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Yale s Frank Slack Talks About RNAi and His Work with microRNAs

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At A Glance

Name: Frank Slack

Position: Assistant professor, molecular, cellular, and developmental biology, Yale University

Background: Postdoc, Harvard Medical School — 1994-1999; Postdoc, Stanford University School of Medicine — 1993-1994; PhD, molecular biology, Tufts University — 1993; BS, molecular genetics, University of Cape Town — 1987; BS, microbiology/biochemistry, University of Cape Town — 1986

Although he grew up and conducted his undergraduate studies in South Africa, Frank Slack left to pursue his career in the US for reasons partly political and partly academic. He conducted his postdoc work under Dale Kaiser at Stanford and Gary Ruvkun at Harvard, ultimately landing a faculty position at Yale.

Recently, Slack took the time to talk to RNAi News about his work and how it relates to RNAi.

How did you get involved with RNA interference?

My broad interest over the past decade or so has generally been in development. Basically, many aspects of spatial patterning development have been quite well worked out, and the genes that control spatial patterning in, for example, early Drosophila embryos and other more complicated organisms, are quite well known and quite well understood.

But, an aspect of development that was less well understood was that of the temporal acts of development — how organs form at the right time during development. So I joined Gary Ruvkun’s lab at Harvard Medical School to study a series of interesting mutants in C. elegans that actually made organs at their own time during development.

These mutants had mutations in genes [that] we term heterochronic genes. These heterochronic genes control how cells behave at particular times during development — in some of these mutants these cells did things that normally their granddaughters would do or, in some cases, they did things that normally their grandmothers would do. They were kind of confused with respect to when they were in development.

Some really pioneering work from Victor Ambros’s lab had shown that one of these heterochronic genes encoded a small non-translated RNA; this gene was called lin-4. It was sort of an anomaly, but it was a very exciting gene because it was the first known example of a small RNA that might function in higher animals to actually control gene expression.

Along with Gary Ruvkun’s lab, Victor had shown that lin-4 was complementary to another gene known as lin-14. The two of them had published back-to-back papers in Cell showing that lin-4 probably regulated lin-14 through an antisense mechanism binding to the 2’ UTR.

These papers caught my interest when I was still a graduate student, and that’s why I went to Gary’s lab hoping to try and understand a little bit more about how these genes function to control the timing of development.

When I was in Gary’s lab, we were characterizing new heterochronic mutants that have similar developmental defects to what were seen in the lin-4 and lin-14 mutants — that is, they made organs at the wrong time. One of these turned out to be a gene known as let-7, which I ended up cloning with Brenda Reinhart in our lab. It turned out let-7 also encoded for a small non-translated RNA and this became the second example of these non-coding RNAs that were controlling developmental timing. I also showed, when I was in Gary’s lab, that a likely target for let-7 was probably a gene called lin-41, which a lot of people now use as controls in their microRNA experiments.

The basic story here is that, through an interest in development biology programs, the Ambros and Ruvkun labs, with a little help from me, basically identified two of these small RNAs, which became the founding members of a large class of RNAs that we now know of as the microRNAs.

That’s how I became involved in microRNAs and, of course, a few years ago a number of labs showed that the microRNA field and RNAi field intersected at quite a number of levels. Even though we primarily work on microRNAs, some of the work that we do is probably going to impinge on the RNAi field, as well.

So, where are you at now? What sort of projects do you have ongoing related to all this?

My lab is still interested in microRNAs … primarily the temporally regulated microRNAs, mainly in C. elegans, although we are moving into mammalian systems to see if some of the things we learned in C. elegans are also true for other organisms.

We’re interested in a number of different areas. First of all, in the case of the temporally regulated microRNAs: How are they actually temporally regulated? — that’s the first thing. So: What are the transcriptional inputs to these microRNAs? That’s an area that hasn’t been explored in much detail in any lab.

The second is that we’d still like to use let-7 and lin-4 as model microRNAs to try to understand how these microRNAs function to turn off their targets — in this case lin-14 and lin-41. So, we have ongoing projects looking for protein factors that might function with the microRNAs, as well as looking at the actual RNA-RNA duplexes for signs of what might be the key requirements of a microRNA target interaction.

Lastly, we very interested in what the targets of some of these microRNAs might be — in particular, the temporally regulated microRNAs lin-4 and let-7, because these target genes are probably going to tell us something about how organs form at the right time, which is the basic biological problem that I’m still interested in solving.

Are you collaborating with anyone on these projects?

At this point, I haven’t started any collaborations with [anybody] in industry. It’s still a fairly fresh area of research and I’d imagine some of the kinks need to be worked out before microRNAs get a serious look as therapeutics.

I do have some collaborations within Yale. Of course, a lot of people are discovering that they have a microRNA that maps really near to their gene or a process that ripe to be controlled by a microRNA, and we’re trying to help them out on those projects.

I [also] have collaborations with bioinformatics groups to try to find targets for some of these microRNAs. As you can understand, these microRNAs don’t recognize their targets with exact complementarity — these are imperfect duplexes, which are a little tricky to identify just by standard blasting techniques or the standard repertoire.

So, we’ve initiated a collaboration with Mark Gerstein’s lab here at Yale. He’s a bioinformatics expert and he helped us — using some of the rules that we were able to provide him for some of the known targets of lin-4 and let-7 — identify additional potential targets of these two microRNAs, which [we] were able to validate using C. elegans genetics. … That’s a paper that we’ve submitted currently.

What about down the road? Are there any projects that you’re thinking about tackling?

There are a few major areas that I can see my lab moving into.

The first is: In C. elegans, some of these temporally regulated microRNAs control when cells will basically terminally differentiate. So, one way to think about these is that they are actually analogous to tumor-suppressor genes in that they tell the cells basically to stop dividing. One emerging theme is that microRNAs are showing up in more and more cases as potential human tumor-suppressor genes, and in some cases as human oncogenes, as well.

We have some interesting evidence from our studies in C. elegans, [which] I don’t want to divulge right now, that show that these microRNAs control genes that, in humans, are also known to be oncogenes and tumor suppressor genes. So, one thing that we’d like to show is that this is indeed true in human cells, themselves — that some of these genes are actually controlled by microRNAs. That’s an area that we’re moving into right now.

A second area is to try to show whether some of these temporally regulated microRNAs are also temporally regulated in mammalian systems. We have, for example, identified mouse homologs to some of these regulated microRNAs and we’ve begun to look in the mouse to see whether these microRNAs are also temporally regulated. In most cases, we see a one-to-one concordance — that is, if it’s temporally regulated in C. elegans, it also seems to be temporally regulated in the mouse, which I think is an amazing conservation of gene regulation and may actually point us to a conservation in gene function, as well.

In terms of what’s going on in the field right now, is there any particular work going on that you see as noteworthy?

One thing that’s definitely being fleshed out right now is the relationship between siRNAs and microRNAs, and the fact that microRNAs and siRNAs can both be found in RISC.

But there definitely seems to be this discord in the field as to how many different RISC molecules exist and whether there are specific RISC molecules for microRNAs and RISC molecules for siRNAs. That’s a very interesting area that needs to be solved.

In C. elegans, there are multiple of these Argonaute/PAZ/Piwi domain proteins, of which AGO1 and RDE1 are actually members of. AGO1 is thought to be part of human and Drosophila RISC, but C. elegans have up to 25 of these molecules suggesting that there might be up to 25 different RISC complexes. That means there could be a lot of different diversity of gene regulation in C. elegans, and maybe even in other species. That would be a very interesting area to try and figure out.

That would bring us, I hope, to understanding how microRNAs function in one particular way and how siRNAs function in another way.

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