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“You get this transfer of silencing from one copy of the gene to another,” says Mello. “And this was permanent, very stable silencing.” Even in future generations, they found, GFP was always turned off.
However, this is only half of the story, Mello says. Equally remarkable was the observation that, in another line of worms, active versions of the engineered genes had become resistant to the transfer of silencing over time and instead activated the silent genes. These observations suggest that in some cell lines, silencing trumps, while in other cases, the on-switch prevails. Importantly, the researchers found, once an on-or-off decision was made, it held true for every descendant of that animal for generations. “The animal is actually remembering which genes are supposed to be on and which genes it wants off,” says Mello.
The connection with piRNA came when Mello’s team repeated the experiments on cells that lacked Piwi, the protein that binds to piRNA molecules, and found that genes that had been silenced in other iterations of the tests were now always turned on. Through a series of experiments, the scientists provided evidence that piRNAs are forever scanning every bit of free RNA in germ cells. They also showed that molecular memories were maintained through two groups of molecules: one that signals activation, another silencing. Both rely on different Argonaute proteins to establish the memories.
When a piRNA successfully binds to RNA, the attached Piwi protein kicks into action: If the RNA sequence has not been seen before, the Piwi turns on a molecular “non-self” pathway that enforces silencing. If the RNA sequence has been seen before, it is recognized by the “self” Argonaute pathway, and Piwi allows the cell to express the gene. The findings were published in two papers in the July 6, 2012, issue of Cell.
While an animal’s immune system is built to recognize foreign particles or cells, the piRNA system offers a second level of protection at the genetic level. If a virus, for example, gets past a worm’s immune system, it can insert its genes into the worm’s genome. The piRNA system can ensure that those viral genes remain turned off—it’s a second line of defense. “Instead of recognizing a structural feature as foreign, the animal is looking at the sequence information itself,” says Mello.
But the system—which Mello has dubbed RNAe, for RNA-induced epigenetic silencing—could also be a way that organisms generate heritable diversity that can be acted on by natural selection. Epigenetic silencing is any form of inherited genetic regulation that allows two organisms with the same set of genes to express those genes differently. Most previously known epigenetic mechanisms are based on protein modifications or chemical tags on chromatin or DNA, but RNAe is based on an inherited RNA signal and is the first epigenetic mechanism discovered to scan new genes by comparing them with a memory of self RNA expression.
“The bottom line,” Mello says, “is that cells appear to have a previously unappreciated level of information technology sophistication, including both an actual memory of every gene that’s been expressed and a constant surveying of information to keep track of what’s new.”