“You may have to clone 15 lncRNAs before you're going to find one that has an effect that you might have been hypothesizing,” says Rinn.Homing in on function
Researchers have managed to decode the functions of a few lncRNAs—perhaps 1% of all human noncoding transcripts, Marques estimates. Some, like Xist and the newly discovered Braveheart, which are implicated in X-inactivation and cardiac development, respectively, are nuclear transcripts that, like HOTAIR, bind Polycomb-group proteins. Another class of lncRNAs, called 1/2-sbsRNAs, are cytoplasmic molecules that mediate RNA decay. And still others, like lincRNA-RoR, act as molecular sponges, soaking up and neutralizing microRNAs.
“There is no unifying umbrella like for microRNAs,” says Leonard Lipovich, a lncRNA expert at Wayne State University. In a recent review on lncRNAs, Jeannie Lee, a Howard Hughes Medical Institute Investigator at Harvard Medical School who studies X-inactivation, includes a figure illustrating some 16 distinct functions for lncRNAs (3).
“It's like you're going through the wardrobe into this crazy, mysterious land, and it's both exciting and scary at the same time,” says Rinn. “You're seeing all these mysterious creatures you've never seen before and yet, you don't know how to navigate it, you don't know what to do.”
Rinn's lab focuses on RNA mapping and functional analysis of long intergenic noncoding RNAs (lincRNAs). They use next-gen DNA sequencing to chart the former; for the latter, it's guilt-by-association: watching for coding transcripts whose abundance rises and falls with that of a specific lincRNA. A match provides evidence that the lncRNA and protein might function in the same pathway, a hypothesis that then must be tested.
The process is high-tech, but crude. “It's almost like looking at Noncodarnia thru an Atari rather than an Xbox,” says Rinn, who tends to pepper his conversation with pop-culture references. Unlike protein researchers, “We're in a low-res form of the field.”
That's in part because there are so few tools available specifically for studying lncRNA. Rinn says the one constant in his career has been the need to use what he calls the “the MacGyver approach,” creating solutions from tools that weren't necessarily designed to address the problem at hand.
In 2008, for instance, his team was having trouble seeing low-abundance lncRNAs against a background of more abundant protein-coding transcripts. So, they used DNA microarrays to enrich noncoding transcripts first, gaining three orders of magnitude in sequencing depth. More recently, his team has focused on informatics, working out methods to filter, analyze, assemble, and disseminate lncRNA data to the broader scientific community.
“What do you do when you're flying blind? You try and make a flashlight,” he says.
A pitch for staying old-school
Building flashlights is a common task in Noncodarnia. Jeannie Lee has had to do that in her lab as well, developing a method to survey the landscape of protein-complexed RNAs called RIP-seq, which she used in 2010 to identify more than 9,000 PRC2-associated transcripts.
Yet like Rinn, Lee wasn't looking to study lncRNAs when she entered the field in the early 1990s. She was studying X-inactivation as a postdoc with Rudolf Jaenisch at MIT around the time that Xist was identified.
“I remember very clearly going to a national conference at which the discovery was presented. And I was blown away by the fact that a long noncoding RNA that's coating the X chromosomes could somehow be involved.”
Once she established her own lab at Massachusetts General Hospital, Lee continued to focus on X-inactivation and very quickly identified an lncRNA antisense to Xist. Called Tsix, the newly identified molecule regulates Xist and together with other lncRNAs enables dosage compensation in XX females.
Nearly a decade later, Lee's lab found a binding site within Xist for the Polycomb complex PRC2, directing PRC2 to the soon-to-be-inactive X chromosome, an observation that ties lncRNAs to X-inactivation and provides a possible solution to an epigenetic riddle.