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University of Alberta s Tom Hobman on Alternate Roles of RNAi Machinery


At A Glance

Name: Tom Hobman

Position: Associate professor, cell biology, University of Alberta

Background: PhD, genetics, University of British Columbia — 1990

MSc, human genetics, University of Manitoba — 1987

BSc, microbiology, University of Manitoba — 1983

After receiving his PhD in Canada, Tom Hobman made his way to the United States to pursue postdoctoral training at the University of California, San Diego. From there, it was back to Canada, where he began at the University of Alberta as an assistant professor. Now an associate professor, and two months away from a full professorship, Hobman took some time to speak with RNAi News about his lab and RNAi-related work.

Can you give an overview of your lab and its focus?

Well, we have a mixed lab, which is an unusual situation in many respects. About half of the lab studies virus structural proteins — we're interested in the interactions between the virus structural proteins and the host proteins, and determining how these interactions affect virus replication and ultimately disease. The other half of the lab works on RNA interference-related processes. It's sort of split down the middle.

We're trying to carve out a little niche for ourselves given the intense research interest in this field; it's really kind of hard to compete with some of the powerhouse labs in the [RNAi] field. We're primarily interested in non-RNAi-related functions of proteins such as Argonaute and Dicer. That's what we're up to.

Was that an outgrowth of work on RNAi itself?

We didn't set out to study RNAi at all. The way that we got into the field was because of my background in membrane trafficking. We were originally interested in isolating novel proteins localized between the Golgi and the endoplasmic reticulum. The first protein we isolated turned out to be Argonaute-2. But when we had purified this protein and cloned the cDNA for it, it wasn't until a few years later that people began to realize that this family of proteins was involved in RNA interference. So we had no idea which direction we were being led in.

We were not really interested in gene silencing at all. That's certainly not our forte and we're not actively pursuing that line of research. But we are interested in where Argonaute proteins are in the cell, how they interact with Dicer, [and] where these interactions take place — that's more what we're interested in.

Can you touch on that work?

Perhaps it might be better to put it in the context of the yeast work we're doing. We've been exploiting yeast as a genetic and biochemical system to study Argonautes and Dicers for a number of reasons. The first of which is the tractability of yeast as an organism. Secondly, yeast is one of the few organisms that has a single Argonaute gene and a single Dicer gene, so this sort of eliminates some of the complexities of [other] systems, most of which have more than one Argonaute and some having more than one Dicer gene.

So that's how we got into yeast. Before Tom Volpe's work was published in 2002, which really broke the field wide open with respect to the role of RNAi in chromosome dynamics and transcriptional gene silencing, we were working on [Schizosaccharomyces] pombe at the same time. What we noticed was that there were severe growth defects, as well as what we thought of at the time as cell cycle defects. Further probing into this, we discovered that Argonautes and Dicer indeed were required for enactment of certain cell cycle checkpoints. However, another component of the RNAi pathway in yeast, RNA-dependent RNA polymerase, does not seem to be involved in these processes. This was our first clue that perhaps Argonaute and Dicer were doing something other than just gene silencing. It's known that RNA-dependent RNA polymerase is required for gene silencing in pombe, and at least one paper -Sigova et al., 2004 — suggests that RNA-dependent RNA polymerase is in fact required for production of small-interfering RNAs in pombe. So this was all leading us to believe that Argonautes and Dicer may have alternative functions that are not directly related to gene silencing.

Could you bring us up to speed in term of where your research is and some of these other roles [for Argonaute and Dicer]?

A paper that we're actually writing up right now, and it will hopefully be of some interest to the field, [involves research where] we're looking where Argonaute and Dicer are — where they spend most of their time in the cell. Certainly in pombe, the most well-studied pathways that these two proteins are involved in involve heterochromatin formation and centromere function. Yet what we find is that both Argonaute and Dicer appear, at least when we're using tagged forms of the proteins, [mostly] in the cytoplasm. What we're trying to figure out is how these proteins gain access to the nucleus, because at least Argonaute has to gain access to the nucleus to guide the chromatin-silencing machinery to where it has to go.

The results we have now differ a little bit from what's out there. Nobody has published anything on Dicer localization in pombe, but certainly it's known now that RNA-dependent RNA polymerase is in the nucleus — and we find that too. But I believe there is just one report — Noma et al., 2004 — showing that Argonaute is associated with intranuclear foci. Yet we're not seeing that in our system. I'm not sure why yet, but we've used two different tagged forms of Argonaute and we see the same thing: most of it's in the cytoplasm.

Can you offer any conjectures on how it gets into the nucleus?

That's a good question. We haven't found any way, either pharmacologically or genetically, to induce concentration of Argonaute in the nucleus. We have tried different cell cycle blocks and we don't really see a lot of Argonaute accumulated in the nucleus either. As I mentioned before, Noma et al reported that Argonaute associates with foci in the nucleus. However, they also mention that they see a cytoplasmic pool of Argonaute. It is my impression from their paper that the majority of Argonaute was in the nucleus, and that is certainly not what we see. It is important to point out that we are using different tagged versions of Argonaute then the group that published before did, and moreover we're expressing the proteins at relatively high levels. So there are a number of differences between the studies. It will be interesting to see what the reviewers say about it.

What was that first paper you mentioned?

It's Noma et al. It's [November] 2004 in Nature Genetics. Shiv Grewal is the corresponding author.

You said you're also looking into the activities of Dicer.

Right. We found that both Argonaute and Dicer are required for enactment of replication and DNA damage checkpoints in pombe, so that if you have Argonaute-null strains or Dicer-null strains they seem to interface with a pathway that ends in hyperphosphorylation of Cdc2, which is a master mitotic regulator. So hyperphosphorylation of Cdc2 results in cell-cycle delay to allow alleviation of replication stress or repair of DNA damage. And when Argonaute or Dicer is not there, the Cdc2 fails to become phosphorylated in response to DNA damage or replication stress. As a consequence, cells that are exposed to replication stress such as hydroxyurea or radiation are much more sensitive to these genotoxic agents than are wild-type cells. However, that is not the case for RNA-dependent RNA polymerase null mutants. These cells do not seem to be as sensitive to these agents. Nor is Cdc2 phosphorylation impaired in RNA-dependent RNA polymerase-null mutants. These findings led us to propose alternate functions for Argonaute and Dicer.

As an aside, there is an RNase type III called RNT1 in Saccharomyces cerevisiae, which is a relative of Dicer in that it's an RNase type III. This RNase is required for normal cell cycle progression — Catala et al., 2004. Interestingly, these authors demonstrated that a catalytically inactive mutant of RNT1 could fulfill the cell cycle-dependent function of RNT1. So there certainly is precedence for RNases having non-catalytic roles in cell-cycle phenomena.

Given that these are all parts of the RNAi machine, and they have these alternate functions, is the thought that there might be some common thread joining it all together?

I'd like to think there's some common thread linking these, but at this point I have no idea what it is. Before we published our paper last year in Molecular Biology of the Cell, [entitled "Ago1 and Dcr1, Two Core Components of the RNA Interference Pathway, Functionally Diverge from Rdp1 in Regulating Cell Cycle Events in Schizosaccharomyces pombe,"] reviewers were concerned that this is some sort of global phenomenon or global defect that was occurring because of disabling of the RNAi system. But we managed to convince them that that is not the case.

What that common linkage is I do not know, but I'd love to know. But the fact that you don't need RNA-dependent RNA polymerase for the cell cycle-related roles of Dicer and Argonaute at least suggests to me that they may be functioning in some parallel [manner]. I'll give you an example, and I know this is against the grain and we certainly have our work cut out for us convincing other members of the field that this is the case: just yesterday in Nature, I came across a paper by Royal et al. where they've now discovered that clathrin is required for the function of mitotic spindles. This is a completely unexpected role for a coat protein like clathrin, which has had well-defined roles in endocytosis for many years. Now people have found that it's involved in congression of chromosomes through its role in mitotic spindles. So it makes me feel a little bit better that if people working on clathrin can convince the field that it has roles unrelated to endocytosis, then we can convince the field that Argonaute and Dicer may have roles unrelated directly to gene silencing.

You can't take anything for granted as far as what we know, huh?

Yeah. One of the perplexing things in my mind right now is where exactly is RISC. I think all along people have assumed that RISC activity is present in the cytoplasm, but a paper came out late last year suggesting that RISC activity exists in the nucleus as well. [This was] Robb et al. in 2004. This begs the question 'Is Argonaute in the nucleus?' Certainly in most cultured cells that we have looked at, very little Argonaute is in the nucleus. To my knowledge, no one has published microscopic evidence that Argonautes are in the nucleus of higher eukaryotes. So there certainly is work to be done in this regard. The geneticists, biochemists, and enzymologists have done a phenomenal job of moving the RNAi field forward over the last five years. However, one piece of the puzzle that is still missing or perhaps overlooked, is a better understanding of the cell biology of RNAi — that is learning about where the functional RNAi effector complexes are localized, the dynamics of these complexes. To me, that is what's lacking at this point.

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