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University of Wisconsin s Susan Mango on Using RNAi in C. elegans

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Name: Susan Mango

Position: Assistant professor of oncological sciences, Huntsman Cancer Institute Center for Children, University of Utah

Background: Postdoc, University of Wisconsin, Madison — 1990-1995; PhD, molecular biology, Princeton University — 1990; BS, biochemistry, Harvard University — 1983

At the University of Wisconsin, Madison, Susan Mango uses C. elegans to research the formation of organs, primarily the pharynx, by embryos. RNA interference is helping her do so.

Recently, Mango spoke with RNAi News to discuss her work and the use of RNAi in the worms.

How did you become involved with RNA interference?

When RNAi first broke, it was so exciting for the whole worm community — and this was before it was a big deal in the general scientific community, before it was even called RNA interference — for two reasons. One was that, from a practical standpoint, it was clear that it was going to be a very powerful tool for people to inactivate genes; so, people were excited from that perspective.

And then, it was the mysterious mechanism. Initially, Ken Kemphues showed that sense and antisense RNA could both inactivate genes — that was the first puzzle. And then, subsequently, Craig Mello and Andy Fire showed that double-stranded RNA was even more effective. Both of those observations had people excited and I think a lot of people were playing around, asking questions [such as]: Did it matter what region? Did it matter what kind of gene? Did translation matter? Blah, blah, blah. So, there were all these questions, some of which we don’t have the answers to yet.

I think [the excitement was due] partly [to] the notion that here was some new mechanism that people didn’t know about — the idea that there have been so many people working in molecular biology and yet, somehow, this whole ancient pathway had eluded us. I think people were just really excited about it, and we were, as well, so we got involved.

I collaborated with Brenda Bass originally; we had just been kind of chatting about RNA interference and we just thought it was a cool phenomenon. I started mucking around with these mutants and the nonsense-mediated decay pathway, called SMG mutants.

So, my foray into RNA interference was the idea that nonsense-mediate decay is a mechanism by which cells recognize a barren RNA, [that] it leads to RNA degradation, and RNAi had those same features where, again, it seemed like one of the main purposes was to recognize the barren RNA and lead to mRNA degradation. Based on that, I was curious to test whether there were any common links between the two.

That was really the first experiment that we got interested in.

Where does this lead up to in the work you’re doing now?

A big interest in my lab is organogenesis. I guess I’d say that’s the main focus.

Most recently what we’ve been doing is a big RNAi screen — so functional genomics — looking at foregut development [in worms], which is a big focus in the lab. We had identified genes expressed in the foregut a couple of years ago, and we have most recently completed a big survey, in part using RNA interference and also teaming up with Mark Vidal at Dana-Farber to do one of these global yeast two-hybrid screens, and really begin to probe what these different genes expressed in the foregut are doing. In this sense, we weren’t really looking at RNAi mechanistically, but using it as a tool to do a big survey.

There have been a lot of RNAi screens for the whole genome, for example from Julie Ahringer’s lab. I think the distinction of our work is both the choice of genes, and the way that we the experiments was to focus on an organ — so, really looking at something uniquely metazoan.

On the one hand, what we’ve discovered is going to be very useful for the future in terms of pinpointing genes because we’ve been able to identify genes with very particular phenotypes that we will now follow up [on] in terms of what they do in the foregut.

It was also interesting to compare what we see in the foregut genes compared with, say, whole genome surveys. There seems to be much more of a reliance on metazoan-specific genes, for example, either by this yeast two-hybrid or by the RNAi screens — it’s put much more of a focus on that class of genes as opposed to genes that are conserved in yeast, for example.

Other work going on there [with RNAi]?

That’s the big RNAi [project]. At the moment, we’ve been very focused on the genomics of the foregut, so we haven’t had a chance to do so much of the mechanistic studies for RNA interference.

Is that something that’s on the burner?

I would love to get back to it if I have time. I don’t know when it will happen, because at the moment we have more projects than we know what to do with.

It would be great [to do] in the future, because there are a lot of questions that I’d be curious to address, and we certainly have tools and reagents and mutants sort of sitting in the freezer, and just haven’t had time to get back to them.

What sort of questions?

This whole link between the nonsense-mediated decay pathway and the RNA interference pathway was something I thought was really intriguing since we showed that components in NMD also played a role in RNAi, but it’s not really clear how. I think that’s going to feed into the ribosomes somehow, but again, it’s just completely unclear how.

I think probing that whole avenue is something that the RNA interference field as a whole hasn’t looked at so much, but I think it’s going to resurface at some point.

Any predictions on the RNAi-in-worms field?

One prediction, which is already starting [to come about] is that there’s going to be a huge number of these … RNAi screens for identifying genes in different processes. It will be a lot more specific than the genome-wide [screens available].

The genome-wide [screen] is a great foundation, but I think that, just as we are looking in a particular biological process — organ formation — a lot of these people are doing these screens for different [reasons]; either to look at things in a particular molecular pathway or a particular biological pathway.

I think it’s just going to explode. It means: Whereas you may have a pathway now of five genes, all of a sudden you’re going to be thinking about 200. I think there’s going to be a lot of that and it’s going to be huge. It’s just going to blast open a lot of biology.

So the genome-wide stuff is just the beginning?

Yes. I think so, because what people are doing now is adapting conditions to be able to really pinpoint genes involved in a particular process, which genome-wide screens really weren’t geared towards.

We have a really good foundation from the genome-wide screens about which genes are essential or genes that might be a starting point in a particular process, [but] I think some of these more targeted screens are going to [take things farther].

For example, Gary Ruvkun’s lab did a screen for fat accumulation in worms. That’s something that wouldn’t have necessarily been picked up in the genome-wide screen but was very powerful for identifying a lot of genes that affected fat accumulation in worms — many of these have homologs or orthologs in vertebrates and some of them have actually been shown to affect fat metabolism there.

So, I think that kind of approach, where you’re really trying to look at some particular process, is going to have a huge impact, and there’s a lot of people doing them now so it’s going to be an explosion. It’s going to be really exciting.

In terms of difficulties or roadblocks [facing RNAi] … have you encountered something that you see as limiting?

I think there are times when it would be really nice to inactivate a gene at a very specific timepoint or in a very specific cell — cell A but not cell B. That kind of specificity is something we don’t have for the most part.

People have tried some approaches to control when and where you get RNAi, but I don’t think they’re that great. So, I think that’s an area [in which], if people could figure out better approaches, [it] would be really great.

A lot of this work is complemented by actual genetic mutations and that’s a really powerful approach, as well — slower, but it’s still quite good.

And then, of course, there’s always the issue of: Do you have a null allele or null phenotype or a partial loss of function? In some cases that doesn’t matter but in some cases it’s absolutely critical. One always has to be careful there, so I think that’s an issue — having good ways to distinguish to what degree you’re knocking something out.

The stronger the RNAi, the better, so really being able to knock genes out, I think that’s also an issue.

With that said, [RNAi] is an amazing tool.

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