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Oregon State s James Carrington Discussion His RNAi, microRNA Work


At A Glance

Name: James Carrington

Position: Director, Center for Gene Research and Biotechnology/professor, botany and plant pathology, Oregon State University

Background: Professor, Institute of Biological Chemistry, Washington State University — 1997-2001; Professor, Texas A&M University — 1988-1997; Postdoc, Oregon State University — 1986-1988; PhD, plant pathology, University of California, Berkeley — 1986; BS, plant biology, University of California, Riverside — 1982

After spending some time at Texas A&M and Washington State University, in 2001 James Carrington landed back at Oregon State as both a professor and director of the university’s Center for Gene Research and Biotechnology. In a place where he says he is comfortable and intends to stay for some time, Carrington is pursuing research into the mechanisms of gene silencing and the role of microRNAs in plants. He recently spoke with RNAi News to discuss his work.

How did you first get started with RNAi?

We got started back in the mid-1990s, prior to when RNAi had a name — it was a phenomenon in plants called co-suppression, or post-transcriptional gene silencing, or gene silencing in general. The reason we got into it was because it was recognized in the mid-90s that this was a naturally occurring antiviral defense response of plants.

First it was shown by a number of groups that if you made silenced plants using transgenes derived from viruses, those plants were highly resistant to infection by viruses. What that gave was a clue that, through engineering these silenced transgenes, we were tickling the natural defense response — certainly, it wasn’t conclusive of that, but it was one of the interpretations for why silenc[ing] plants using transgenes was so effective as an antiviral strategy.

The second piece of data, from a few different laboratories again, was that during the natural course of virus infection of wild-type plants, phenomena very similar to RNAi were being induced. This was shown by infecting plants with viruses and then showing that the infected plants mimicked a state of RNAi against the virus.

The third bit of evidence, which is where we entered the field, again along with a few other laboratories, was showing that viruses encode suppressors of RNAi.

With those sets of observations established in the mid-1990s, a framework for RNAi in plants as an antiviral response was very much solidified. We’ve been in the field ever since for those reasons.

Could you talk a bit about the work you’ve done since then?

From the mid-90s to the current day, we’ve focused on a few different things. One of the themes that’s been maintained in the laboratory is looking at RNAi-related processes during the course of virus infection. So, we’re very interested in: One, the basic mechanism of RNAi in plants. Two, how plants have diversified their RNAi systems for a number of different specialized functions, antiviral silencing being one, development being another, and chromatin structure being another.

And then, for the past several years, we have been very interested in microRNAs, in addition to RNA silencing as a defense response. Our interest in microRNAs arose out of our initial attempts — [which are] now going on three years — to identify natural targets of RNAi in plants. So, if you scroll back to the year 2000 or so, it was just becoming clear that there were natural targets of RNAi-like processes in eukaryotes — so, some of the initial microRNA targets from animals were being discovered and the first initial microRNAs from C. Elegans had been characterized.

Plants had all of these RNAi processes that we knew about, therefore we asked the question: What are the natural targets of RNAi-like processes in plants?

What this led us to — first, through the sequencing of libraries of naturally occurring small RNAs — is that plants have large populations of endogenous siRNAs and they have, like animals, populations of microRNAs that are involved in suppression or negative regulation of genes involved in development, primarily.

Would you talk a little bit about how you’re approaching these different aspects of your research?

First of all, there’s an overarching project in the lab, and that’s to understand how the RNAi-related pathways have diversified. There, we’re using a genetic approach, going in and knocking out different sets of factors that have proliferated and specialized in these different RNAi functions.

For instance, there are four Dicer-like genes in Arabidopsis. One obvious question is: Are these specialized Dicer-like enzymes for different RNAi pathways or have they proliferated for some other reason. So, we’ve gone in and knocked out all of the four Dcls and asked: Which RNAi functions are lost? What we find is that there’s one enzyme required for most microRNA biosynthesis, there’s another Dicer-like enzyme that’s specialized for most classes of endogenous siRNA biogenesis pathways, [and] there are other Dicer-like enzymes that probably play redundant roles in antiviral silencing.

We’ve done the same thing for RNA-dependent RNA polymerases that are involved in the siRNA-generating pathways that are in plants.

That’s one of the overarching approaches to looking at all of the pathways. That’s kind of a genetic overview of factors specialized in these three different areas.

On the antiviral side, we’re still very interested in how viruses suppress the RNAi pathways. We’re finding that different RNAi suppressors of viruses look evolutionarily unrelated to one another, yet many of them function at the same point in the pathway. What we’re finding is that several of the strong viral suppressors interfere with assembly of RISC complexes by either binding to or inhibiting a specific step in the integration of siRNAs into the RISC complex. [This] involves a little bit of biochemistry and some of the typical arrays of techniques that most people are familiar with.

On the microRNA side, again the questions are very basic — what does the microRNA population look like in Arabidopsis and how do they function? There, we’re in a biologically fortunate position because the microRNA targets in Arabidopsis and other plants are relatively easy to recognize because of the high degree of sequence complementarity with microRNAs.

[For] the initial findings there, we credit David Bartel with making some of the key observations showing us how to computationally recognize microRNA targets in plants. That only gets you so far, though, because some targets are not predicted and we don’t know about some microRNAs — if you don’t know about the microRNAs, you can’t predict their targets. So, the approach that we use for identification of new microRNAs is library cloning with some specialized genetic resources.

What we do for making microRNA libraries is use mutants that are deficient in production of endogenous siRNAs, because when you make the microRNA libraries a “contaminant”, in the library are the endogenous siRNAs that come from places like heterochromatic regions, regions of the genome rich in transposons and retro-elements — these things that are not microRNAs, we’re interested in them but not for the same reasons that we’re interested in microRNAs.

So what we use are mutants in the RNA-dependent RNA polymerase and Dcl genes that are deficient in siRNA — we have a genetic enrichment for cloning microRNAs.

That’s one approach. The other approach is computational, looking for hallmarks of genomic sequence that look like microRNAs and then going in and validating that a microRNA comes from that particular locus.

One the function side, and that’s where I think the frontier is over the next few years, [we’re examining] the role of microRNAs in plants. Here, we’re using functional genomic-type technologies. One is to take microRNA genes and over-express them, and ask: What happens? A prediction that we make is that microRNA targets will decrease in abundance, because one of the things we’ve found is that, unlike animal microRNAs, plant microRNAs generally degrade their targets in an siRNA-type guided mechanism. If we over-express microRNAs, we can down-regulate their targets and so we can use techniques like expression profiling to give us the complete picture of what happens to transcript abundance and therefore make some inferences and refined hypothesis about what the targets are.

Other approaches are to systematically eliminate microRNA target sites from known targets and ask: What’s the biological consequence of doing that?

We’re not the only ones doing these sorts of things. In many ways, these are obvious experiments that need to be done.

Do you have any projections on the field, on miRNA research?

Once targets are identified and the biological consequence of microRNA-guided regulation is better understood, once much of the current activity is done and we have a picture of the biological role of microRNAs, the next set of questions that are quite important and readily addressable is how the microRNA system is integrated with other cellular systems.

There are some hints about where microRNAs are acting. There are some hints that microRNAs are affiliated with the translational apparatus on polysomes, for example. I think there are some really interesting questions on how the microRNA system functions as a sensor and a suppressor as part of the normal process of gene expression. There is a frontier waiting to be pushed back there.

In plants, we have probably a few more directions to go in than in animals because RNAi-like processes have proliferated more in plants and play a much more prominent role in, say, regulating chromatin, in suppressing viruses, and as a developmental mechanism. Understanding how all those systems are diversified or integrated, I think, is a very interesting set of questions.

Are these three different areas completely functionally specialized? We have genetic data that says that they’re not completely independent, that there are some shared factors between these different branches of RNAi.

So, understanding how the microRNA formation and effector complexes are integrated with the chromatin siRNA biogenesis and effector complexes, and the antiviral biogenesis and effector complexes, is a very interesting question. I think we’ll learn a lot about how these systems function and how they’ve evolved by understanding how these different branches work in plants.

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