Epigenetic regulation is locus-specific— some genes are turned on while others are turned off. Yet epigenetic factors show little if any sequence-specificity. Lee suggests that lncRNAs can bridge that gap, because they can fold up to bind proteins but are also inherently sequence-targeted.
“When you're studying epigenetics and you need locus specificity,” she says, “you have to look to long noncoding RNAs.”
Today's researchers have RNA-seq and RNAi to tackle that job, not to mention databases overflowing with lncRNA sequence data they can search at the click of a mouse. But when Lee started her lab in 1997, there was practically nothing upon which to build. “Imagine to yourself, you've set out on a career to figure out how X-inactivation works, and there's only one molecule,” she says, laughing.
Lee had to make do with good old-fashioned genetics, making knockouts in embryonic stem cells and looking for changes in phenotype. As a postdoc, she used random deletions to identify a 450 kb sequence of the X-inactivation center that is both necessary and sufficient for X-inactivation, and it was in this sequence that her lab found Tsix.
Today, a popular choice for querying lncRNA function is genetic knockdown, using either siRNAs or shRNAs. Lee's lab uses these approaches, too, but also remains true to the technique that served her so well in the past. “I want to make a pitch here for the importance of making traditional knockouts to demonstrate function,” she says. “A lot of these knockdown approaches to lncRNA biology yield phenotypes that do not ultimately agree with the knockout,” though both approaches have their place in functional studies, she emphasizes.
To get a sense of the difficulties researchers face in Noncodarnia, look no further than Case Western University biochemist Saba Valadkhan.
When Valadkhan started her lncRNA project in 2006, there were no deep sequencing or large-scale transcriptomic data available. “We had to rely on a very small number of accidentally discovered long noncoding RNAs to choose one for our studies.”
Not wanting to step on anybody's toes, Valadkhan searched the literature for interesting but “abandoned” lncRNAs. She settled on a bone morphogenic protein-induced transcript first described in 1998.
But working with that transcript, she says, has been a challenge. “We really didn't even know what to worry about and what would be okay.” For instance, when cloning the transcript, what would happen if extra sequences were tacked onto the 5′ or 3′ ends? “They can potentially change the secondary structure of the RNA in these regions... or they can be binding sites for proteins that shouldn't be bound to this RNA.” Knockdowns weren't obvious, either. Where, for instance, should she target the interfering RNAs?
Function was a problem, as was the dearth of knowledgeable colleagues. And so, when the data started rolling in, Valadkhan's team wasn't sure how to deal with it. They didn't know, for instance, how much of a phenotypic response was real as opposed to noise. The only thing they knew for sure was that their RNA seemed to be doing something amazing.
When overexpressed in myoblasts, the transcript caused the cells to “change shape strongly and [start] to grow little processes.” The team had no idea what that meant, but the postdoc doing this work thought the cells kind of looked like neurons. “Lo and behold they were screaming with neuronal marker expression,” Valadkhan says.
This was in 2007, she says, shortly after Shinya Yamanaka introduced the world to induced pluripotent stem cells. Nobody doubts the power of transcription factors to alter cell fate, yet Valadkhan's discovery that noncoding RNAs evidently do the same, remains unpublished.
“The reviewers hated it,” she says. “They said this must be an artifact.”
Reviewers asked for control after control. They demanded knockout mice. They even questioned Valadkhan, whose background is in “hardcore RNA chemistry,” on her ability to culture cells.
Six years later the paper is once again under review. “Again they have asked for quite a number of things,” she says, “but at least the tone of the reviewers has become more and more positive with every submission. I think it's partly because we are adding a lot more controls and data,” she allows. “But partly also because people are appreciating that these RNAs can really do these kinds of things.”
For too long, Mattick says, the broad molecular biology community has acted as if it had the conceptual framework of cellular genetic regulation more or less figured out, thanks to promoters, enhancers, and transcription factors. “They decided they understood the system 50 years ago, after just a decade or so of initial work, and haven't really changed their minds since.”
That view seems finally to be changing, Mattick says.
It looks to be a fine day in Noncodarnia.