Now Showing: RNA Activation
RNA is supposed to silence genes, not boost gene expression. So why are scientists seeing just that?
fter getting the data back from the very first experiment at her new job, Rosalyn Ram, a lab technician at the University of Texas Southwestern Medical Center at Dallas, was convinced she had messed something up. The results were decidedly "weird," she recalls. Her lab heads, the husband-and-wife research duo David Corey and Bethany Janowski, had already shown that synthetic DNA molecules with protein-like backbones, known as peptide nucleic acids, could block gene transcription. And as a long shot, in October 2004 they had tasked the new lab tech with trying to do the same with small RNA molecules, fully expecting it not to work.
But it did work: Like the peptide nucleic acids, the RNAs targeted to the same promoter also silenced gene expression at the level of transcription. "When [Ram] saw the silencing, she thought she had done something wrong," says Janowski. "She didn't want to show me the data because she thought it was supposed to be a negative result."
The data just didn't make sense: Single-stranded peptide nucleic acids bind directly to unwound DNA at the transcription start site, and double-stranded RNAs were thought only to target messenger RNA (mRNA) to prevent translation—a well characterized process known as RNA interference, or RNAi. So how could they both be causing the same effect? With this one finding, "all those things that you thought you could predict just flew out the window," Janowski says.
"It took months before we convinced ourselves that the results were real," says Corey. But eventually they did. They tested several different double-stranded RNAs—each 21 nucleotides long, just like standard, small interfering RNAs used in RNAi. All of these RNAs perfectly matched regions of the DNA promoter but had little to no overlap with the gene's mRNA, to ensure the RNAs were acting on transcription, not translation. Ultimately, in September 2005 Corey and Janowski showed that introduced RNAs could inhibit transcription by as much as 90%.1 Interestingly, the findings confirmed work by Kevin Morris, now at the Scripps Research Institute in La Jolla, Calif., who had published the first evidence of this phenomenon in human cells a year earlier.
But Corey and Janowski also noticed something in their data that, if correct, would be even more unbelievable than what Ram saw on her first day at the bench. A few of Corey and Janowski's seemingly "inactive" RNAs that did not reduce gene expression reproducibly enhanced transcription by around 25% to 50%. Such relatively small changes weren't enough to say definitively that the researchers had observed gene activation, "but it planted the seeds in our minds that maybe activation could be occurring," says Corey. To investigate the trend further, the researchers switched to a cell line with much lower background activity levels of their gene of interest, the human progesterone receptor, and the data became glaringly obvious: The same RNAs that did not inhibit transcription could now trigger as much as 10- to 20-fold increases in gene expression. The activation effect was real.