Scientist Stories: Craig Mello, RNAi & Rethinking Gene Expression, Evolution, and Medicine

Good afternoon. It's a great pleasure to welcome you all in the name of the Center for Genetic Medicine to the 2008 Richard A. Scott Lecture. And I would like to especially welcome Mrs. Ann Laxand Scott and Dr. Scott's brother Andy Scott. This lecture series is funded by a bequest from Richard A. Scott and generous donations by his wife. And the goal of the lecture series is to bring internationally recognized scientists to Northwestern who have made major contributions to cutting-edge genetics research. Previous Scott Lectures have included Susan Lindquist, David Altshuler, Aravinda Chakravarti, Dr. Botstein, and Dr. Brown, among others. And it is a special pleasure today to have Dr. Craig Mello, Nobel Vice-Printer 2006, among us. I would like to call now on Dr. Jim Cramer, who is going to introduce Dr. Mello formally.

Well, it's certainly my great pleasure to be able to introduce Dr. Craig Mello as today's Scott Lecture presenter. Craig is truly an outstanding scientist in many ways, not only because of the intellectual impact, I think, of his work, but also because of the very generous and interactive way he deals with other scientists. I mean, he's very much a contributor to the community of scientists in the C. elegans field, but also in the broader field of science. And I think an example to look up to. I first met Craig in 1982 when he started his graduate work in David Hirsch's lab at the University of Colorado. Craig was working with Dan Stinchcomb, another postdoc in the lab, on developing DNA transformation for C. elegans, which was something that was critical to make the system really viable. Dan left Colorado and took a position at Harvard, and Craig moved with him to complete his PhD work there. Where he also was mentored with Victor Ambrose, another worm researcher who has been involved in some of the same avenues of microRNA that Craig ended up in. Now, Craig's efforts at developing a transformation system went quite well, and I remember visiting Craig on one afternoon there at Harvard. He helped me generate transgenic lines that actually established the molecular identity of a genetic locus that we were working on at the time. So if I could do it in one afternoon, he had to have a pretty good system working, is my attitude. And his generosity went beyond that in that he actually let me sleep on his couch that night before I left for the Gordon Conference the next day. And just in case they don't have a place for you tonight, Craig, my couch is available. So the work Craig did there was published in a paper in 1991, and the methodology for generating transgenic nematodes is done the same way now as it was when Craig started it back then, and probably the most cited paper in the worm field now, other than maybe Brenner's original paper based on that methodology. So he went for his postdoc work the Fred Hutchison to work with Jim Priest, who I think arguably is probably the most insightful developmental biologist working in worms. There's others like this that you could argue, but he has this sort of innate view of how an animal, an organism develops that I think nobody else seems to have. And that worked quite well for Craig. He published multiple cutting-edge papers identifying genes involved in Cell fate and polarity determination in the early C. elegans embryo, and it did very well with it. And these are Cell, Nature, all those sorts of things. He joined the faculty at University of Massachusetts Medical School in 1994, where he's been, and he still is— and he holds the Blaise University Chair in Molecular Medicine, and in 2000 became a Howard Hughes Investigator. He's continued his work on the genes that are involved in early development of the embryo, and actually, again, he's still at the cutting edge of what's going on there. But somehow he got sidetracked by some experiments that didn't quite work out the way one would have expected. And these are based on using single-stranded RNA to do what was considered antisense RNA to interfere with gene function. And the conundrum was that the control experiment of injecting the sense strand of the RNA, which should have had no effect, worked just as well to knock down gene function as the antisense strand, amongst other things. So most of us sort of shrugged our shoulders and said, well, I don't know, I mean, you know, it works, it gives you a phenotype, so why worry? But Craig, to his credit, and Andy Fire really pursued this as potentially a novel way of understanding some mechanism people didn't— had no knowledge of. And in fact, that was the case. And they uncovered this entire system that exists in all plants and animals that does what's considered— they're named now RNA interference or RNAi. And it's been shown that it's involved importantly in multiple areas in developmental biology as well as in host defense. And I would say that the potential of manipulating the RNAi system for impacting human health is substantial, obviously. So I think there's a great future in what this technology will do. So based on this sort of groundbreaking work that Craig performed, he's received many awards, which I listed a few. In 2005, the Louis Rosenstiel Award, the Gairdner Foundation Award, and the Massry Prize. In 2006, the Ehrlich and Darmstadt Prize, the Janssen Award, and finally the Nobel Prize in Physiology or Medicine, which he was awarded with Andy Fire for his work on the RNA system. So join me in greeting Craig Mello.

Thanks, Jim. Well, it's a real pleasure to be here, and it's great to see Jim again. He didn't snore or anything that night. And so I want to— let's just sort of start off right away here with my title. This is sort of the real title here to my talk is "How a Worm Won 5 Nobel Prizes in Medicine." And it's kind of a fun opportunity I've had over the last year and a half or so to go talk to people who've never heard of nematodes or, you know, they think worms are what you fish with and stuff like that. Kids in high school and everything, and it's been really fun to be able to tell people that they're related to worms and things like that, and then they have to listen to you because you've got some big prize. But it's actually the worms that have— that really are the prize winners. And here's Andy and I, and I like to show this picture first of all to thank Andy, because without him I wouldn't have gotten there. And also because this is one of the few pictures in existence where we're not talking to each other. We got there essentially by talking to each other and collaborating and sharing ideas openly, something that a lot of times people are nervous about doing in science, a little bit, you know, worried that someone would take your idea and scoop you on it and stuff like that.. But in fact, Andy and I working together to try to develop this technique for introducing DNA back into this little worm at a time when no one had been able yet to put DNA back into any animal at all. It was just, you know, we knew we had to be able to do this, but it hadn't really been worked out yet. And I remember trying lots of different things and working really hard at this, reading all of Mario Capecchi's papers where he was injecting into vertebrate cells during those early days and trying to figure out how to transform this animal with DNA using a— it's a tiny animal too. It's only about a millimeter long, about the size of a comma on a printed page. And you have to do these injections to put the needle inside the animal under a microscope, and it's really quite challenging. So everything we tried wasn't working, you know, and we became very relaxed about sharing our ideas because we knew that they were always wrong anyway, so, you know, what have you got to lose? But the great thing about Andy was, you know, he was full of ideas, a great collaborator, great person to talk to, and together we really did get our DNA transformation working very well in C. elegans so that we could put DNA back and fix what was wrong and really confirm the forward genetics, which was already very powerful. So just a plug for, you know, sharing your ideas, being open with people, talking. 9 times out of 10, you're going to benefit from that, and then 1 time out of 10, someone's going to scoop you because of it. But, you know, you're really going to benefit in the long run if you do that. Now, one of the funny things that happens when you get recognized with this kind of award is you do get invited to the White House. So I had to put this up here, caution, politically sensitive slide coming up, because it turns out that the slide that I'm gonna show after this one is actually insulting to just about everyone who I show it to. It upsets everyone, whether you're a Democrat or Republican. But the fact is, I had to really do some soul-searching because this administration, the Bush administration, has been so terrible for science. It's just been horrifying. I mean, at a time when there's so much that can be done with genetics and medicine, that, you know, and they're flatlining the budget so that we're actually declining with inflation. But I thought this is a chance to try to change that. So I decided after all to go. And, well, that's not me. That's Richard Nixon. So what could be worse than meeting Richard Nixon? That's not me either. That's Elvis. There. The president was out of town. So there were Andy and I and 5 other American Nobel laureates went there to meet the president, and we got to meet Dick Cheney. But, and so I put this up here because I didn't vote for these guys, but voting, you know, really seriously, voting isn't enough. In a democracy, vote, your vote is, is really only as good as your education. And if you really want a democracy to work, you have to do a good job of educating your young people, your leaders, whoever. And so I took this opportunity to go there as an opportunity to try to educate them. And I knew that when I got there, they were just gonna shake my hand and I'd be out the door. So I wrote a letter, a one-page letter,. And I tried to describe to, you know, George and the Vice President if he read it. I was told that the letter was on the President's desk, believe it or not. But here's some of the highlights from my letter to the President. RNAi, well, first of all, it was discovered during his administration. No other president has had an opportunity to act on RNAi and the Genome Project, which together create this opportunity to intervene in disease at a genetic level. I tried to get him excited about it and like take credit for it, like it happened under my administration. And I pointed out that it raises no ethical concerns because it's a natural process in all of our cells. You know, I didn't tell him no embryos are destroyed, but I thought I was being clever. I asked him to mention it in the State of the Union address. That didn't happen. And I appealed to his patriotism, told him that if we want to stay ahead in biomedical research, we better invest, because the DNA sequence of the human genome is up on the computers in China and India, and they are investing and they're catching up. But to this date, really, there's still no response to that letter at all. But I did take the opportunity of sending the same letter to every member of Congress as well as to every member of the Massachusetts legislature and the new governor of Massachusetts. And the response, at least from Massachusetts, has been really overwhelming. And in fact, we're gonna build this new building right here shown in green with partly with money from the state, $90 million investment from the state. And these are all the existing buildings. So we're almost increasing by like a third the amount of research space available. And what we're going to do here is try to develop medicines based on human genetics, a lot like this center that you guys have built here, the same sort of concept to try to translate the new discoveries of genetics into treatments for diseases, either through gene therapy or potentially through RNAi-based therapy or stem cell or all of them. So that's exciting. But anyway, here's the worm, the real prize winner. And the— these worms are really tiny, simple little animals. They have only about 1,000 cells in the adult, and yet they have the same diversity of cell types that we have: neurons, muscles. And clearly we shared a common ancestor with worms, probably 700, 600 million years ago. And we— the thing that we always do is we always underestimate living things. That little animal has a lot still to teach us. It's already taught us a lot, but it has a lot still to teach us. It is a very amazing little creature. And people always often make the mistake of just saying, oh, that's worms, you know, or worms are not humans, why do you work on a worm at a medical school? And I think the whole discovery of RNAi is partly because people tend to take that attitude, that these model systems are, you know, somehow fundamentally different from humans and therefore we shouldn't be working on them, or at least we shouldn't invest as much in them. There's a bias out there, but they really, because of all of the The many mechanisms inside these animals are so similar to the ones inside our cells, and they're much easier to work on. The life cycle of these animals is amazing. They go from a fertilized egg to an adult in 3 days, and in 3 more days, they produce 300 progeny by self-fertilization. Amazing, amazing, easy to culture and use in the laboratory. So I'm going to describe RNAi to you, and so I thought just in case there's some people here who are not scientists, I would show you a few movies that illustrate sort of everything you need to know about RNAi. And the fun thing about them is they're each really very short, and they were designed for totally lay audiences, so they're kind of fun to watch. The first one was designed by CBS News And it aired on public TV— not on public, on TV primetime— when the first human trials of an RNAi therapy were described. And they wanted to have a news segment where they could show, you know, how it works. And so this is 15 seconds long, believe it or not. It takes me an hour, sometimes an hour and a half, to explain RNAi. And they can do it in 15 seconds. It's just incredible. They are so good at what they do. So I thought I would just show you this. And it's so quick that I actually looped the model part of the slide a few times. You'll see coming up here. Here's the double-stranded RNA. Now watch carefully. These are the defective genes here. I always warn people, you know, because I can tell from the laughter that next time you see something like something from, you know, the realm of physics or microprocessors explained on the primetime TV that is outside your field, you better not believe it. This is just incredible. And I've given this I've shown this slide to so many audiences, a lot of them are not biology audiences, and the scary thing is that they don't laugh. But I can get them to laugh by pointing out that defective genes don't look like cheese puffs and that RNA can't chew. But other than that, this model is actually pretty accurate. And it really does capture the really amazing thing about RNAi, and that is that it is something really, really amazing and cool and active. It's something that the cell is doing. It's not just something that happens. The cell is responding to the double-stranded RNA. And it's— although it's not chewing up the DNA, it's— the double-stranded RNA is triggering a mechanism that is really, really incredibly cool. And that's the theme of the talk that that I'll actually give if I ever get to it. But then the next movie was made by PBS for NOVA Science Now, and I'm just going to show a 1.5-minute segment from that. And this has a soundtrack, so it'll sound a little loud.

So the theory is that long ago, cells developed a secret defense system which we will call the cop. What the cop does is when viruses invade and create showers of murderous recipes, The cop looks and thinks, "Hmm, some of these have a very fishy shape." It's a chemical difference which comes down to some of the viral recipes are 2 pages instead of 1, and one side is a mirror image of the other. But the point is, to the cop, there's something not right about this shape. So when they see it in that shape—

They say virus.

They say, "Uh-oh." Uh-oh. And the cop, hiya, destroys the recipe. When you say it destroys, is this— should we think like kung fu kind of thing? Is it like, hi, sort of deal?

Yeah, a little enzymatically, a little thermodynamics. Enzymatically? Enzymatic kung fu, maybe.

Yeah.

The cop destroys not only the oddly shaped version, whenever he sees that recipe, oddly shaped, regular shape, that recipe in any form must be destroyed to defeat the virus. And the interesting thing is, until 1998, Nobody knew that cells had this defense mechanism.

I was just looking at my remote audience. Hi over there. Can you see me? Yeah, hey, there's like a 1-second delay, I guess. Yeah, so the funny thing— this is another— obviously, this one is not trying to produce a scientific model for how RNAi works. Even my 7-year-old knows that there's not actually a little cop inside of every one of her cells. But it's a nice analogy, and I think it helps people understand that RNAi could be thought of as a sort of an innate immune response, a way that cells can mount a response against a virus that might have a double-stranded RNA during its life cycle. So I kind of like this movie, and I know my 7-year-old got it because she was sick one day and she came to me with this sort of like accusing voice, and she says, Daddy, how come the cop isn't working? Like it was my fault or something. And then finally, this movie is one that I kind of like because it shows you the sort of Star Wars version of the cell. We're going to fly inside the nucleus here, fly along the DNA, the supercoiled DNA inside your nucleus. And here's the DNA, it's going to open up and the RNA polymerase is going to transcribe. And I like this one because if the audience didn't laugh when the DNA looked like cheese puffs, now they get to see the DNA actually has this structure to it. And it's actually kind of similar to the RNA. The RNA is a copy of the DNA information. That's splicing going on there. And this information, of course, is going to go out into the cytoplasm here in a moment. The cytoplasm C here will encounter the chefs, which actually look like the ribosomes. They're not the little chef in the PBS thing. Ribosome is gonna jump on here and start reading the code, putting in amino acids, and then the scientist is gonna inject in here in a minute some double-stranded RNA. Here it comes. Now that Nature magazine got it wrong. This should be an A-form helix. That's okay. Here's Dicer dicing it. That's an enzyme that chops it into pieces. This isn't gonna chew. I think that's where CBS went wrong. That goes away. Now this enzyme complex will search for matching sequences and cut them. And that process can happen over and over and over again repeatedly in the cell, allowing this enzyme, which is holding a small piece of genetic code, to find and silence similar sequences that are located elsewhere in the cell. So it's a way that the cell has invented for using genetic information in this enzyme complex to search out and destroy or regulate complementary sequences in the cell. It's a lot like the internet. You know, there's so much information in the cell that you've got to have some way of sorting through it. On the internet, if you want to use it for anything, you have to be able to search it. So we've developed these search engines where you enter in a few letters of the alphabet, really one or two words is enough usually to find whatever it is that you want to find on the internet. Cells invented the same thing probably 2 billion years ago when the first argonauts, these slicer enzymes, started holding onto little pieces of RNA and using that to find complementary sequences in the cell. How does it work, and how did we get onto this mechanism? So it really started back in 1994 when I was starting up my lab, and I learned about an experiment done in another lab by Sue Guo, graduate student in Ken Kempfueh's lab. And Jim alluded to this in the introduction. She was trying to do antisense, and she did the control which at the time was injecting the sense strand. So antisense is the strand that's complementary to the message. So it'll base pair with the message. And the idea is it'll interfere with the process of reading the transcript and translating it into protein. It'll just sort of block that. So the sense RNA, because it can't pair with the sense RNA, shouldn't do that. But she reported that it— that both were causing this weird, very potent silencing effect. And everyone started using it. And I was starting my lab. We had— the worm genome was already very, very far along back in 1994. We were sort of a pilot project for the human. And so we had this great resource, all the genetic information for this animal. And now we had a way to turn off genes one at a time. It went from being really, really, really slow hard work to find a new gene and to analyze its function to being something that you could do in, in a day. So you could inject RNA into this animal, the next day you can come back and see hundreds of embryos in which you've inactivated that gene and study the functions of the gene very, very rapidly. And it was really great too because nobody else could do this yet, only worked in the worm. At the time. And so we were using it just to study gene function, and I was— because I was a new PI working in the lab alone, I sort of like missed the timing. And I told you these things develop so rapidly that I had injected into one animal and I came back a few days later and there were hundreds of progeny on the plate. I had missed one whole generation. I'd been gone 3 days or something and came back and I looked at my experiment anyway, and I noticed that the silencing that I had attempted to induce in the animal I injected one generation ago was occurring among the progeny of that animal a full generation later. Now, those of you who've worked with RNA before know that it's not supposed to last, especially not after you've injected it into an animal. It's supposed to go away pretty quickly and be digested away by the enzymes in the in there. So I remember looking at this. It was late at night in my lab. I was the only one in this whole lab by myself, and I just had this chill run down my spine because I knew something really weird was happening. Very weird, but cool. So it was heritable. The silencing was transmitted to the progeny. And because I had worked so long and hard on getting DNA transformation to work, I realized, you know, that getting a heritable effect that easily from injecting an RNA molecule was really, really strange. Okay, and it was absolutely the phenotype of the gene that I was targeting. There was no question about it being some wishy-washy thing where there was like a bump on the side of the animal. This was a Notch signaling pathway that I had knocked out. It was complete phenocopy Amazing. Okay, so that was step one. And I then I told Andy about that. I told a lot of people. I talked to a student. I got this graduate student interested in working on this. The graduate student, I started teaching him how to inject. And because it's hard to inject into the germline of the animal, that's— we were trying to silence a gene whose activity was required in the germline. If you're trying to inject DNA and you want germline transmission. You'd better inject the DNA into the germline or you get no transmission at all. So of course we were meticulously injecting the RNA into the germline of the worm to get silencing of a germline gene. That's what we thought you had to do. Well, the new student is— Sam Driver was trying to inject into the germline, and because it's hard, he was missing. I was watching on the monitor and I said, Sam, This is so weird, this phenomenon. Maybe it's going to work anyway. So let's just— we won't do anything. You missed— mark down that you missed the germline. You just injected into the intestine of this animal instead of the germline. But let's just come back and check it tomorrow anyway. So he checked it the next day. Sure enough, it had silenced the genes in the germline. So we were injecting into the intestine and seeing silencing of a germline gene. So the next thing Sam did was diluted the RNA basically 1,000-fold. So we went from like 1 mg per mL to 1 microgram per mL. And it still induced silencing, and it still caused an inherited silencing effect. And so we made a conclusion at that point that not only was this an active process, the animal was not only actively or enzymatically silencing, but it was amplifying the injected material and causing silencing that could spread from one cell to another, from one tissue to another throughout the animal. Something really, really cool was going on. And the other thing that was, of course, cool about this is that this phenomenon must involve lots of different worm genes because it was an active response in the animal. Keep in mind, we had assumed that the reason the sense and antisense would both work was because they were being copied and amplified to make more copies, and that the silencing was probably only mediated by the antisense strand because that's the only one that could carry sufficient sequence identity or information. And yet the amplification would possibly explain that. In the course of discussing these weird results with Andy, Andy suggested that the double-stranded RNA might in fact be the trigger, or a good trigger for this might be the double-stranded RNA. And sure enough, when Andy purified the double-stranded RNA and injected that directly, we got really strong silencing. And it was even possible to dilute it even further. So we didn't know any— at that point, we published a paper in 1998 describing the induction of this kind of silencing with double-stranded RNA. And to be honest, even though that paper was, you know, in retrospect an important paper, at the time we were not at all happy about it. We weren't satisfied with that at all because it was purely phenomenology. All we knew was that we were— something— inject double-stranded RNA and something cool was happening inside the organism, but we had no idea what was going on. But we did have a great way to find out. We had genetics. Remember, that's why the worm was such a great model system. We could ask the animal what genes are important for this process. And we imagined that all of these things in blue must be encoded by worm genes. These functions must be encoded by worm genes. So let's try to find those activities, those proteins and genes involved, and maybe when we find those, we'll understand the mechanism. And so Hiroaki Tabara had just joined the lab, and he had come to the lab to do the screen shown over here, which basically was a screen for maternal effect lethal mutants, and basically genes required in the mother for the production of embryos that were viable. So he wanted to find genes important for embryogenesis. And the funny thing is that he actually did that screen, but he did it absolutely in reverse. Instead of looking for genes that cause a maternal effect embryonic lethality, he started with a double-stranded RNA that triggers embryonic maternal effect embryonic lethality. So he fed animals— this is something even more amazing— Lisa Timmins in Andrew Fire's lab was didn't even tell Andy, but because she thought if you can inject in their intestine, why not just feed them the double-stranded RNA? And so she made bacteria that expresses double-stranded RNA and then fed it to worms, and it worked really well. In fact, now you can buy different flavored bacteria with one— each bacterial strain has a different double-stranded RNA segment, so you can silence every gene in the worm essentially by feeding them different bacteria. But what Hirowaki did is he made a double-stranded RNA targeting an essential maternal RNA. He fed it to the worms, and if the worm's wild type, it's going to make all dead eggs that all have the same maternal effect lethal phenotype. If he gets a loss-of-function mutation that knocks out the RNAi, it's going to be viable. So he's basically doing the screen that he planned to do exactly opposite to how he had planned to do it in terms of looking for these instead of for these. And the thing is it worked really well. He got hundreds of mutants. And we started cloning the genes and working out the pathway. And then just— I'm not going to go into that, but one— the first gene that we found turned out to be the COP, encode the COP. It's a member of this conserved family of proteins called the Argonauts. And we— and Hirowaki amazingly was able to set up that screen in 1998, and we were able to clone the gene by August of 1999. And we actually are still cloning some more— many of those genes involved in this mechanism, but this first one turned out to have a highly conserved core domain that I'll talk about in a moment. And the crystal structure was done in 2004 by Jeejun Song and Limor Joshua-Tor, and there have been elaborations on this structure as well by several other groups. But basically what this enzyme does is it holds onto a small piece of RNA, holds it very tightly at the 5' end actually, and allows it to base pair here with the messenger RNA, pushing the messenger RNA into this groove where it can be cleaved at this catalytic center. And so it's basically an RNA H-like fold, and this piece of RNA in purple can be reused, and the message or the target RNA can be cut and removed, and this can be done repeatedly. And now I stuck this up here, it dices, it slices, because the earlier step I told you is the Dicer-mediated step. And this pathway really is pretty well-conserved in quite a variety of organisms. And a lot of this was worked out in Drosophila. The Dicer gene was first described by Greg Hanin's group, but Dicer is also found in worms and is involved in this process as well. And I'll just give a little bit of an overview of that in a moment. But one of the fun things about this field is that, you know, back in 1999, we didn't know it yet, but all— many, many different groups working in different organisms from plants to fungi and even other people working in C. elegans were already working on the RNAi process in Tetrahymena even. It was just all of these parallel bodies of research were ongoing and suddenly began to converge around the genetics of the common— the commonality became apparent from the genetics. So as the genes were identified, it became clear that we were working on the same thing. And it was so fun to be watching this happen. It was like, you know, I like to compare it to the late-night commercials for the Ginsu knife where they tell you, you know, it dices, it slices, It does all these incredible things. And you, just when you want to buy it, they always say, "But there's more." And that's what it's like. You know, it's just been, you can't, you think it can't get any better than this and then it does. So here's a conserved gene silencing mechanism and everybody just finished sequencing the genome project from the human and the fly and the worm and the plant. And, you know, what are we going to do with all that? And here's this mechanism for turning genes off that the cells already had, and you think, oh, it can't get any better than this. And then it turns out that this pathway is also required for development, and that was something that came out of pursuing this family of RDE1-related proteins. So RDE1 was the first gene Hirawaki found, and and because the genome was completely finished in the worm, we could look at it and say, well, what other genes are related to RDE1 in the animal? And it turns out RDE1 has a big group of relatives here. There's about 15 right here. There's 2 over here, and there's 2 here. There's a couple more over here. This is a family tree of the RDE1 proteins. So how close you are to RDE1 indicates, you know, the closeness, the similarity at the protein level. Humans, it turns out, have 8 members of the RDE1 family. They have 4 over here, and they have 4 over here. And the worm has members of this subgroup that are very closely related to these human proteins, and the worm has members of this subgroup that are very closely related to this family. So the common ancestor of worms and humans already had these two different branches of the Argonaut family, so a very ancient gene family. And plants have members of this family, fungi, so it's an ancient family. But what's really cool about it was for the worm, here we have RDE1, and Hirowaki's analysis of RDE1 showed that when you knock that gene out, completely null mutants lacking the activity of that protein, are viable and healthy, and they look just totally fine in the laboratory, suggesting that, you know, this gene is only required for RNAi. They have no RNAi response to foreign double-stranded RNA no matter how you deliver it— by feeding, by injecting, even by transcribing the RNA directly. So this is required for the induced— experimentally induced RNAi response. But we wondered what, you know, so if this is for RNAi, what are these genes for? And, you know, maybe if we knock these out, we'll learn something about what these human proteins do. And so Ola Grishak, a graduate student in the lab, decided to do this experiment. She knocked these genes out, and that's when we we discovered the link between RNAi and another pathway discovered in worms, the microRNA pathway. And I don't have time to go into this, but it just turns out that there are naturally occurring RNA encoding genes. LIN-4 and LET-7 were the first two, but there's now 100 in the worm. There are hundreds of these in the human. They produce short RNAs. They go through a dicing step. And they make small RNAs that regulate 3' UTRs of target genes. And when Alla knocked out ALG1 and 2, these Argonaut homologs, she had to knock them out, both of them, because they're very similar, partially redundant proteins. She saw a block in the processing of LIN4 and LET7 and a reduction in the mature form, and she saw the same effect when she knocked out DICER, which had recently, at that at that point just been implicated in RNAi in Drosophila. So these pathways not only were coming together from different species, but it turned out that the worm— or, you know, this is actually a funny story too. The LIN-4 gene was worked on by Victor Ambrose, who Jim mentioned I overlapped with at Harvard. We had no idea, of course, that Victor was trying to clone a microRNA. He just thought it was an interesting gene involved in the process that he was studying, and only after I had left the lab in 1993 did he clone this gene. And it's funny because everybody thought, oh, that's just a worm thing, you know, that's what a weird thing worms do. They make these hairpin RNAs that— because Victor had all of this in his '93 paper. So, but what happened then, of course, with RNAi and with the identification of the Let-7 gene, which has a human homolog that's fully conserved. Then there was this bonanza of looking for microRNAs in all the different genomes that have been sequenced. And of course, the human, as you know, has a lot of these as well. So RNAi is related to a gene regulatory mechanism, the microRNAs, and they have lots of interesting roles in development, in cancer, in all kinds of processes. Almost like a transcription factor. And the funny thing is, you know, like the movie says, we didn't even know these things existed until sort of the RNAi pathway came along and helped to lead to their discovery. And they're not necessarily their discovery, but their universality, the fact that these exist in humans, not just worms. So getting back to one of those amazing things that we noticed early on, inheritance the transmission of the silencing from one generation to the next. And this is something that we've remained interested in and we're still trying to sort out. But early on, we had a suspicion that this inheritance might involve these really interesting structures that are present in the germline at all stages during C. elegans germline development. So this is a 4-cell stage embryo. These are the nuclei here in blue. And you can see this cell here with all these, you know, beautiful little green dots inside of it. That's the only immortal cell in this embryo. That's the cell that has been, you know, making more worms over the last, you know, 100 million, 300 million, I don't know, go back as long as you want, billions of years. Of evolution, this lineage has had no breaks in it until, of course, we killed it when we fixed it, unfortunately. But I mean, it's an amazing thing. If you look inside this cell, it has these amazing little structures. These are RNA protein granules that were first identified by Susan Strome and Bill Wood in worms and were also identified in plants and, I mean, in Drosophila and a variety of other metazoans. I don't think they're in plants. But basically, these structures have RNA and protein, and they track with the germline. Could they be involved in the transmission of these small RNAs? So this was an experiment done by Darrell Conti in the lab. This is just another picture showing these RNP granules during times in development when the germline is transcriptionally active. These structures sit on the nuclear envelope and they seem to be associated with nuclear pores. And when transcription stops, they seem to fall off. So there may be some association of these structures with the actively transcribing germ nuclei. And one idea is that they might be storage granules where messenger RNAs go that are going to be stored for later use during embryogenesis. That don't need to be translated right away. And there's a lot of transcripts of that nature that are expressed in the germline. So might they also have a role in these kinds of gene silencing mechanisms? And it turns out this picture actually shows wild-type animal expressing a GFP that is expressed in— that has a promoter that drives expression of its cognate gene in all tissues. But when you make a transgene using this promoter, it's only expressed in the soma. It's silenced in the germline through a process called transgene silencing, which is related to the RNAi pathway. In PIGL1 mutants, this is the gene product that I showed you in the previous picture that localizes— the PIGL1 protein localizes to these polar granules. It's an RRM-containing protein. Protein. Animals which have a mutation in this exhibit expression. You can see here's the germ nuclei. In wild-type, there's no expression. In the PIGL1 mutant, you can see these nuclei are expressing the transgene. So there's a defect in an RNAi-related pathway in PIGL1 mutants. I don't know if you can see that. The lights are a little bright. Maybe we'll need to lower them a little bit for for some of the later slides. Here's PIKL1 mutants showing that if you look for RNAi— here's a wild-type animal, N2, and a PIKL1 mutant targeting a gene that's expressed in the soma. And what we see is that if you look for silencing in the soma, PIKL1 mutants exhibit a full 100% silencing of the target. If you look for transmission of silencing, you can see that wild-type animals transmit the silencing efficiently, but the first-generation progeny of a PIGL1 homozygous animal fail to exhibit transmission. I should add that PIGL1 mutants are semi-fertile. They're a little bit— there's a little bit of sterility in the strain. That's actually a gene cloned by Jim Kraemerlet, too. Now it turns out that it— POS1 siRNAs is a maternal RNA that we're targeting. If you target wild-type animals, you can see siRNAs that accumulate. Those siRNAs can be detected in embryos of a wild-type animal. So you can— you basically feed double-stranded RNA to a hermaphrodite, you harvest the eggs, and you look at the small RNAs that are present inside the embryos. Produced by the mother that was exposed to the double-stranded RNA. You can see siRNAs are present in wild-type but are missing in the PIGL1 mutant, again suggesting that the PIGL1 defect, this RRM-related protein that localizes to the p granules, might have some role in the transmission of these siRNAs from one generation to the next. So while we were doing that, Weifeng Gu in the lab postdoc, noticed something interesting down at the bottom of one of his gels, and that's shown here. These are mutants that are deficient in RNAi, and here's wild-type, and here's a Dicer mutant. And what he noticed is that right here— I don't know if you can see my pointer in the other room, but right here there's a band that's missing in the mutants. So these mutants are defective in RNAi. But what was interesting about this is they're missing a band even though we're not exposing them to any foreign double-stranded RNA. These are just wild-type worms living in the laboratory under conditions where they're not exposed, as far as we know, to any foreign double-stranded RNA trigger. And yet they have this abundant— well, this is— take my word for it, it's pretty abundant. Small RNA species, probably on a molar ratio equivalent to tRNA. It's very abundant in the germline. And there's even another thing down here that is harder to see, but these are also present. And so Weifeng started analyzing these to try to find out what they are, and he realized they have a 5' triphosphate on the end. And I'll tell you a little bit more about that in a moment. But first I have to explain where that 5' triphosphate is coming from. And to do that, I'll just sort of go through this model that explains how RNAi works. Remember I told you it's amplified. Well, that amplification involves an RDRP. This is RDE1. It interacts with DICER, and it gets loaded with siRNAs that are derived from the trigger double-stranded RNA. So worms eat double-stranded RNA. In, for example, the RNA that they get gets processed by DICER. It gets loaded onto RDE1, and then RDE1 goes searching for targets. And when it finds a target, presumably it cleaves that target, and it recruits RDRP. And interestingly, this primary siRNA complex is very low abundance. So these are actually subthreshold for detection based on any technique that we've been able to use. And yet these RDE1 siRNA complex are interacting with siRNAs derived from the ingested double-stranded RNA that has somehow made its way into target tissues in the animal. So there's a transport mechanism going on that that we haven't worked on. There's a little work that's been done on that, but it's quite interesting. But they're very low abundance. And then these RdRPs amplify the silencing signal, making more siRNAs. But the interesting thing is that Dicer is not required again. So when Dicer processes these guys, it leaves a 5' monophosphate because that's what the ribonuclease 3 domain does, but these guys have a triphosphorylated terminal nucleotide, and that's one of the features that distinguishes this endogenous class of small RNAs. They ultimately get loaded onto another Argonaut family, the worm AGOs, and I'll show you those in a minute. And they do this second loading step without involving Dicer apparently. And we don't have any idea how the RDRP enzyme loads the Argonaut. And I should point out that humans don't have this RDRP. It helps explain why the worm is so good at doing this type of silencing. So here is the family tree of Argonauts again. And it turns out the WAGO Argonauts, this expanded group of Argonauts, is shown here, and these are the Argonauts that are interacting with the small RNAs that Weifeng identified on his blot. And this one in particular is one that I'll talk about in a moment. Well, here it is. This is the GFP tagged onto this one, this Argonaut shows this distribution pattern. These are the same P granules that I showed you in the earlier picture. The localization is identical. I'm not going to show you the data for that, but it basically— this Argonaut tracks with the germline where it localizes in these P granules at all stages. And so Wei Feng wanted to find out what the endogenous small RNAs are, so he devised a way of cloning them. This is just a cloning technique that he used. He identified He made a cDNA library of these small RNAs and cloned them and identified millions of small RNAs that match the genome. And the interesting thing that he learned is that these are antisense to genes. They not only— they also target transposons and repetitive sequences, but amazingly, many of the protein encoding genes in the animal have naturally occurring small RNAs that are targeting them. And that was quite intriguing. And furthermore, this just shows you that in the 11 million or so sequences so far analyzed, we've already hit 75% of the genes that the animal has total in the genome, about 22% we don't have any hits. Some genes have thousands of these endogenous small RNAs that target them. And this is just an example. Here's a messenger RNA, K02 mRNA. In wild type, the mRNA is expressed at a low level. In the mutants, it's expressed at a high level. And here's the siRNA that's present in the wild type but missing in the mutant. So this is a naturally occurring species of small RNA that targets an endogenous worm gene. And here's another example. This is an example of a gene that has thousands of these endogenous small RNAs targeting it. And in this case, miR-243 has a perfect match in the 3' UTR for this gene. It mismatches only by one at the 3' end of the microRNA. And it has— it turns out that this microRNA triggers the production of all of these, what we call secondary siRNAs. And this is work from Elaine Youngman in the lab. This is the only example of a microRNA triggering the production of these secondary RDRP-dependent siRNAs. And I'm not going to— I'm not going to go into it, but it's an interesting example of how these small RNA pathways can interact with each other because it turns out that in this case, this microRNA is doing this in conjunction with RDE1. So RDE1 is getting loaded with the microRNA and using it to identify this target. But this is the exception. So it's the only microRNA in the worm that seems to do this so far. It's not the way that these endogenous secondary siRNAs, these triphosphorylated siRNAs, are generated normally. So it looks like the animal— and I should add that the mutants that Weifeng is working on are sterile. They're temperature-dependent sterile. So whatever this mechanism is, it seems to be important for fertility. So there seems to be a general mechanism for silencing many worm genes or regulating many worm genes that's very active in this germline and that involves these endogenous gene-targeted small RNAs. And interestingly, the Argonaut and the small RNAs presumably as well are localized to the pea granules in the animal. Now I'm going to just briefly touch on this Argonaut and then these two, and then I'll be done. But I wanted to just give you some more examples of ongoing studies that are leading us back towards these amazing little germline structures. And this is work from Julie Claycomb working on Caesar-1. She calls it the emperor of Argonauts, and you'll see why. It's pretty cool. This is wild-type up here, and what you see here is a tubulin GFP and a histone GFP, and that's the metaphase process. You can watch the cells dividing in this movie. This is wild-type. You can watch these cells go through mitosis. Watch there, you can see the chromosomes segregating. Beautiful, everything works great. In the Caeser1 mutant, there's a real catastrophe. There's really no good metaphase here, and the chromosomes are lagging and seem to be caught in between the poles. Interesting phenotype. And it turns out that Caesr1 mutants is— the gene is absolutely essential. If you knock out Caesr1, the animals are sterile. Completely. And actually, the eggs that are made undergo this chromosomal missegregation phenotype. So if you look at the kinetochore, you can see in wild type that the kinetochore is aligned on the poleward faces very beautifully at metaphase. But in the caesar1 mutant, the kinetochore proteins are all throughout the chromosome and don't ever become oriented on the poleward face. We don't know yet how this works, whether it's a direct effect or an indirect effect, and we're still in the process of identifying the small RNA species that interact with caesar1 to mediate perhaps this centromeric organization function. We suspect it might be a direct effect based on some of the nice work that's already been done in pombe. Now this picture shows where Caesr1 is located inside the embryo. This is Caesr1 localization at the 28-cell stage where this is the germline cell and you can see the P granules here. This is the PIK1 localization shown here and the merge. And then if you look at PIK1 localization in wild-type and Caesr1 mutants, I'll blow that up. Here's wild-type. This beautiful perinuclear distribution of PIGL1 protein. That's the first protein I talked about. It's not an Argonaut, but it's a component of the P granules themselves that was previously identified. Now, the question Julie asked is, what happens when you mutate Caeser1? What happens to the P granules? And interestingly, they seem to fall off. Periphery. This is a germline of a Caeser1 mutant, and you can see that the P granules have aggregated and have lost their beautiful perinuclear distribution. So there's something fundamental about the Argonauts in this case and its role in organizing the P granule as a whole. And so this is the second Argonaut that I'll show you, localizes to the P granule and has some role. In fertility and in chromosome behavior. All right, and so finally I'm going to talk about these piwi Argonauts, which are actually the most mysterious of them all. And this is work from Pedro Batista, who, you know, got interested in this 21U band that's shown here. You can barely see it on the gel, but he wanted to figure out— this was identified by Dave Bartell's group, and the question was, is this 21-nucleotide-long RNA species that they identified in their sequencing, is it in the RNAi pathway, or is it just 21 nucleotides long and somehow unrelated to the RNAi pathway? So the 22 Gs all start with a G at the 5' end. The 21 Us all start with a U at the 5' end. We're not really sure why. But the amazing thing about these is that they— there's several amazing things, but one of them is that they all— there's 15— over 15,000 of these that we identified along with Dave Bartel in a collaborative effort defined among 30 million small RNA clones that we've sequenced. And they map to chromosome 4 in two broad regions. This is a 2 million nucleotide base pair long region of chromosome 4. And there's a 3 million base pair region. And almost all of these 15,000 small RNAs map to this one chromosome in these two regions. And there's so many of these within these regions that there's another 21U locus every 300 nucleotides for 2 million base pairs. Of this chromosome, and we don't know why. And I'll show you this. You can see if you blow up, you know, just this one little region of chromosome 4, you can see a regular old-looking protein encoding gene. These are the exons and the introns, and inside the introns you can see there are 21 Us that are in either strand. They're also in the intergenic regions. They don't overlap with the coding region. In general, they're always in the intron and non-coding region. Here's another example where you can see a big protein encoding gene, and within the introns you can see 21U loci generating loci here, and then between genes you see them here. And so what are these doing? Why— you know, there's just many more questions than answers at this point. One of the curious things about these loci is that they have an upstream motif that's conserved. So they look like they have like a little, maybe a promoter element or an element that might be involved in their processing. But they don't have a hairpin-like structure. They can't fold into a microRNA precursor. They don't require Dicer for their biogenesis. I guess I'm going to show that in a moment. And they don't show conservation, even with a closely related worm, Briggsii, you see conservation of the upstream motif, but not of the 21U itself. So quite mysterious. What could these be doing? So here's just showing that they don't depend on Dicer, whereas a microRNA does depend on Dicer. So far, prg1 is the only gene absolutely required for the biogenesis. That's the Argonaut itself, is so far the only gene in this pathway. We don't know what the polymerase is that drives the production of these. We don't know how they get processed or loaded onto the Argonaut. So the idea that Argonauts always interact with Dicer products is wrong. It turns out that vertebrates also have these, as I'll tell you in a moment. So out of 15,000 some loci, we've only been able to find a target for one, and that's here. So here's a transposon element, and there's a locus right here, a position in the— near the inverted repeat that is perfectly complementary to a 21-U RNA. And that 21-U RNA, when you mutate PRG1 and you lose the 21-U RNAs, you see the loss of these— these are 22G siRNAs that are derived from the inverted repeats. So there's a 21U here, and we noticed that this species of small RNAs from the repeat region seemed to be missing in the PRG1 mutants. Now we know from sequencing the PRG1 immune complex that it interacts only PRG1 only interacts with the 21U RNAs. It doesn't interact with these. These are in the 22G class, and there are hundreds of these siRNAs that match to TC3. So what we believe is happening is that the 21U is functioning, in this case, like the microRNA did in the previous example from Elaine's work, where the microRNA 243 triggered the RDRP-dependent production of the triphosphorylated siRNAs from that gene I showed you, which was that rare example. In this case, the 21U seems to be recruiting RDRP to make siRNAs that target the inverted repeats of the transposon. Now, invertebrates, the pi— we call them— I've got a phone call on my computer. It's not my computer. It's your computer over here. So I don't know how to shut that. All right. So anyway, the— it's probably somebody telling me I'm over time. How do I stop it? Help? All right. They hung up. All right. End call? All right. It's annoying to me. I don't know about you.

Oh. See, there's always something you don't know about in biology.

I just mutated it. Oh, no. I think I ended the phone call to the remote site. Someone might want to dial them back in. I feel bad. Maybe they were calling because they got cut off or something. Anyway, so this looks like another example where the 21U RNA is recruiting RDRP, and that's driving the production of these secondary siRNAs that target these inverted repeats. And interestingly, Eric Miska's group has shown that TC3, among all the various transposons in the worm genome, is the only transposon that's activated in the mutants that lack the 21Us. So they seem to be playing a role in transposon surveillance, but only of this one transposon. And it's quite a mysterious pathway. So the other thing that's interesting about these 21Us is considering their sequence diversity, there's 15,000 of these, and they don't target anything but themselves perfectly, with the exception of the TC3. So what are the other guys doing, and are they important for some biological mechanism? It turns out that PRG1 mutants are sterile at high temperature. And we're trying now to figure out why they're sterile and investigate it further. And there are a lot of models for how they might be playing some general role in the germline. One model that I think is very attractive is that they might be used in the germline to uncouple transcription and translation. As you can imagine, the germline is transcribing a lot of genes that are required later on for embryogenesis. In the case of C. elegans, they can run most of development off of maternally loaded RNA and protein. And so one model is that the animal has come up with a way of uncoupling transcription and translation by simply imposing a general negative regulatory step involving Argonauts. So one possibility is that the PRG1 21-U RNA complexes use that sequence diversity to silence all the genes, all of the messages that are transcribed, and they might use a mechanism like microRNAs where they have a seed region match. And because of their great numbers and the sequence diversity, they could essentially silence all genes. And then when the right developmental time occurs, you could have derepression of that general silencing and activate the translation of these maternally loaded RNAs. And that might be something that they're doing, might be why they are required for fertility. Another possibility, and this one is basically an untestable model at this point, that they could be targeting the silencing of pathogenic transcripts derived from viruses that the worms encounter in the environment that are not resident in the worm genome. So we can't see them in the laboratory because they don't leave their DNA in the organism. Unfortunately, we haven't been able to identify a virus yet for C. elegans. Now this is where Prg1 localizes, and if I can have the lights down a little bit, you can see that it has the same perinuclear distribution again as P granules. In fact, it overlaps completely with P granules It's present at all stages in the germline with the exception of the mature sperm, which is also the only time in the germline that the P granule itself is not visible. Interestingly, though, the sperm can transmit RNA silencing. So even though the— at least this Argonaut, this PRG1 Argonaut is not there, it may be that there's another Argonaut that is in the sperm. We just haven't found it yet, or there might be some other mechanism of inheritance. They also track with the germline at every stage. Here's a 4-cell stage and a 2-cell stage. So here's the third Argonaut protein interacting with yet a different, very interesting novel species of small RNA that tracks with the germline. And this is beginning to make us suspect that these P granules may have some general conserved role in the germline, and that maybe these related structures present in other animals have a similar function, and that they might be controlling gene expression in some general way. I already talked about that sort of global suppression model. Now this is not just a worm thing. Interestingly, even before we got onto this topic, what were called piRNAs or piwiRNAs were identified in vertebrate cells from the mouse testes primarily. And here, I've been trying to convince you that you can see this band, and maybe you believe me, maybe not. But I know you can see this one. And the interesting thing about these is, again, these are, in this case, larger 30 nucleotides. They're extremely extremely abundant. You can see them in these gels. This is just an ethidium stain gel. So you're basically just looking at total RNA. You know, tRNA is up here, and you can see these piwi RNAs down here. They're extremely abundant. And the interesting thing is they also, in the mouse, seem to have a really wide diversity of sequence. And they derive from loci that produce thousands of piwi small RNAs whose targets are unknown. In many cases, there are known targets in the vertebrate. Many transposons are targeted by a subset of the piwi small RNAs. But there's another subset called the meiotic piRNAs whose sequence diversity is huge, at least hundreds of thousands of these small RNAs exist in the mouse, and their targets are unknown. So they may be doing something very important in spermatogenesis. And here's from zwi. This is zwi from zebrafish, which is a member of this green family, the green branch of the Argonaut family that includes PRG1. The other piwi-related argonauts. And you can see that in zebrafish, it localizes, as was shown by Howing et al., to the germline, where it localizes in these perinuclear structures. These structures also contain this helicase called WASA, which is one of the key components of these granules first identified in Drosophila. So it looks like a conserved mechanism exists in germ cells where these cytoplasmic structures contain argonautes and presumably small RNAs involved in some way in germline functions. Now, I show this slide. Don't look at the hat. I don't know why there's a hat there. But one of the interesting things about all of these mutants So the mutants that Wei Feng was working on are all temperature-sensitive sterile. The Argonauts in that pathway, the WAGO Argonauts, if you mutate them, you can actually knock out 12 of them and make a 12-fold mutant. The animal's homozygous viable, but it's temperature-sensitive sterile. So if you raise the temperature up to 20 degrees, they're completely sterile. They're viable at 15. PRG1 mutants are viable at 15, they're sterile at 25. PIGL1 mutants are viable at 15, they're sterile at 25. There's a germ— so the basic mechanism is there's a germline process that's temperature sensitive. And it's interesting because I think you'll— those of you who are males probably realize that this is not a good place to put the testes in— you know, this is the best argument against intelligent design that's out there. In fish, the gonads, the male gonads, are next to the heart. That's where your testes should be, tucked away inside the body cavity, safely protected. But when endothermy arose a couple hundred, 300 million years ago, the common ancestor probably had a problem. It's the— it turns out the internal core body temperature of a human is lethal to sperm. Spermatogenesis needs to occur at a lower temperature, and it's probably— this is just speculation, but it's probably true that it was too hard for these organisms to change that process for some reason, and so they squeezed the testes outside the body cavity and developed all of this sort of convoluted mechanism to maintain the temperature of the testes to permit sperm development. And we suspect that the Argonauts and the small RNAs, the piRNAs, are hyperexpressed in the testes in the mammal because they're trying to cope with the change in temperature at which they're being asked to function. In the mammal. And that same process that may underlie the temperature sensitivity of all of the mutant lines in the worm that are deficient in this mechanism. So it'll be interesting to sort out what that process is, but it may have to do with programming chromatin that has to occur during maturation of the sperm or something like that. And I think one of the themes I'm going to try to wrap up here— I'm sorry it's taking me a little bit longer than usual, is that it's not just about the DNA. There's more information in cells, that there's actually information in the interaction between the DNA, the chromatin, and these kinds of small RNAs. And as I told you earlier, we can induce silencing experimentally in this worm, and that silencing can be transmitted in the germline, without any underlying mutation, we can carry out that inheritance forever. So we've passed 30, 40 generations worth of animals where the silent gene stays off. It reverts at high frequency. So it's not a DNA mutation. It's an epigenetic silencing that can be transmitted for multiple generations, presumably because these small RNA pathways can lead to some sort of a feedback mechanism that allows them to be reinforced indefinitely. So you can— that's an example of almost Lamarckian type of inheritance, an inheritance of a trait acquired during the life of an animal. I think I'm just going to stop there because I've already said all this and take your questions if there are any. But the point is that there's information perhaps in the three-dimensional interaction between the DNA, the chromatin, and the RNA, and that that information may be acted on by evolution. It would be interesting to see if that's the case. I've already acknowledged the people who did the work, so I'll stop there and take any questions. Thank you. I think we lost the remote site, but if there— anyone has a question there, they can call me on the phone.

Great, thank you very much for this fascinating excursion into the RNA world, and I hope you all remember to vote. Yeah, and educate. Communicate and collaborate. So I think this is really essential. Before we go to a few questions, I would also like to thank Dr. Michael Kennedy for organizing this event. Mike, would you please briefly stand? Truly, Mike's efforts— and he is the director of our outreach and education program, so we take this seriously. We haven't outreached the Vice President yet. Good luck. And I would also like to thank the staff of the Center for Genetic Medicine for helping to organize the event.