In a Perspective in Science, Peter Fraser wrote a very nice summary to our work. However, I thought it might be helpful to back up even further and introduce the problem we tackled.
Introduction: I am currently a PhD student at MIT in Health Sciences and Technology. My goal, shared by much of the biomedical community, is to understand how our bodies work on a molecular level so that we can rationally design drugs to improve human health. I work on a very early step of this process, which is to understand the molecular components of our cells and how they interact with each other – the wiring of the cell, if you will.
This wiring, it turns out, is extraordinarily complex – so complex, in fact, that we don’t even have a list of all of the wires. I study a class of molecules in the cell called large (or long) non-coding RNAs (lncRNAs, pronounced link-RNAs). LncRNAs are large (>200 RNA bases), 5′ capped, and often spliced; in many respects, they look like the messenger RNAs (mRNAs) that transmit genetic information from DNA to protein (see above). However, lncRNAs do not code for protein and are thought to play functional or structural roles in the cell as RNA molecules. Although we’ve known about a handful of lncRNAs for more than twenty years, we discovered in the years following the Human Genome Project that there are in fact thousands of different lncRNAs encoded in our genomes. Subsequent functional studies determined that many of these lncRNAs play crucial roles in the cellular circuitry, similar to protein-coding genes. In other words, we know lncRNAs are important because when you delete one, the mouse dies.
When I started my PhD, we had pinpointed thousands of new lncRNAs in our genomes and realized that many of them may be critical to human biology and medicine. The next question that we set out to tackle was: How do lncRNAs work?
Methods: To answer this question, we needed a method to examine lncRNAs in their native cellular environment. If lncRNAs are in fact controlling cellular processes, they must be interacting with other components in the cell, such as other RNA molecules, proteins, or even specific regions of a chromosome. If we could identify these interacting components, we could use this information to figure out how lncRNAs achieve their various functions. To accomplish this, we would need to purify a lncRNA from cells and identify the other cellular components that co-purified with our target lncRNA.
This experiment would be straightforward if we were trying to discover the molecular components that interact with an uncharacterized protein: we would raise an antibody to the protein of interest, and use these antibodies to bind and capture the protein from cell extracts. However, this method does not work for lncRNAs because antibodies cannot recognize them.
To solve this problem, we developed a method called RNA Antisense Purification (RAP). RAP takes advantage of the fact that we know the sequence of our target lncRNAs; we simply design other nucleic acid molecules with a complementary sequence that will hybridize to and capture our target lncRNA. Although conceptually simple, developing this protocol was very challenging! I won’t go into the details here; if you want to learn more, visit our RAP web page.
Results: As an initial foray into studying the molecular mechanisms of lncRNAs, we decided to test our method on the canonical lncRNA Xist (pronounced “exist”), which is one of the few lncRNAs that has been studied for a long time. Xist was discovered over twenty years ago because it is responsible for silencing one of the two X-chromosomes in female mammals in a process called “dosage compensation”; females silence one of their two X-chromosomes in order to balance the expression of X-chromosome genes with male mammals, who have one X-chromosome and one Y-chromosome. The gene encoding the Xist RNA is located on the X-chromosome is activated randomly on one X-chromosome early during embryonic development. The Xist RNA spreads out from its gene locus to cover the X-chromosome, turns off gene expression, and packages up the X-chromosome DNA into an inactive bundle visible under a light microscope (the “Barr Body“).
Despite this knowledge about Xist, we did not know exactly where on the X-chromosome Xist actually bound, and we did not understand how Xist spread across and accessed the entire chromosome. We used RAP to purify the Xist RNA and its associated cellular components, and we sequenced the DNA that was bound in a complex with the Xist RNA. Unexpectedly, we found that Xist bound broadly across the entire X-chromosome, which differed dramatically from the pattern of binding of a similar lncRNA (roX2) in fruit flies. We also discovered that Xist spreading takes advantage of the three-dimensional conformation of the X-chromosome; rather than binding to specific high-affinity sites across the X-chromosome, Xist simply reaches out in three dimensions from its gene locus and spreads to these nearby sites (which may not be close in linear sequence).
This last point is worth considering in more detail, because it in fact takes advantage of a special property of a lncRNA: a lncRNA can function at its site of production in the nucleus, allowing it to use its specific position in the genome to target nearby sites in three dimensions. This function is unique to lncRNAs as opposed to proteins: proteins are produced in the cytoplasm from messenger RNAs and thus have no memory of where their gene is located in the nucleus.
Whew! To summarize, we demonstrated an important ability of Xist that might apply to many other lncRNAs. We are now in the process of studying many of the hundreds of other lncRNAs encoded in our genomes to identify additional mechanisms by which lncRNAs control cellular processes. In the future we will be able to use this information to develop drugs that target specific lncRNAs or interfere with their interactions with other cellular components.
Edit: For a more whimsical summary of our paper, visit Sick Papes: http://sickpapes.tumblr.com/day/2013/07/09