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Robert Horvitz has shown in worms that microRNAs work in families; knocking out just one has little, if any, effect.
To find out what microRNAs do biologically, scientists have been knocking out the known microRNA genes in worms. In December 2007, Horvitz, Ambros, Bartel, and their colleagues reported in PLoS Genetics on a new collection of engineered worms in which 95 of the 115 microRNAs that had been clearly identified in C. elegans were knocked out.
“To our surprise, the worms in general seemed perfectly okay,” Horvitz says. The researchers looked for worms with abnormal behavior or anatomy but, apart from those with deletions in lin-4, let-7, or lsy-6 (identified earlier by Oliver Hobert, an HHMI investigator at Columbia University), they could find only one—a worm that was constipated. This worm's “abnormally long defecation cycle” was caused by the deletion of two microRNA genes, miR-240 and miR-786, both of which are conserved in other species.
“The microRNAs are in families,” Horvitz explains. “If you knock out the entire family, defects can appear,” but knock out just one microRNA and in most cases the worms look normal. To see a change, he says, it takes not just single or even double knockouts, but at least triple, and perhaps six or seven knockouts in a single animal. “We can do this in the worm, but imagine how difficult it would be to do it in a mouse!”
It takes much longer—about a year, as opposed to a week—and costs a great deal more to produce a mouse with a specific gene deletion. Nevertheless, the Sanger Institute in Cambridge, England, is creating a library of knockouts of each of the 500 microRNAs identified in the mouse genome so far. This resource will serve as a counterpart to the National Institutes of Health Knockout Mouse Project for protein-encoding genes. Eventually, stem cell lines of each mouse in each library will be available to scientists who want to use these rodents as models for human diseases.
Meanwhile two studies—one led by Matthias Selbach and Nikolaus Rajewsky of the Max Delbrück Center for Molecular Medicine in Berlin, Germany, and the other by Bartel and Steven Gygi of Harvard Medical School—have measured how proteins change after a cell encounters a specific microRNA. Using a new technique called SILAC (an updated version of mass spectrometry), they examined several thousand proteins and concluded, in the September 4, 2008, issue of Nature, that a single microRNA can repress the production of hundreds of proteins—but for each protein this repression is relatively mild.
Yet even small changes in protein expression can make a huge difference, says Phillip Zamore, an HHMI investigator at the University of Massachusetts Medical School. Zamore is comparing highly selective microRNAs with those that control many genes at once. He points out that animals in which researchers have knocked out a gene—such as the fruit flies he studies in his own lab—are well fed and live in temperature-controlled environments. But what if a fly that is less than perfect is put in a normal environment? “In real life, flies experience noonday sun and cold nights. And their proteins are distributed in gradients that change with temperature,” he says. “Something may look like a small defect in the lab, but you know what would happen in the wild? The fly would die.”
Photo: Paul Fetters