At A Glance
Name: Rob Martienssen
Position: Professor, Cold Spring Harbor Laboratory
Background: Assistant professor, Cold Spring Harbor Laboratory — 1989-1995; Postdoc, University of California, Berkeley — 1986-1989; PhD, genetics, Plant Breeding Institute — 1986; BS, genetics, Cambridge University — 1982
After completing his undergraduate and graduate studies in England and a postdoctoral stint at UC, Berkeley studying maize genetics with Bill Taylor and Mike Freeling, Rob Martienssen landed at Cold Spring Harbor Laboratory among some of the leaders in the RNAi field.
Recently, Martienssen spoke with RNAi News about his RNAi and microRNA research.
How did you get involved in RNA interference?
We were working in plant development with a mutant called Argonaute because of our interest in stem cells, and also in patterning of lateral organs in plants. A mutant, actually several mutants, that we picked turned out to be allelic to Argonaute, and that’s really how the direct interest in RNAi started. But I’ve been working on transposable elements since I was a graduate student, and it turns out that both of those aspects of plant biology — both transposable elements and various development cues — depend on RNAi.
While we were working on Argonaute in plants, it was discovered that an Argonaute homolog in C. elegans was required for RNAi — this was actually the first RNAi-defective mutant in C. elegans, called RDE1. That led us to think that maybe the development defects we saw in Argonaute were actually due to RNAi defects. We’ve done a great deal of work on that since and actually we recently had a paper in Nature last February, which demonstrated that one of the alleles of Argonaute that we received resembled very closely a mutant called Phabulosa, in which the dorsal-ventral polarity — the up and the down of the leaf, if you like — was reversed. We suspected that maybe this other gene, Phabulosa, might be a target of Argonaute. It transpired from other people’s work that it was, or at least it was a target of RNAi.
We showed that the microRNA that regulates that gene is actually unevenly distributed in the leaf, so in wild-type plants it’s normally on the lower side of the leaf and in Argonaute mutants it’s throughout the leaf. This accounts, in part, for both the genetic interactions we described in that paper, as well as the polarity defect. We hypothesized on the basis of that that microRNAs in plants might actually act as developmental signals. It’d been previously shown in worms that microRNAs are heterochronic signals — that they’re involved in developmental timing — but we showed for the first time that they could also determine spatial patterns of development, and that was exciting for us. It was the combination of many years’ work on Argonaute.
Before we knew the Argonaute gene was involved in RNAi, before the work had been done in C. elegans, we realized that there were many Argonaute homologs in plants. This, of course, makes genetic dissections of the pathway quite difficult because of redundancy and overlapping functions and so on. On a genome search we realized that the fission yeast Schizosaccharomyces pombe has only a single copy of the Argonaute gene, so we set about knocking that out. Shortly after we’d done that, the breakthrough came that revealed that Argonaute was involved in RNAi.
So we started looking at silencing phenomena of various sorts in fission yeast, and we discovered that centromeric silencing was actually affected by Argonaute very strongly. This was counterintuitive because centromeric silencing was well known to be regulated transcriptionally by what is known as the histone code.
The histone code was worked out first, I suppose, in Drosophila, then later in mammalian cells, and has subsequently been worked out in plants. Basically, there are some modifications of histones that are associated with heterochromatic silencing. What we showed in fission yeast was that the mutants in RNAi actually affected this pattern of histone modifications, the histone code, in the centromere. The reason it was able to do that, we discovered, was because the centromeric repeats, which fission yeast has in common with all other high eukaryotes — humans, plants, everything — these centromeric repeats were actually transcribed. That hadn’t been noticed before because they’re turned over extremely rapidly by RNAi, so the transcripts don’t accumulate. But in these mutants we could see the transcripts very clearly.
It turns out that transcription of these repeats and their turn-over by RNAi acts as a signal for heterochromatic modification of the repeats. This is all in this rather simple organism, fission yeast. We and others have done more work in fission yeast to try and dissect exactly how that pathway happens, but in broad terms: It has always been known that repetitive elements, including transposable elements, are modified epigenetically both by histone modifications and by DNA methylation. These epigenetic modifications silence them, but it’s always been a mystery as to how that silencing could be so specific. What was it about transposable elements that allowed the cell to recognize them as being different from the rest of the cell, the rest of the nucleus, the rest of the DNA?
It is, in fact, the small RNAs that turn out to be crucial for that. We just recently had a paper in last week’s Nature …
That was my next question.
Right. What we did was profile these epigenetic modifications throughout the plant genome, and found that DNA and histone methylation were restricted to transposable elements. We compared that to the distribution of small RNAs and it fits almost perfectly. So the small RNAs are somehow programming these modifications. In a curious twist, two mutants that have been known for quite a long time and … control DNA methylation … not only lose DNA methylation but also lost many of the small RNAs that correspond to these sequences. We believe that [one of these called] DDM1, which actually encodes a chromatin remodeling enzyme, might in some way use these small RNAs to remodel the chromatin and keep it silent.
We believe, then, what we discovered in fission yeast — that small RNAs are used to program heterochromatic modifications of specific sequences — is actually a general mechanism that may apply well outside yeast. Actually, it’s been recently shown … that mammalian and vertebrate cells do exactly the same thing in the sense that small RNAs from heterochromatic sequences, and RNAi in general, seem to be involved in programming the heterochromatin in mammalian cells too.
Can you talk about what’s going on currently in the lab?
We’re currently looking at centromeric repeats in plants, and we’ve found that they’re also subject to these mechanisms in very similar ways. In high eukaryotes centromeric repeats are interrupted by transposons, real transposons, and these also, we’ve shown, play a role. So, transcripts that come from transposons also program the double-standed RNA in the centromeres. I suppose in a broad view, we think the transposons are likely to have this ancient function in chromosome structure and organization, even as far as centromere function itself because the RNAi mutants that we isolated in fission yeast actually have defective centromeres.
So this whole marking process that goes on under the control of RNAi is actually a biological process that’s required for centromere function and seems to be intimately associated with transposable elements, at least in plants. We can think, then, of transposable elements not just as parasitic — they certainly are very damaging to the cell if they jump around and interrupt genes — but in the context of heterochromatin, they are actually very valuable because they allow chromosome segregation and centromere function.
We’re pursing that sort of idea in plants, and we’re also continuing our work on the developmental aspects of RNAi in plants. We’re, again, looking for things that might be in common. One thing that we’ve been thinking a lot about is the fact that in animals — that is to say C. elegans, Drosophila, and probably in mammalian cells, in the mouse — as well as in plants, RNA interference mutants often have very strong stem cell phenotypes; they fail to propagate stem cells. Now, you could argue that stem cells in plants and animals are rather different, and certainly they are, but they have at least comparable functions. And yet, no one has found any microRNAs in common between plants and animals.
Arabidopsis has probably in the order of 100 to 150 microRNAs, [and] C. elegans and mammalian cells have similar numbers, but none of them are in common. So, if the stem cell phenotype really does reflect something that plants and animals have in common with respect to RNAi, we rationalize that it must be small interfering RNA, potentially from the centromeric repeats because that seems to be a common thread, a very ancient role for RNAi. We’re looking into that.
You mentioned mammals. Have you ever worked with mammalian cells?
We’ve done some work in collaboration with some of our colleagues at Cold Spring Harbor: Dick McCombie, Scott Lowe, Greg Hannon. And we’ve done a little bit of work on looking at methylation patterns of transposons. I’m certainly interested to do more. It seems that we’ve got a lot of good things we can do together.
Well, Cold Spring Harbor seems to be the place to be for this sort of work.
It certainly is. It’s really been an exciting time. By using these very simple model systems we can easily get to many of the genes that are important. Actually, Arabidopsis has a fantastic track record. Argonaute and the first Dicer homolog to be shown to have a developmental function were all isolated in plants actually before they were known to have anything to do with RNAi, which is ironic because important aspects of RNAi were discovered in plants early on.
Plants are really a good place to get at some of these fundamental mechanisms, and it would certainly be very gratifying to have [the research] applied to the more biomedical side of things.
On that note, what about things down the road? Are there areas that you’d like to extend into?
I think it’s very interesting to compare methylation profiles in mammals and plants. Obviously, many animals don’t have much in the way of methylation: Drosophila and C. elegans don’t have very much. But mammals do, and so do plants, and yet their patterns of methylation differ — we’ve shown that at sort of the anecdotal level. It would be interesting to see what the basis for those differences really is. That’s the sort of thing that interests me at the moment.
Then, as I said, on the developmental side, we’re pursuing the stem cell connection, which I think could be extremely valuable.