Doudna lab, University of California, Berkeley
Name: Ian MacRae
Position: Postdoctoral fellow, Doudna lab, University of California, Berkeley
Background: PhD, biochemistry/molecular biology, University of California, Davis— 2002
BS, biochemistry, University of California, Davis — 1996
In the Jan. 13 issue of Science, Jennifer Doudna and colleagues at the University of California, Berkeley, and the Lawrence Berkeley National Laboratory published a research article detailing the crystal structure of an intact Dicer enzyme.
"The PAZ domain, a module that binds the end of dsRNA, is separated from the two catalytic ribonuclease III domains by a flat, positively charged surface," the researchers wrote in the article. Finding that the distance between the PAZ and RNase III domains matches the length spanned by 25 base pairs of RNA, they concluded that "Dicer itself is a molecular ruler that recognizes dsRNA and cleaves a specified distance from the helical end."
Recently, Ian MacRae, a postdoc in Doudna's lab and the paper's lead author, spoke with RNAi News about the findings.
Let's start with an overview of the lab and the kind of work you do there.
Jennifer [Doudna] first started her lab [as] one of the very first people to look at structures of non-coding RNAs. She is a pioneer in the field, looking at how these things fold for their functions — in particular she looked at ribozymes. That was the first five years of her lab, at least. Then as time went on she branched out into looking at the structures of other RNAs, and also RNA-protein complexes.
I was interested in RNAi [as a student] at the time and was thinking of studying it from a structural standpoint. Since she was a pioneer in the structure of RNAs … I thought [her lab] would be a good lab to start out in.
Could you talk about the Science paper, how you started into the work, and the findings?
I just picked a question — this [started] three years ago and there were a lot of questions floating around RNAi. I wanted to keep my question very structural, and one of the most fundamental questions was: How are these 23-nucleotide siRNAs produced structurally? I focused on Dicer, partly because at that time that was really the only RNAi component that there was a clear enzymatic assay for. We just started cloning different Dicers from all different organisms and making many different truncations, and seeing what it really took to make a Dicer. Then [we took] constructs that worked and set them up in crystallization trials.
It turned out that this Giardia [intestinalis] Dicer, which is really small compared to other Dicers, was the one that crystallized first. So that's the one we've been pursuing. …
The whole goal was to crystallize a Dicer protein, and so really we just took a shotgun approach — we just tried basically every Dicer that people had found and different versions of it. The first challenge is just making enough protein to try and crystallize because it's sort of a hard protein to produce in general, and to do crystallography you need large amounts of very pure protein.
So the first challenge was just finding different variations that we can make on the right scale. The second was the crystallization itself. After screening through everything, the only one that satisfied both these criteria was the Giardia Dicer so we were stuck with that one because it was the only one that worked — so far.
But it was actually kind of nice in a way because it's much smaller [than other Dicers] so the structure is a lot simpler to like at. It's a very streamlined mini Dicer, and it works just fine in vitro so we know it's a real Dicer. [Additionally], there's not a lot of extra junk hanging out that you're not sure [the purpose] of — it's very simple.
Can you touch on the findings of the paper … [and] anything you found surprising?
The findings give the overall architecture that I think is going to been seen in all Dicer enzymes, and it really sets up how the measurement of these small RNAs and how they're produced.
The structure itself was actually quite intricate. Dicer has many different domains, and typically with proteins that have many domains they're sort of modular and don't all interact with each other. But actually the way Dicer is woven together, it actually folds back onto itself several times, which was a surprise to me and explains why a lot of times when you try to make truncations of Dicer it doesn't work anymore — because this thing is all folded together. The structure actually agreed very well with biochemical data that came out the year before from the [Witold] Filipowicz lab. They had proposed a model for human Dicer that was basically what the Giardia Dicer looked like. So I think this validates the idea that this is going to be the structure seen in all Dicer enzymes.
Where does this lead you, then, in terms of your work?
The basic question of how does [Dicer] measure is pretty clear to me. Now there are a few new directions to go in. The structure was solved without RNA, so one direction [to go in] would be to ask: What are the exact interactions between the protein and the RNA? The structure suggests that Dicer probably bends in several places to accommodate different sequences, so that is one thing we're looking at — the very detailed, specific interactions between the protein and RNA, and how the two influence each other. Another direction is to start addressing other functions of Dicer, for example how it participates in the handoff of the RNA … into RISC. I see Dicer having two functions: one is to cut the RNA and process it into the right [sized] piece, and the second is to participate in RISC loading.
Do you have plans to look at other Dicers?
Yeah. We have all these other constructs we're looking at … [and since] our next big goal is to get a structure of a Dicer bound to the RNA, [we'll] hopefully [do it with] a larger Dicer like a human or Drosophila or C. elegans.
But you think that ultimately they'll all be pretty similar?
I think they'll have the same overall architecture, but [larger ones] will have a lot of extra stuff for other purposes. Some Dicers may be processive, so instead of letting go of everything, they slide down and keep cutting on the same RNA without letting go. Giardia Dicer is not, but there is some evidence that [others] may be.