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Carnegie Institution s Brenda Reinhart Discusses Her Work With MicroRNAs

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At A Glance

Name: Brenda Reinhart

Position: Postdoc, Carnegie Institution

Background: Postdoc, Whitehead Institute — 2000-2002; PhD, genetics, Harvard University — 1999; BS, cell and molecular biology, Cornell University — 1989

Having worked in the labs of such notables as Harvard University’s Gary Ruvkun and the Whitehead Institute’s David Bartel, it is no surprise that Brenda Reinhart is including in her second postdoctoral effort the study of microRNAs. Recently, she spoke with RNAi News about her research.

How did you get involved with microRNAs?

I actually came at them kind of fortuitously. It was that I was working in Gary Ruvkun’s lab [at Harvard] on my graduate thesis, and I was basically taking a genetic approach to studying the genetic factors that regulate the timing of cell division in Caenorhabditis elegans. One of the genes that we had identified as a mutation that actually altered the sequence of developmental events and cell division in C. elegans turned out to be let-7, which was the second microRNA that was identified and turned out to be the first one that was conserved in evolution. [This] led us to believe that there would be a much greater family of microRNAs in other species.

Can you talk a bit about how this led you to working in plants?

When I, and especially Amy Pasquinelli, in Gary Ruvkun’s lab had shown that let-7 was conserved in basically … in the animal kingdom, the question grew as other people [continued] working in the field. David Bartel’s lab [at the Whitehead Institute] and other labs had been working on cloning methods to actually directly clone more microRNAs to begin to look at the sequences and numbers that would be in different species.

The question became: How far out in evolution were microRNAs going to exist? So, how ancient a role were they playing in the development of multicellular life? It just became an obvious approach to then go to David Bartel’s lab and try to clone more small RNAs from plants, for which we chose Arabidopsis thaliana, and also Schizosaccharomyces pombe as the species that was more closely related to the animal, to get a view of what small RNA populations were and whether microRNAs also existed in plants and fungi.

One of the reasons we also chose Arabidopsis thaliana and Schizosaccharomyces pombe was simply that their genomes clearly had homologs of Dicer and Argonaut family members, which we knew should be involved in small RNA production and function.

Can you talk about your findings and work in the Bartel lab?

When we had done the initial clonings, we were actually very pleased because there were immediately sequences that were coming out of our cloning procedure in Arabidopsis thaliana that looked like microRNAs. They were sequences that were in the range of 21 nucleotides, which fit with the length of the mature product of a microRNA; they had a local region adjoining genome sequence, which should be able to fold back into a stem loop with the mature microRNA sequence in a double-stranded region that could then be cleaved by Dicer; and they also were clearly in regions of the genome that were outside obvious protein-coding genes and other structural RNAs. So [the sequences] clearly fell out of this pool that we were working with as candidates for microRNAs in Arabidopsis thaliana.

Then we also showed on top of that that [the sequences] were definitely processed by Dicer; there was a mutation that was already existing in Arabidopsis thaliana that was at the time called Carpel Factory — it’s now known as Dicer-like 1. We simply looked at the level of the microRNA expression and found that they were lower in the mutant background relative to wildtype. Basically, that initial finding — that there were, in fact, things that fit all of our criteria for microRNAs in Arabidopsis thaliana — convinced us that there was a much greater family.

That’s what I’ve been pursuing in Kathy Barton’s lab [at Carnegie].

Would you give a little detail about your work at Carnegie?

One of the reasons I came to Kathy’s lab is that they have a lot of expertise at Arabidopsis thaliana development and genetics. The next question I had, now that we knew there were microRNAs, was: What processes were they regulating within the plant?

Matt Rhoades, a student in David Bartel’s lab, had actually taken the sequences we had identified as microRNAs and had done some computational analysis with the Arabidopsis messenger RNAs to look for potential regulatory targets of our microRNA pool. He had actually come up with targets for the majority of our microRNAs, just based on complementarity to the target. One of the ones that had fallen out was a transcription factor called phabulosa and a homolog of it in Arabidopsis called phavoluta, both of which had gene-of-function mutations in the locus, which would be disrupting the region that is complementary to the microRNA.

Both of these were associated with a misregulation of the locus; there’s an increased amout of gene activity, which fits with the idea that you actually needed the microRNA regulation to actually control the level of the gene activity from phabulosa and phavoluta.

My question, when I came to Kathy’s lab, was just to look at the details of this system. While at the time, and this was a few years ago, people were really focused on gathering a lot of information about the microRNA family as a whole, their targets as a whole — focusing on questions such as: How many microRNAs are there in Arabidopsis? Do they have one target or multiple targets that they’re acting to regulate in the plant? We were more interested in what was happening with this particular pair, and that’s what I was interested in following up on in Kathy’s lab.

Now that we knew we had a regulatory pair, what could we tell about the details of that regulation? Some of the questions are quite obvious: Are we controlling the phabulosa and phavoluta loci on a transcriptional or translational level? Others are a little less obvious: Why are you doing this at all? Why are we trying to control the level of gene activity using microRNAs, rather than transcriptional control at the locus?

I’m interested in looking at the details for how phabulosa and phavoluta are regulated, kind of in a bigger issue because I don’t believe that it’s just microRNA regulation that’s going to be setting up the proper pattern of activity for these proteins within the plant. I think it’s going to be one of several levels of regulation to kind of fine tune the gene activity.

Somehow in all of this I didn’t mention what phabulosa and phavoluta are doing, which is probably a key point for this. They are genes that are expressed within the primordial of the leaf, so the cells which are basically separating from the meristem and being designated for leaf tissue.

The inner half of the leaf primordia towards the center of the meristem is the adaxial half and will give rise to the future top of the leaf. The portion that is on the outside, that is located more distal from the central portion of the meristem, is abaxial half that’s going to give rise to the underside of the leaf. Phabulosa and phavoluta in a wildtype plant are expressed in the adaxial half of the leaf. In dominant mutants that disrupt microRNA binding, that activity seems to spread to the abaxial half as well, so if you looked at the RNA levels, you actually now have elevated levels of phabulosa RNA in the abaxial half of the leaf when you’re disrupting microRNA activity. And then, the leaf itself appears to be radialized, because if you look at the characteristics of the leaf, it appears to be that the dark green waxiness and tissue type that appears on the top of the leaf is now around the entire circumference of the blade.

The questions that are important for knowing how phabulosa is regulated are: Precisely when is the onset of expression within the leaf primordia? How are we restricting it to the proper domain of expression? How is the microRNA playing into this system?

When people were first thinking about what microRNAs were doing in plants, it was observed that you had a more extensive homology between microRNAs and their targets in plants than in animals. The few cases at the time that were known in animals for microRNA-based regulation tended to be translational control. They’d have good pairing between the microRNAs and the targets at the ends of the microRNA with mismatches between the target and the middle region of the microRNA. In plants, we had more extensive homology throughout the entire region, and that superficially looked much more similar to the type of target that an siRNA in RNAi would have to its target.

So when you first look at this, the simple answer seemed to be that maybe microRNAs in plants are acting more like siRNAs and be directing cleavage of their target. So, we could look at it as they’re acting to destroy the RNA transcripts in the cell.

To put that back in the context of the regulation of phabulosa: The idea might be that the microRNA is actually acting to keep phabulosa expression out of the abaxial half of the leaf domain. So the wildtype expression is in the adaxial half, and you want to limit it to that portion so that you can properly pattern the top of leaf. The role of the microRNA would be to keep it off in the other half, which seems to fit very well with this increase of the phabulosa messenger RNA in the abaxial when you’ve made a mutation in the microRNA binding site of phabulosa and you’ve disrupted that microRNA-based regulation.

That turned out to be a little simplistic when you look at the details, because the levels of phabulosa messenger RNA in a gain-of-function mutant are actually elevated throughout the entire leaf primoridum, not just in the abaxial half where you want phabulosa off for proper leaf development, but it’s also elevated in the adaxial half where it’s normally expressed.

So it’s not a simple clearing mechanism. The question is now: Is it a general dampening system to regulate the overall level, and if that’s happening is it something that is just kind of dampening levels overall but without an actual goal for a particular level of gene activity? Or is it now feeding in, trying to get particular concentration of the phabulosa protein in the cell for activity?

We’re hoping by looking at some of the details of this system, we can also figure out just how crucial the microRNA regulation is, because I think we don’t have all the full information we could possibly have in this system. There’s still this kind of glaring question of why you want to use this mechanism to begin with. Is it just a very general way for affecting the overall level of gene activity, or is it that somehow you’re actually trying to use a highly regulated system so that you can really fine tune a level of gene activity to a particular critical point.

For me, those are the types of questions that are best answered in vivo in an organism where we have a well-defined target and a very nice assay for activity, which is the developmental output for the leaf itself.