Biological dark matter: newfound RNA suggests a hidden complexity inside cells.

It started with worms that just would not grow up. In the early 1990s, Victor Ambros and his colleagues were conducting a gene hunt. In particular, they were searching for the gene that was mutated in a perplexing strain of Caenorhabditis elegans, the small nematode whose development many biologists study.

This genetic change Ambros hunted had apparently disrupted the worms' developmental timing.

In normal strains, worms pass through four larval stages as they mature into fertile adults. But members of the mutant strain get stuck at the first stage. They would molt, but instead of moving on to the second larval stage, they simply repeated the first stage. The larvae kept growing larger but never became full-fledged adults.

Ambros' team painstakingly homed in on the gene responsible by adding pieces of DNA from normal C. elegans back into the mutant worms. If a DNA sequence restored full development, it presumably harbored a working copy of the gene that's defective in the mutants, reasoned the investigators. In 1993 at Dartmouth Medical School in Hanover, N.H., the hard work of Ambros and his colleagues paid off with the elusive gene's discovery.

It was a "heroic detective story," says Sean Eddy of the Howard Hughes Medical Institute at Washington University in St. Louis.

The story had a surprise ending, too. Unlike most genes, the one identified by Ambros' group doesn't encode a protein. It spawns a small molecule of RNA--a chemical relative of DNA--that somehow turns off other genes that play a role in worm development.

This odd finding stood alone until a few years ago, when a team led by Gary Ruvkun of the Massachusetts General Hospital in Boston found a gene that controls C. elegans' transition from the fourth larval stage to adulthood. This gene also creates RNA that regulates the expression of worm genes.

Although Ambros hadn't found genes in other organisms similar to the one he'd identified in C. elegans, Ruvkun and his colleagues discovered that many animals have versions of this second RNA-encoding worm gene. His team found such genes in flies, mollusks, fish, and even people. The researchers speculated that the RNA produced by the gene is a universal regulator of animal development, perhaps an important controller of a caterpillar's metamorphosis into a butterfly and a tadpole's into a frog.

Inspired by such research, biologists have now begun to systematically look for so-called RNA genes." DNA whose final product is RNA instead of protein. Several groups, including one led by Eddy, recently surveyed the DNA of the bacterium Escherichia coli and uncovered dozens of such genes. Just a few months ago, Ambros' team and two other research groups reported that worms, flies, and people contain dozens of previously undetected genes that spawn RNA instead of protein.

These investigators argue that the many intensive searches for protein-coding genes have ignored or missed genes for small, stable RNA molecules that have cellular functions. The RNA genes found so far are "just the tip of a huge iceberg," says Ruvkun.

The biologist goes as far as to compare the RNA-gene findings to a humbling discovery on a much larger scale. Astronomers studying the effects of gravity on galaxies found to their astonishment that the universe contains large quantities of so-called dark matter, mass that still eludes observation. In the Oct. 26, 2001 SCIENCE, Ruvkun speculates that "the number of genes in the tiny RNA world may turn out to be very large, numbering in the hundreds or even thousands in each genome. Tiny RNA genes may be the biological equivalent of dark matter--all around us but almost escaping detection."

RNA genes have already attracted commercial interest: A biotech firm is testing whether some of the newfound bacterial RNAs play a role during infection and might therefore be targets for new antibiotics. If that's not provocative enough, some scientists suggest that RNA regulation of gene activity and other cellular processes could explain the diversity and complexity of plants and animals as compared with bacteria.

RNA has long stood in the shadow of DNA. Both chemicals consist of molecules called nucleotides. In DNA, two strands of nucleotides pair up to form the double-helix structure discovered by biologists James Watson and Francis Crick. In contrast, RNA usually consists of a single strand of nucleotides, although that strand can sometimes fold back on itself and create double-stranded regions.

The "central dogma of genetics," a phrase coined by Crick, argues that information in a cell flows from DNA to RNA to protein. A cell reads the information encoded in a gene's DNA and makes a strand of RNA. This messenger RNA, or mRNA, travels through a cell to sites of protein synthesis called ribosomes. These microscopic factories then read the mRNA to determine what amino acids to string together into a protein.

Yet biologists have long known that RNA does more in a cell than convey protein recipes. For example, RNA strands are important parts of those protein-making ribosomes (SN: 8/12/00, p. 100). In fact, some researchers speculate that life began solely with RNA molecules, an idea known as the RNA-world theory (SN: 4/7/01, p. 212).

Unlike the RNA genes recently identified by Amros and his colleagues, the genes for the RNA in ribosomes were discovered several decades ago. After all, a cell produces 10 millions copies of every ribosomal RNA. Moreover, each of these strands is at least 13,000 nucleotides long--large enough for relatively straightforward detection in the laboratory.

To unearth much smaller RNAs, such as the 22-nucleotide C. elegans strand that Ambros initially identified, biologists have had to develop new search methods. To pick out traditional genes, scientists had developed computer programs that scan DNA sequences for distinctive protein-coding sequences. Those programs, however, are ineffective at finding genes for RNAs.

"Everything is biased towards proteins," says Stephen R. Holbrook of Lawrence Berkeley National Laboratory in California.

He and his colleagues are trying to fix that. They recently tested a computer program that they call RNAGENiE on the genome of E. coli. Armed with knowledge about most of the bacterium's known RNA genes and rules regarding RNA structure, the program spotted other previously recognized RNA genes, Holbrook's team reports in the Oct. 1, 2001 NUCLEIC ACIDS RESEARCH. RNAGENiE also identified several hundred potential RNA genes that researchers knew nothing about.

These genes "are an undiscovered kingdom that's slowly revealing itself," says Holbrook. His team plans to further refine RNAGENiE so that it can inspect the more complex genomes of yeast, plants, and animals.

Several research groups have scanned the E. coli genome using other methods. One of the most powerful is known as comparative genomics. Its success rests on the idea that evolution preserves what's important. In other words, if two or more species share an identical stretch of DNA, it probably does something important. Otherwise, over time, mutations would scramble the sequences in each species.

Biologists have used this principle to identify possible protein-coding genes, but it also works for RNA genes. A team headed by Gisela Storz and Susan Gottesman of the National Institutes of Health in Bethesda, Md., recently demonstrated that approach in E. coli. By comparing several of the bacterium's intergenic regions--parts of the genome empty of protein-coding genes--with those of some closely related bacteria, the researchers identified 59 potential RNA genes. They then verified that 17 of the genes in the bacteria produce RNA strands ranging in length from 45 to 320 nucleotides.

Like several of E. coli's known RNAs that regulate gene activity, many of the new RNAs bind to a protein called Hfq, the researchers reported in the July 1, 2001, GENES AND DEVELOPMENT. To Gottesman, that's evidence that the newfound RNAs also influence gene activity in the bacterium.

"There's a level of RNA regulation that we didn't realize was there," she says. "It was just invisible."

In the Sept. 4, 2001 CURRENT BIOLOGY, Eddy and his colleagues described a similar comparative-genome scan. Matching E. coli's DNA against that of four other bacteria, the researchers identified 275 potential RNA genes. To test the predictions, the biologists followed up on 49 of the candidate genes and determined that at least 11 of them produced RNAs of unknown function.

From the overlap seen in these and various other groups' results, Eddy estimates that E. coli has 50 to 200 RNA genes. Its protein-coding genes number about 4,000, he notes.

If biologists are going to exploit the newfound RNAs as targets for novel antibiotics, they need to figure out the function of each one. That assignment interests Ibis Therapeutics of Carlsbad, Calif. Funded in part by the Department of Defense, which is looking for new ways to combat biological warfare, this biotech firm develops small molecules that can dock inside RNA molecules and interfere with their function.

The newly discovered bacterial RNAs represent potential targets for Ibis' drugs, says David Ecker, the company's president. Ibis has begun to create bacteria with mutations in their RNA genes and examine whether the mutant microbes infect mice as effectively as the unaltered germs. If a gene mutation reduces a bacterium's capability to produce illness, the gene's RNA product could provide a good target for a drug, explains Ecker.

The hunt for new RNA genes also goes on beyond the world of microbes. According to a preliminary analysis by Eddy's team, biologists should soon be able to expose most human RNA genes by comparing the human genome to the mouse genome.

Several research groups have already turned up one new family of RNA genes in flies, worms, and people. In the Oct. 26, 2001 SCIENCE, three teams describe dozens of RNA genes similar to the two initially identified in C. elegans. Researchers have dubbed the RNAs produced by these genes as microRNAs.

Thomas Tuschl of the Max Planck Institute for Biophysical Chemistry in Gottingen, Germany, and his colleagues unearthed new genes by sifting through all the RNA produced in fruit fly cells and human-cancer cells. They developed techniques to pick out RNAs about 24 nucleotides long, ones that normally would get discarded in experiments because of their small size. Tuschl's team identified 16 novel microRNAs in fruit fly embryos and 21 in human cancer cells.

Working with C. elegans, David P. Bartel of the Whitehead Institute for Biomedical Research in Cambridge, Mass., and his colleagues followed a similar strategy. They sorted through the worm's RNA for molecules 21 to 25 nucleotides in size and identified 55 microRNA genes. Many of these, the researchers found, vary in activity during the worm's development.

Ambros and his Dartmouth colleague Rosalind C. Lee also found microRNA genes by examining novel small RNAs made by C. elegans. Moreover, they compared the worm's genome to that of a closely related nematode. All told, the two researchers discovered 15 new genes encoding microRNAs. At least 10 of those vary in abundance during larval development, suggesting that they too may regulate the timing of development.

All three groups discovered that mammals, insects, and worms share some of the same RNA genes. One intriguing gene is active in human-heart tissue and in the developing mouse embryo.

Bartel suspects that there may be as many as 200 microRNA genes in C. elegans, which would represent about 1 percent of its genes. He also points out that there may be many other classes of RNA genes that investigators have yet to uncover.

How important are all these newfound RNAs and their genes to human development and health? That won't be clear until scientists reveal the functions of the RNA. Eddy speculates that scientists may have searched in vain for genes causing some diseases because they considered only protein-coding genes when an overlooked small RNA gene is the culprit.

RNA genes may be even more important than Eddy suggests, according to John Mattick of the University of Queensland in Brisbane, Australia. In a radical theory developed with University of Queensland physicist Michael J. Gagen, the geneticist proposes that small RNAs account for the diversity and complexity of eukaryotes--the animals, plants, and other organisms whose cells keep their DNA in a pouch known as the nucleus.

Mattick notes that biologists have been surprised to find that the number of protein-coding genes in an organism doesn't seem to reflect its complexity. Worms and flies, for example, have roughly the same number of such genes, which is only about twice the number counted in yeast and some bacteria. Moreover, people may have only twice as many protein-coding genes as flies and insects do and the same number that some fish have.

Perhaps the complexity of higher organisms lies in RNAs, not proteins, Mattick and Gagen speculate. They note that in a traditional gene, not all the DNA encodes the protein. When a cell reads a gene's DNA sequence to create messenger RNA, it initially creates a longer-than-needed strand of RNA. To then finalize the mature messenger RNA, enzymes cut out segments.

Mattick contends that these excised pieces of RNA, as well as the other RNAs formed by the genes turning up in current studies, form a vast molecular network that regulates a cell's overall activity. According to his calculations, about 98 percent of the RNA produced in a eukaryotic cell don't encode a protein.

"This will be the big story in genomics over the next few years," Mattick predicts. "You would have to be blind not to see that noncoding RNAs are a vastly unexplored world."