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Chester Beatty s David Barford Discusses Structural Biology and RNAi

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At A Glance

Name: David Barford

Position: Professor, Section of Structural Biology, Institute of Cancer Research, Chester Beatty Laboratories, London

Background: D.Phil, molecular biophysics, University of Oxford — 1988

BSc, biochemistry, University of Bristol — 1984


After receiving his graduate degree, David Barford completed his postdoctoral research at the University of Dundee. For the next three years, he was a fellow at Cold Spring Harbor Laboratory, but eventually found his way back to England to serve as a lecturer in the department of biochemistry at Oxford.

Now, he works at the Institute of Cancer Research at Chester Beatty Laboratories, investigating the structural mechanisms underlying cellular functions. Recently, he spoke with RNAi News about his research and how it extends into the RNAi field.

Could you give an overview of your lab?

My lab is investigating the structures of proteins, of macromolecules. We do X-ray crystallography and now also some electron microscopy. We're a structural biology lab. Our main interests are in understanding cell regulation of signal transduction pathways and mechanisms. My early work, during my graduate studies, was to understand the control of protein function and regulation by protein phosphorylation, and we worked on glycogen phosphorylase.

I moved on to the enzymes that modify other proteins — basically kinases and phosphatases, which are involved in modifying proteins by protein phosphorylation events. Now we're working on a number of others involved in oncogenesis and cell-cycle regulation, including kinases [and] also ubiquitin ligases. The work on RNA interference really came from the idea that gene repression by RNA interference is needed for regulatory processes, and we wished to understand how these small siRNAs could actually regulate cell processes. [We also were looking to answer questions like,] 'What is the molecular mechanism for the RISC and RITS complexes?'

About two years ago we started our work on analyzing Argonaute proteins. At that time, there really was not much known about the molecular mechanisms by which RISC can recognize messenger RNA target strands and catalyze cleavage processes — it wasn't really known which component of RISC was the endonuclease. We thought that a structure-based approach would provide a lot of insight into which component of RISC might be involved in cleavage and also the recognition of the guide RNA strand and target RNA strand. That's when we started the structural work on the Argonaute proteins.

Maybe it's just coincidence that you were at Cold Spring Harbor Lab and there's a lot of RNAi work going on there? Did you start working on RNAi on your own or did you have some dealings with someone in the field?

No, we didn't have any collaboration with anyone at Cold Spring Harbor on RNAi, or anyone else actually. I really turned to [RNAi] spontaneously. We have a group here, [and] don't really have any extended collaborators. We do all the work in-house.

I was doing a piece earlier this year, an overview of 2004, and I was talking to Phil Zamore at UMass. He mentioned to me that work you published regarding Argonaute proteins last year stood out in his mind as a highlight of 2004. I think he was referring to an EMBO paper. Can you give an overview of the findings of that paper?

It's very generous of Phil to mention this. This paper reported the structure of the Piwi domain of an archaeal Argonaute-like protein. We published this after the group at Cold Spring Harbor — Greg Hannon and Leemor Joshua-Tor — published the Pyrococcus furiosus Argonaute protein. We also discovered that the Piwi fold has a domain within it, which is very like an RNase H-like domain. That implied that Piwi might be involved as the endonuclease activity of Argonaute. Argonaute was therefore the endonuclease slicer activity of RISC. That was one finding, but it came out after Leemor's paper, so we were not the first to report it. We actually found it independently around the same time, but we published it later.

What was unique about our paper, and which maybe stands out a little bit, was that we … did a very careful structural comparison between the Argonaute sequences in eukaryotes and our Archael Piwi domain sequence. We could really highlight the conservation between Archael proteins and eukaryotic proteins. From this we spotted a site on the Piwi domain, which we thought may be involved in binding the 5' end of the guide RNA. We looked for this because of the literature that showed you need the 5' phosphate of the guide RNA for RNAi processes, and also that you get cleavage of the target RNA at an assigned site relative to the 5' end of the guide RNA.

We identified the structure of a conserved site within the Piwi domain, [and what] was quite interesting was that it had a metal ion bound to it. That implied that the Piwi domain itself could bind to siRNAs, because the metal binding site had the structural features consistent with it being an RNA-binding site. We actually found that the Piwi domain did bind to siRNA and that was the first time that had been reported. It turns out that this site I'm referring to … does bind to the 5' nucleotide of the guide RNA. This actually involves C-terminal residue of the Argonaute Piwi protein, and it involves the carboxylate group of C-terminal residue, which coordinates the metal ion, which in turn coordinates the phosphate group.

Our paper which came out recently [in Nature] is a follow up to [the EMBO] paper because we had the idea that the Piwi domain would bind to the guide RNA, and therefore position the target RNA into the right position at the active endonuclease site of the Piwi domain — we wanted to get a complex of the RNA bound to that Piwi domain.

That's the Nature paper.

Yes. We had the idea that Piwi domain would bind, so we took the same protein — the Archaeoglobus fulgidus protein — and we co-crystallized that with a range of different RNA oligonucleotide duplexes, one of which co-crystallized quite nicely. From that we got a structure.

It turned out that the site I referred to just now, which we reported in our EMBO Journal paper, was indeed the site that binds the 5' phosphate of the guide RNA. Also the phosphate of the third nucleotide of the guide RNA binds to that site by a metal ion. What's interesting about this work is that we showed this binding actually causes an unwinding of the duplex RNA, so the first nucleotide of the guide RNA is not base paired to the complementary base on the target strand. We predicted, and it turned out to be correct, that this binding then does position correctly the target messenger RNA strand within close proximity of the RNase H catalytic site of the Piwi domain at the right position for cleavage.

So, where does that lead you now? What things are you investigating currently?

One of the big questions that remains [involves] the details of the catalytic site of Argonaute, which is an RNase H-like domain. So far there's no structure of any RNase H-like enzyme bound to RNA substrates. Human Argonaute2 has been shown to be a slicer whereas Argonaute1, 3, and 4 don't appear to have any slicer activity. It's been proposed by Greg Hannon and Leemor Joshua-Tor that a DDE motif might be the catalytic site of slicer activity of Argonaute.

Argonaute1 and Argonaute3 don't catalyze RNA cleavage, so there must be something special about Argonaute2 that allows it to cleave RNA, which is not present in Argonaute1 and Argonaute3. That's still the big question: What really defines the activity of Argonaute2?

A big [goal] for the field is to get a complex of a eukaryotic Argonaute protein bound to an RNA duplex. That's clearly a target for other groups, as well as mine.

Another interesting question is related to a paper that recently came out in Cell by [David] Bartel's group, which showed that there is some propensity to have adenine bases surrounding the site of cleavage. That would imply, I think, that there could be some sequence specificity involved here, which is actually recognized by the Argonaute protein. Argonaute2 may recognize the bases within the RNA duplex around the active site to promote cleavage. So what we want to do is understand the structure of the duplex RNA bound to the active Argonaute protein.

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