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function. “We’re calling long noncoding RNAs a class, when actually the only definition is that they are longer than 200 bp,” says Ana Marques, a Research Fellow at the University of Oxford who uses evolutionary approaches to under- stand lncRNA function. As a conse- quence, working out the function of novel lncRNAs is very difficult. “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 illus- trating 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-asso- ciation: 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 conver-

Vol. 54 | No. 6 | 2013

sation 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.

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-in- active X chromosome, an observation that ties lncRNAs to X-inactivation and provides a possible solution to an epigenetic riddle. Epigenetic regulation is locus-spe-

cific— some genes are turned on while others are turned off. Yet epigenetic factors show little if any sequence-spec- ificity. Lee suggests that lncRNAs can bridge that gap, because they can fold up to bind proteins but are also inher- ently 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 overf lowing with lncRNA sequence data they can search at the click of a mouse. But when Lee started her lab in 1997, there was practi- cally 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 genet ics, making

old-fa shioned John Rinn, aka @noncodarnia. 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.”


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-inacti- vation, 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 impor- tance 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.

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