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Web Conference with Dr. Craig Mello Web Conference with Dr. Craig Mello - 00:00 So welcome to my Cell Physiology class, and this is-- - Hi there. - Hi! - 00:09 So I really appreciate your time, and I know you've had a lot of requests to meet and talk. So in the interest of time, we'll just get started with the questions about RNAi. And you should know that my class has read your original 1998 paper in Nature, critically analyzed it, tore it apart, and found absolutely nothing wrong with it, of course. But so, the first question is, just briefly tell us a bit about yourself, your career path over the years, and specifically what led you to the discovery of RNA interference. Thanks a lot for joining us. - 00:48 Okay, sure. Well, it's hard to figure out where to start with a question like that, you know. I started out, as a kid, being very interested in all kinds of different sciences. My dad is a paleontologist. My mom was an artist. And they did a good job, I think, of keeping that sort of natural curiosity that you usually, I think, most kids have growing up. They helped, kept that alive for me but you know I always knew I wanted to have a career where I would spend my time trying to learn more about the world, how it works. And I remember as a kid being really captivated by the notion of deep time, 'cause my dad was a paleontologist so the exposure to the museum, the Smithsonian Museum when I was a kid and basically that, seeing the history of life and thinking about the history of life and the origins of us and all that, the human condition, how we got here, those were all fascinating to me and I wanted to be able to spend my career as I went through college I realized that we could essentially see evidence of our evolution in our history, in our relatedness to other living things through the genetic material, the DNA and through molecular biology we could actually learn a great deal about living things. And even make medicines like insulin that's basically a human hormone that's produced in bacterial cells, that that could be that the bacterial cells could read the human Web Conference with Dr. Craig Mello genetic code. And I read that actually in a newspaper when I was a high school student, and that is what made me want to become a scientist, or not a scientist but a molecular biologist and that's what I pursued ultimately. In college I guess I majored in biochemistry and then in graduate school I went on to essentially do genetics and molecular biology and that sort of led up to my work as a C. elegans researcher. C. elegans is really a simple model organism that had been developed by Sydney Brenner back in around the early '70s as a genetic model system, essentially for its simplicity. It has only 1,000 cells, whereas humans have about 10 trillion, so I guess I started in 1982 at graduate school working on this system at a time when no one had ever injected DNA into it successfully. I remember I told you I got interested in molecular biology having read about the ability of the bacterial cells make and that process of putting a gene into bacterial cells you know was something that hadn't been worked out for other types of organisms really. I mean you could do it for bacteria and yeast but you couldn't really do it for more complex animals. The earliest work on flies and mice delivering DNA into those types of organisms was just beginning. And C. elegans hadn't been transformed as we call it with DNA at that time, so I got interested in that and began working on that. Meanwhile Andrew Fire was also working in that same area, developing techniques for injecting DNA into C. elegans and so the two of us were working in different locations on the same problem. And over the course of the next few years, Andy and I got it working for C. elegans to the extent that you could now inject DNA very easily and reproducibly get the genes that you're injecting into the animal to be expressed. And all that technology, I don't know if you've had that in molecular biology already, but that technology, putting the promoter on the gene, in front of the gene so you can drive it in the animal, in the right tissue, and so on is all sort of worked out now. But it was a bunch of technology that Andy and I were developing during that time, got to know each other very well and develop the techniques for injecting. And these animals are really small, they're only a millimeter long so the injections are done under a microscope and so we were doing that kind of work together and developing technology is, first of all, it's extremely important for biology but second it's also Web Conference with Dr. Craig Mello something that's very difficult because when you fail, usually you don't know why it didn't work. You know, no one's ever done this before and you're trying to get it to work. And so Andy and I talked a lot about solving problems and how to get this really critical tool working for the organism. So we developed trust and a relationship during that time that I think was key to our later collaboration on RNAi. In RNAi, the interference phenomenon was first noticed by Su Guo and Ken Kemphues when they did injections, essentially trying to do antisense, which is a technique where you basically, putting lots of complementary RNA to the transcript and try to inhibit it. They noticed that it worked whether they used the antisense or sense and they were using the same injection technology that Andy and I had developed for DNA delivery only they were injecting RNA. And so when Andy and I naturally were already the best at that procedure and we both became interested in essentially trying to understand what was happening. And we began our collaboration then and there. I could go on and give my hour long seminar but I think I probably should let you ask another question, but that's basically how it led up to that. - Great. Thank you for the history there. We'll start student questions now. And each student will come up to the computer here. Read the question to you, and then we'll move on. So our first student is Jesse. - Hi Dr. Mello. - Hi Jessie. - 08:14 Can you please describe your current model of RNA interference, and what types of approaches your lab has taken in the past few years to achieve this model? - 08:26 Okay. Models are yeah, they're really important. That's a good question. Basically we're always trying to come up with new models. We have probably eight or nine models at any given time. And quite often they are contradictory and they couldn't Web Conference with Dr. Craig Mello both be right. So you basically have a model that's a working model, you know, all the parts of it fit, the pieces fit together, you draw little circles and arrows and everything and it all makes sense. But you realize it's a work in progress. So that's where we are right now with RNAi. And in C. elegans RNAi works a little bit differently from other organisms, other animals, in that there's an amplification step involved in the silencing which probably helps explain why RNAi was discovered in C. elegans and also related phenomena were discovered in plants and fungi because those organisms all have a very potent amplification step where they respond to foreign double-stranded RNA and they have an enzyme called RNA-dependent RNA polymerase that amplifies silencing signals. So we had basically in our model, we have these enzymes called Argonautes. I don't know if you've read about those. Certainly in the 1998 paper there's no mention of them because we hadn't discovered them yet. 1999 we identified the first gene involved in RNAi in C. elegans and it turned out to be this highly conserved gene which had been identified as a developmentally important gene in plants. And it had been named by the plant people as Argonaute as the plant gene name. We named it RNAi-deficient gene number one. Kind of a boring name, Rdg1. And our name didn't catch, their name caught. The Argonaute name is what people usually refer to this group of enzymes as, and these are enzymes that hold on to the little RNA, they're called the siRNA and they use it to hunt for and search out targets in the cell by base pairing. Base pairing is a very efficient way of identifying a target because as you know there's a lot of information in each of those interactions between the base bair interaction so by a high degree of sequence specificity it allows even a short RNA to uniquely identify almost any gene in the genome. So the mechanism basically involves this protein called an Argonaute that holds on to the small RNA and uses the sequence information in the small RNA to identify other sequences that can base bair with it, forming those hydrogen bonds that make up the Watson-Crick base pairing in the DNA, only it's an RNA-RNA base pairing interaction in this case which is really very similar, even a little bit stronger than the DNA base pairing interaction.
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