestrated primarily by RNA. In 2006, HHMI investigator Craig Mello shared the Nobel Prize in Medicine or Physiology with Andrew Fire, a professor of pathology and genetics at the Stanford University School of Medicine, for one of the most important of these discoveries.
Mello began working with RNA in graduate school as part of his research on the developmental genetics of the tiny worm Caenorhabditis elegans. In the 1980s, researchers began using a kind of RNA known as antisense RNA to block the function of particular genes. Antisense RNA, like most of the RNA found in cells, consists of a single strand of nucleotides. The sequence of nucleotides in antisense RNA is complementary to that of the single-stranded messenger RNA generated by the targeted genes, meaning that the two molecules can bind to one another. When antisense RNA binds to messenger RNA, it blocks the passage of information from the gene to the protein-manufacturing machinery of the cell.
In 1994 — the same year Mello finished a postdoctoral fellowship at the Fred Hutchinson Cancer Research Center and set up his own laboratory at the University of Massachusetts Medical School — a graduate student at Cornell named Su Guo made a puzzling discovery. As part of an experiment using antisense RNA to block the function of a gene, she injected C. elegans with sense RNA, which should not bind with messenger RNA because it has an identical nucleotide sequence. Surprisingly, the sense RNA was as effective as antisense RNA in blocking the gene’s function.
Mello was intrigued by this odd result and began investigating it in his own laboratory. One day he injected antisense RNA into C. elegans and then became so distracted that he did not check the experiment until after the injected worms had reproduced. Amazingly, he saw that the activity of the target gene was blocked not only in the injected worms, but also in those worms’ offspring — even though there was no obvious way for the effect to pass from one generation to the next. Shortly after that, another happy accident occurred. A new graduate student, Sam Driver, was learning to microinject RNA into C. elegans and kept missing the target. But the effects of the RNA spread from one cell to neighboring cells, again with no obvious mechanism to account for the effect.
Mello often talked with Fire, who was then at the Carnegie Institution of Washington’s Department of Embryology in Baltimore and who also had been investigating the unexpected effects of antisense RNA. One day Fire made an off-the-wall suggestion. Maybe the preparations of single-stranded RNA also contained small amounts of double-stranded RNA, and the double-stranded RNA was causing the effect. The idea seemed improbable. Double-stranded RNA was expected to be relatively inert in a cell, whereas single-stranded RNA would bind to complementary strands. But when Fire isolated double-stranded RNA from the preparations of single-stranded RNA, he found that the double-stranded RNA had a much stronger silencing effect than either the antisense or sense single-stranded RNA.
Mello, Fire, and their colleagues published their results in Nature in 1998, calling the effect RNA interference (RNAi), after consulting with other worm researchers. The paper triggered an avalanche of new discoveries. Mello, Fire, and other researchers uncovered an elaborate mechanism that explains not only the original findings but many other unexplained occurrences of gene silencing in organisms ranging from petunias to humans.
Furthermore, the mechanism they discovered turned out to be part of a much larger system that regulates the expression of many genes in multicellular organisms, including humans. When double-stranded RNA is introduced into a cell, a protein known as Dicer cleaves it into double-stranded fragments 20 to 25 base pairs long. The fragments are then incorporated into a molecular structure known as RISC (for RNA-induced silencing complex), which degrades messenger RNAs that have the same nucleotide sequence as the RNA fragments, blocking the action of the gene that produced the messenger RNA. RNAi appears to act in cells as a defense mechanism against viruses that rely on double-stranded RNA to reproduce and as a way of controlling jumping genes known as transposons, which can interfere with the function of DNA.
As soon as it was discovered, RNAi was put to work as a powerful research tool. Researchers use it to silence genes one by one to determine the effects of each gene on a cell’s growth and function. The same technique can block the action of genes that have gone awry in disease. Today, RNAi is being tested for its ability to treat respiratory infections, macular degeneration, hepatitis, cancer, and many other illnesses.
Photo: Robert Carlin