Jidong Liu tackles the many unanswered questions of RNAi.

Text and Interview by Ruth Williams

The development of RNA interference (RNAi) as a tool for knocking down genes has revolutionized the study of gene/protein function for biologists. With relative ease, researchers can knock down their gene of interest by introducing double-stranded RNA into their cell system. Indeed that's precisely how Liu first came across RNAi. But Liu then became hooked on the question of how it works.

When dsRNA is introduced into cells, it is first chopped into shorter, 21–26-nucleotide pieces called siRNA, which then get incorporated into an RNA-induced silencing complex (RISC). With the siRNAs on board, the RISC complex can target, cleave, and degrade particular mRNAs. Liu and collaborators discovered the RISC component responsible for the cleavage step (1, 2).

Liu has also made headway in determining the mechanism of endogenous RNAi, in particular the role of processing bodies (P-bodies)—cytoplasmic depots for many RNAi factors (3, 4).

Liu recently moved to the Sloan Kettering Institute in New York to set up his own laboratory. He is in the first flurry of grant writing and staff hiring, but he nonetheless found time to chat about his P-body passion.

When did your first interest in science develop?
 My father is a chemist, and when I was a kid he would bring me to his lab. I spent a lot of time there. Then, when I was in high school, I became increasingly interested in chemistry and biochemistry. I decided to go to college with biochemistry as a major.

Where was college for you?
 I went to Nankai University in Tianjin, which is my hometown and one of the largest cities in the northeastern part of China.

And then you moved to the states?
 Yes, back in '95. I did my Ph.D. studies in biochemistry and biophysics with Dr. Yue Xiong at UNC Chapel Hill.

What made you leave China?
 At that time in China, most of the universities didn't have enough resources to carry out cutting-edge biomedical research. Like lots of my classmates, I applied to graduate programs in the U.S. to get further education.

Your Ph.D. studies were on ubiquitin-mediated degradation. So how did you become interested in RNAi?
 For one of my last experiments as a graduate student, I used RNAi to knock down my favorite gene and study its function. It helped me graduate!

At that time I realized how powerful the technique was. But people didn't know the mechanism involved. That got me thinking.

You graduated in 2002 and moved to Cold Spring Harbor. What was the main premise of your postdoctoral project?
 Back then, people knew that siRNAs could lead to the degradation of the target mRNAs, but they didn't know how. I joined Greg Hannon's lab at CSH as a postdoctoral fellow to study the mechanisms of RNAi and took advantage of my previous training in studying protein function.

The first approach was to look for proteins that directly interact with the siRNA. We found that one of the RISC components, Argonaute, binds directly to the siRNA. Then, later on, we found out that Argonaute itself functions as the slicer enzyme that cleaves the target mRNA.

Once cleaved, the mRNA is defective and will be degraded. That's how siRNA leads to the degradation of its targets.

That led to a publication in Science very early in your career. Would you say that was the highlight of your career so far?
 The Science paper was obviously a quite exciting and important finding, but on the other hand, we were really expecting to find a new enzyme that cleaved the targets. Argonaute had been known for several years; we just didn't know its exact function. Obviously, you always hope to find a novel protein so you'll have more to work on. But instead it was kind of the end of that chapter. So in a way, it was a little disappointing. You shouldn't write that…[laughs].

What else did you work on as a postdoc?
 We wanted to find the endogenous/physiological function of the RNAi machinery. The first endogenous small RNA (currently known as miRNA) was discovered back in 1993 in C. elegans. However, the real significance of the discovery was not realized until 2000/2001 when several groups reported the presence of large numbers of miRNAs in both invertebrates and vertebrates. Such conservation suggested they were important.

Sequence specificity of RNA silencing is based on base pairing between the small RNA and the potential targets. In the case of siRNAs, the targets are perfectly matched. But for the miRNA, you don't necessarily have perfectly matched targets.

If the targets are not perfectly matched, they cannot go through the cleavage and degradation steps. However, those targets can still be silenced by the miRNA. So the question was, How does miRNA silence gene expression?

 It's still the biggest unanswered question. It's related to siRNA-mediated silencing. They both use the same core protein components: the Argonaute proteins and the RNA-induced silencing complex (RISC), but the mechanisms are different.

What are the theories?
 Some studies have shown that miRNA can lead to mRNA degradation just like siRNA does, although it doesn't cleave its targets. It's possible that miRNA and Argonaute could lead to deadenylation of the target mRNAs, which would then lead to degradation of the mRNA. Other studies have shown that there's no degradation at all but that miRNA instead represses mRNA translation. Almost all the steps of translation including initiation, elongation, and termination have been suggested as the mechanism of miRNA-mediated silencing.

We showed miRNA could lead to the sequestration of target mRNAs into cytoplasmic bodies, and this prevented the mRNAs from being translated. Other studies suggested sequestration is not required, but that binding of Agonaute/RISC to the mRNAs is sufficient to silence them. Everyone seems to agree that miRNA will silence gene expression, but the exact mechanism is still highly debated.

These cytoplasmic bodies you mentioned are P-bodies? 
 Yes. We decided to look at where the protein components that carry out the silencing localize in the cell. We found, back in 2003, that argonaute proteins localized into very specific foci in the cytoplasm. At that stage we didn't know what the foci were. It was just a very interesting observation.


P-bodies are thought to be the sites of silencing, as they contain the RISC factor Argonaute (green).

People first observed similar foci back in '97. They found that a nuclease that degrades mRNA localizes into specific foci in the cell. But again, at that time, nothing was known in terms of function. Later, Roy Parker's group showed that mRNA decapping and degradation occurs in these P-bodies.

The formation of P-bodies is RNA dependent. So, it's a complicated situation: you need mRNA to form those foci, but if the foci's purpose is to just degrade mRNA, then if they do a good job, you shouldn't see them, right?

So when you see them, either they are not efficiently degrading those mRNAs so you see the accumulation, or these foci may function to sequester and store the mRNA, not degrade it. The P-bodies could just sequester mRNAs away from the translational machinery.

Could they do both?
 I think whether mRNA will be degraded might depend on the protein complex associated with that particular mRNA. Obviously, we haven't figured out what proteins decide these fates yet.

But that could explain why there are so many different reports: some groups see mRNA degradation and some groups don't. Perhaps the P-body functions like a sorting machine for either degrading or protecting and sequestering mRNA. Of course, miRNA is just one branch that can bring mRNA into P-bodies. There are several other pathways, like nonsense-mediated or ARE-mediated RNA decay. These could provide the mRNA substrates and trigger P-body formation. Clearly, in these cases, mRNA will be degraded eventually.

What are the next big P-body questions?
 What are the components of P-bodies? Are all P-bodies the same or different? How do they assemble together and what regulates their assembly? Also, how does sequestered mRNA get back into cytoplasm for translation?

Wow, those are pretty big questions. How do you hope to answer them?
 From the cell biology point of view, we have a long list of P-body components. We want to study whether these colocalize in the same P-bodies, what are their particular functions, and how do they modulate the silencing activity of miRNA?

We are also trying to purify P-bodies. Right now there is no biochemical way to purify them, so it's hard to argue as to what percentage of the RISC or silenced mRNA is in the P-body as opposed to in the cytoplasm.

Also, a major question for the miRNA field is, What are the real genes that they repress? Because miRNAs are not perfectly matched to their targets, genome database searches give you hundreds of potential hits. There are very few confirmed miRNA targets at this point.

If we can biochemically purify P-bodies, and if our idea about sequestering is right, then the important targets should be there in the P-bodies.

That's a lot to keep you occupied! 
 Yeah, the P-body finding—that it could function as both the degradation sites for mRNA and the storage sites for translationally repressed mRNA—I think is exciting. It kind of opens up a new field, a new direction for research.

Liu, J., et al.
Song, J.J., et al.
Liu, J., et al.
Nat. Cell Biol.
Liu, J., et al.
Nat. Cell Biol.