In a new study, researchers from the University of Chicago describe how short RNA molecules, known as Piwi-interacting RNAs (piRNAs), perform their function of finding foreign genetic sequences to silence them without targeting endogenous genes. Their findings have been reported today in Science.
Nearly every animal has piRNAs. In the current study, the researchers were studying piRNAs produced by cells in the reproductive system of the nematode worm C. elegans. They wanted to find out why C. elegans has such as large number of piRNAs—over 15,000 of them—and what purpose they serve.
Knowing that Piwis recruit a set of smaller secondary RNAs corresponding to a target sequence, the team created a synthetic piRNA with a sequence that didn’t exist in the worm to track where it created its marker. What they found is that piRNAs need a fairly close match to a portion of the sequence, but can tolerate some mismatch on the rest. They also found that the large number of unpaired Piwis in worms gives them a repertoire of many possible sequene combinations that they can use, giving flexibility in the number of foreign genes they can target.
Next, the team wanted to identify how the piRNAs avoid false positives. The researchers created piRNAs that recognize well-known nematode genes. This set of piRNAs didn’t silence or affect the function of the endogenous genes, indicating the genes were resistant. What the team found was that endogenous genes have additional repeating sections of A and T nucleotides that mark them as “self” genes.
This study explains a mechanism that is known to affect fertility in animals. Previous research has shown that if Piwi genes develop mutations, the piRNA system can fail to detect viruses or transposons that inject foreign DNA elements into the germ line. These changes can lead to infertility and other issues.
The study also helps solve an issue that has been a hurdle for scientists studying C. elegans for decades. When researchers want to study a specific gene, they often make slight modifications so a protein tag that produces fluorescent light can attach to the proteins produced by their gene of interest. While this works in many cells, the piRNA system shuts off production of those proteins in the germ line. The team was able to edit those sites just enough so the piRNAs can’t find them anymore, but the genes will still function.
"When I first presented this at a conference, after my talk I was surrounded by like 30 scientists asking for help, so they can use our algorithms to study their own favorite targets in germ cells," said Heng-Chi Lee, PhD, assistant professor of molecular genetics and cell biology at UChicago and senior author of the new study. "It solves a very practical hurdle that has haunted C. elegans biologists for the last 20 years."
Image: This is a picture of germ line cells of C. elegans showing PIWI and piRNAs (red dots) located outside of germ cell nuclei (blue circles) to scan for RNA passengers that move from nuclei to cytoplasm. Image courtesy of Jordan Brown, UChicago.