Craig Mello (2007) - RNAi and development in C. elegans

I think we should try to start, it is nine o’clock and I wish you all welcome to the start of the lectures for this year. The first session has already an alteration because Sir John who was supposed to be the third speaker could not come. For that reason we have made the following decision. The three remaining lectures will each one be extended by about five minutes. In addition after the first lecture we will try an experiment and do questions from the audience. And with these questions and the elongations of each of the talks we will be back in normal time again. We don’t have questions after all talks but since the first talk is by the freshest Nobel laureate, from last year, we decided that we do it after the first talk. And then also the coffee break will be after the first talk and this question round. So we will have coffee between 10.05 and 10.40. And we therefore restart ten minutes early to allow for the extension also to the second and third speaker. With these words we are then ready to start. The first speaker is Professor Craig Mello from the University of Massachusetts, the Medical School in Worcester, USA. And he got the award, as I just said, last year. He got it “for their discovery of RNA interference - gene silencing by double-stranded RNA”. Please, Professor Mello. Thank you. Thank you, thank you Hans. So it’s a pleasure and a real great honor to be here. I look forward to having an opportunity to have many conversations and discussions with everyone here during the course of the next few days. So let me go ahead and I’ll get started. I’ve sort of retitled my talk. RNAi, rethinking gene regulation, evolution and medicine, or how a worm won five Nobel Prizes in medicine. I’m going to go ahead and start my talk with a sort of an unusual first slide here. I don’t know if you—can we have the lights down a little bit. This is Dick Cheney and I at the White House. When you win a prize like this one of the special benefits is you get to go meet leaders of your country or people around the world who have important roles in politics. Can I have the lights down on the stage. I think it’s too light. Can you see that alright. You can see I’m not standing too close to him. His approval rating right now in the US is about 7%, he’s the Vice President, but a lot of people think he’s the smart one. But the important thing and one of the reasons I show this is, it’s very important to vote and get involved in politics, you know, no matter whether you voted for him or not. Now if you can’t vote in your country and some of you probably can’t, it’s certainly important to educate everyone that you talk to, educate people about what’s going on in the world, talk to people, it’s very important. And one of the issues that I think is important for us to discuss here is whether it might be wise in the future to always have the Lindau meetings be interdisciplinary. Because I think one of the things that’s happening to science is, it is already very interdisciplinary. Biomedicine for example is very dependent on physics and chemistry, I think. But also political issues are very important. So I think it’s important to have a very interdisciplinary focus at the university as well as in settings like this to try to get that dialogue going. I went to Washington DC, hoping to be able to tell the Bush administration how important RNA interference is as a technology and how well it fits with the genome sequencing project, which together are really changing, along with other great technologies, the way that we do and practice medicine. And opening many opportunities for new types of medicine, linking human disease with genetics. Unfortunately all we did was have a very brief meeting in the White House and shake hands. I wanted to tell them that RNAi was discovered under their administration, that the genome sequence was finished under this administration. And I think I will still go back and talk to them again if they’ll listen. But you know, fortunately we had a change in the Senate, in the House recently in the US and things politically are definitely changing. This is Ed Kennedy and I together at a congressional visit that we had recently. There’s a lot of hope I think that the US will turn things around in the near future. And begin investing in science more broadly again. Right now, if you don’t know, the US is really very tight with funding for medical research. Now I’d like to show this slide, this is Andrew Fire and I, for several reasons. First of all, if it wasn’t for Andy, I wouldn’t be here today. He’s a tremendous colleague and a great friend and we worked together on our science, we didn’t compete. In fact, our collaboration began back in the late ‘80s. We focused at that time on delivering DNA into the organism, that we both love and work on, a very tiny worm. I’ll show you a picture of it in the next slide. But the worm is so small, it’s about the size of a comma on a printed page. And so, to inject the DNA into the animal, Andy and I had to work out ways of inserting the needle into this tiny animal under the microscope. And it was something that had never been done, of course. So working together—actually we worked independently at first—but once we got to know each other we began sharing ideas. Because it was very difficult breaking in this new technology and figuring out how to handle this very tiny animal and inject efficiently into the animal. Together we described a transformation procedure for delivering DNA that worked very, very well. And really, it was a lot of fun to work with Andy. And the other reason I like this slide is because we’re not—what we’re doing here of course is, we’re on the stage—we’re not talking. You can see Andy’s mouth is closed and so is mine. But what we really won the prize for was for talking to each other, for sharing our ideas. And for not being afraid, for trusting each other and not being afraid of being scooped. And I think that’s something that we really need a lot more of, a lot more openness and willingness to share ideas. Thank you. Now here’s C. elegans swimming back and forth in the laboratory on a culture dish. These animals are extremely beautiful. As Sydney Brenner noted when he chose this organism to study, they’re essentially transparent. So you can look right through the animal and see all the cells inside and see all kinds of detail. The other thing that’s incredible about these animals is, they’re so simple in terms of their cellular complexity. They have only about 1000 cells compared to a human where we have 10 trillion cells. So they’re a really elegant system. But when you talk about worms to people, like let’s say my neighbors in Shrewsbury, Massachusetts where I live, they would always, you know, they’d be very polite at first. Oh, you work at a medical school, that’s nice. What do you work on. Worms. And then their eyes would sort of glaze over. Why would anyone work on a worm at a medical school. They just couldn’t get it. And then, thanks to Hans’ phone call, all of a sudden my neighbors, when I tell them I work on a worm, they actually listen to me. And I think that, if anything, that has been the most satisfying thing since October 2nd, is to be able to actually tell my neighbors about worms and evolution and have them actually listen. So why is it that worms have turned out to be so important. And yeast for that matter, have turned out to be so important to human biology and medicine. And this is something that the general public does not appreciate, especially in America. I think it’s less of a problem in Europe and Asia. So to answer that question I’m going to go all the way back to the Big Bang. And I took this slide from John Mather, who I’ve gotten to know quite well, he also shared the physics prize with George Smoot in 2006. And so when we were in Stockholm together the press was always asking us. What does your discovery have in common with your discovery. You know, they discovered the Big Bang, we discovered RNA interference. How do you connect those two. It’s quite a challenge. But what they did is, they used a satellite called COBE to map the cosmic background radiation that’s just everywhere in outer space. And this is the map that they put together. And what they noticed is that everywhere you look in outer space there’s a little bit of left over radiation. They measured the temperature in the spectrum and from that they calculated an age of 13.7 billion years ago for the Big Bang. And so the connection that we came up with, or that I think is really interesting to think about, is that life exists on a cosmic scale. Life on this planet arose about close to 4, 3.5- 3.8 billion years ago. During the time that life has been evolving on this planet, actually just since the common ancestor of plants and animals, the galaxy that we’re in has rotated about four times around its axis. Life itself is remarkable in its durability and the fact that it really does exist on a cosmic scale. So, let’s look at a little bit of the history of life. Here’s the common ancestor of worms and humans. Now on this side I’m showing the planet earth going through what’s called a snowball Earth event. And there’s geological evidence in the rocks, in the earth’s crust for glaciation events of this type where the earth was completely covered with ice all the way to the equator. And I think this is a very interesting concept from a biological standpoint because of what these kinds of events would do to the common ancestor of worms and humans. So here we are, the common ancestor, a sophisticated, really good, little organism capable of doing all kinds of things. It has Hox patterning, it has all kinds of fascinating things going on inside of it like RNA interference, working just great. But it has got a big problem. The earth itself doesn’t have any land. So, where is this little guy going to live. Either thermal vents or perhaps little cracks in the ice, in the ocean. So this kind of event actually happened twice and both times there were massive extinctions. So there was evidence for an extinction here and an extinction here. And then, right after that event, we have the biologic equivalent of the Big Bang. We have the Cambrian explosion. And there has never been an adequate explanation I don’t think from the biological side for the Cambrian explosion. But I think from the geologic side these glaciation events may provide a real compelling possible explanation where, what happened here was finally the ice melted, the earth became habitable, the land masses and the shallow seas around the land became habitable and you had this real amazing diversification of life. The rapid appearance of different animal groups including the group that gave rise to humans and the groups that gave rise to the nematodes. And in fact if you go back further, the origin of plants and animals would be down off the bottom of this page. But plants and animals all have RNAi and that is the thing that I think, I feel like even my neighbors get it. You know, even the people who don’t know anything about biology, they’re afraid that. Oh, we can’t be really related to monkeys. They actually get it. And I think that it’s partly because of the President we’ve had in the US that now people are accepting of this. But the fact is that we really are—you know, as Nietzsche said, we’ve made our way from worm to man but much within us really is still worm. And I think that’s a really important aspect of what I can do as a biologist to sort of get across that message of our common ancestry. Not only are we related to worms but everyone in this room is closely, closely related to each other. So mankind has to get over these nationalistic barriers and start thinking about each other as brothers and sisters. Now here’s what the little worms encounter sometimes in their real environment. There turns out there are hundreds of little fungi that live in the soil that will feed on worms. And they’ve even devised these little traps, like this one here in which you can see this poor nematode has been lassoed. And I’m going to show you this little movie where you can actually see this happen. These are worms that have already been captured. But watch this one here. It’s going to swim through there and it just closes right on his tail. Here, watch this one too, there’ll be another worm coming in here. This is what they’re exposed to naturally in the environment. These animals encounter all kinds of predators. And watch, they’re just closed. I don’t really know how the fungus can sense the nematode. But it has a trigger mechanism that senses the motion. And when that happens the worm gets trapped. And then the fungus sends hyphae into the animal’s body and digests it from the inside, a terrible fate. And this drama plays out every day as you’re walking to work. There are 10 to the 9 worms per cubic yard of soil. And you don’t even realize that they’re struggling to survive as every day. A lot of you have seen this movie. In fact I was talking to several of the students from China, I think everyone in China has now seen my talk. So this is my—supposed to be— my funny slide. I have to wait for CBS News to do another documentary or something on RNAi because they provide such great material. This is the CBS News 15 second explanation of how RNAi works. Watch carefully. Here’s the double-stranded RNA. Now here are the defective genes. I can see some of you didn’t see this already, I’ve shown this so many times, I love it. Many people didn’t know that defective genes look like cheese puffs. And probably you didn’t know that the RNA can actually chew. But this is why Andrew Fire and I knew that we had to work on this mechanism. It was such an exciting problem. I’ll come back to this at the end, there are some elements of this that are actually correct. And in fact, the thing that I think is really interesting is that—even though, of course the genes don’t actually look like cheese puffs—RNA can, RNAi can direct the chromosomal elimination of DNA. If you look at the Tetrahymena as an example where the RNA is guiding chromatin modifications that then are recognized by enzymes that catalyze the cleavage of the DNA and the elimination of the DNA during the process of macro nuclear formation in the Tetrahymena. The other thing about this of course is that they’ve illustrated it as an active mechanism. And I’ll come back to that in a moment. This is another movie from NOVA. So, in this case another nice little movie. This is from NOVA scienceNOW and the movie itself is 15 minutes long, so quite a bit longer than the CBS segment. It’s actually a fairly nice movie because it gets across, even for children the concept of RNA interference as a surveillance mechanism that can silence viral genes in this case. And there’s clearly some evidence that in multiple different organisms RNAi can serve that kind of function. But even my seven year old daughter doesn’t believe that there’s a cop inside of every cell, so I think this is a safe analogy or metaphor if you will. Whereas the CBS metaphor I think is a little pseudo scientific and people might actually believe that RNA can chew and that could be bad. But this one I like a lot. Now, here’s one more movie and this is from nature. This is just like the Star Wars version. We’re flying into the nucleus of the cell here and now we’re flying along the DNA. The DNA is all super coiled there. And now it’s opened up and the polymerase is going to get on here and start making a message. This is transcription occurring. This is a movie that is very useful for high school kids and lay people who aren’t familiar with basic biology. That’s the capping of the message. This is the RNA getting spliced here. Now it’s going to be transported out of the nucleus, that’s polyadenylation. And the message is going to go out, here it comes. Now, a scientist is about to inject the RNA into the cell. If you watch, you’ll see. Oh, here’s the ribosome making protein from the message. Here’s the scientist injecting the double-stranded RNA, it should be an A form helix, not that. It’s getting cut, that’s Dicer which cuts as… Now, here’s the slicer enzyme or argonaute and this will go away, it won’t chew, don’t worry. The beauty of this is it can use—this protein complex can use the sequence information here to find perfect matches. And it can do that catalytically, so it can go on and on and on and silence thousands of transcripts. It really is remarkable. But one way that I like to describe RNAi to the lay audience is. Imagine having the internet, but no way to search it, you have no search engine, no Google, no way to find anything. That’s really the way I figure your genome would be if it wasn’t for a mechanism like RNAi. RNAi uses a little search sequence, small sequence, just like you would type into the window of your Google browser to find genes or sequences, messenger RNAs or even DNA in the cell and regulate it. And the beauty of that type of mechanism is it can coordinate related—the regulation of related genes that are separated in multiple places in the genome. So, the reason we wanted to study RNAi is we noticed that it was remarkably potent. And in fact, when you inject RNA into the worm, the first thing we noticed is that the silencing effect could be inherited. So it could be transmitted via progeny that were carrying the silencing effect producing effective progeny after a full generation. And it could even be transmitted via the sperm and effect progeny after a subsequent generation. And if you keep selecting for effected progeny this silencing effect can be inherited, apparently indefinitely in C. elegans. And this is something that we’re still very interested in understanding because this long term inheritance must reflect some sort of change at the chromatin level, most likely. It’s also systemic in that the injected RNA can spread throughout the animal. So you can put RNA into the intestine for example and the silencing effect can be observed inside the germ line. So there’s a way that these RNA molecules can move from cell to cell. And that’s something we need to learn a lot more about. When we noticed these phenomena we realized that there was an active response in the animal. As I said, the movies I showed you all have some active process. Either the cop or the RNA chewing, there’s some response in the animal to the double-stranded RNA. And for example there appears to be a transport mechanism, the silencing mechanism itself and then the amplification mechanism or the inheritance mechanism. And we still don’t understand many of these steps. But when we began working on this in C. elegans, there was an obvious approach to take to try to understand these mechanisms. And that is to use genetics. The first person to do RNA interference genetics was Hiroaki Tabara in my lab. And he set up a very elegant screen back in 1997, early 1998 in which he mutagenized animals and then placed them on bacteria expressing an essential worm gene. Now the worms are so sensitive to double-stranded RNA that they can eat it in their food and experience silencing as a result. Silencing that’s so potent that if you target a worm gene that’s essential, it will kill every egg in the wild type worm. So a gene for example required for embryogenesis can be targeted by feeding this animal bacteria that expressed the double-stranded RNA. They eat the bacteria, they silence the gene, all the eggs fail to hatch. So Hiroaki set up a very simple screen where he just looks for animals after two generations where the progeny are viable and by doing so he found lots of interesting genes involved in the RNAi mechanism. And I’ll just skip way ahead because I know I don’t have a lot of time today and tell you about the cop. The cop gene was—just so happens—was the first gene that Hiroaki identified. He named it RNA deficient gene number 1 or rde-1. And it’s now called after the first plant gene that had been identified in this family as a developmental defect in plants. The enzyme is shown here in grey in its protein structure, crystal structure that was done by Leemor Joshua-Tor and Ji-Joon Song in 2004. What you can see here is the guide RNA, the silencing RNA and the messenger RNA base pairing inside this groove in the enzyme. The base pairing event that occurs here pushes the messenger RNA up against the catalytic centre. This domain encodes an RNase H related fold that then cleaves the messenger RNA. And this is then discarded and the siRNA is allowed to then go on and silence more genes. We call these short interfering RNAs or siRNAs. They can catalyze multiple reactions like this. The thing that is funny about the RNAi field is the people who work in it obviously watch too much TV, especially the late night TV. Probably when they come home from lab the only thing on late at night are those shows that have lots of commercials for things like the Ginsu knife. Because we named the enzyme that’s upstream, is called Dicer and this one is called Slicer, so it dices and it slices. And if you’re from America, you’ve seen these commercials where they’re selling you this knife that will cut all kinds of different ways. But the thing that’s funny about those commercials is they always end—when you’re about to buy the knife, they always say there’s more, then they try to sell you the steak knives that go with it. And that’s the way it’s been working on RNAi. Every time you think it can’t get any cooler than this, something really, really interesting comes along and that’s what it’s been like. So we cloned rde-1 back in 1999. And for Andy and I that was like the eureka moment for us. Before that the fact that double-stranded RNA could trigger silencing was just phenomenology. It was interesting, but we didn’t know for sure that it was conserved or if it was just a worm thing or whether other animals would have the same kind of response. But when we cloned rde-1 we found that it was a highly conserved gene with multiple homologues. And this is just the family tree of rde-1 related proteins in the different animals. So, these are worm homologues here in red, these are other worm homologues over here and more worm homologues over here. Now it turns out humans have eight copies of the rde-1 gene, there’s four here and there’s four human genes over here. So, if you look at this, what you see is the black group of argonautes are the oldest branch of the family. These even have plant homologues in this area over here. But there are animals—almost all metazoan animals have two families. this one over here and this one over here which we call the Pee-wee family of argonautes. So the common ancestor of worms and humans already had at least two argonaute genes. When we cloned rde-1, because of the genome sequences we got all of this other information. And this is the beauty and the power of this genomic era that we’re in. You clone one gene in a model system like C. elegans and all of a sudden you have all the genes in all the organisms that have ever been sequenced. But this allowed us to ask the question. What did these highly conserved genes do. And so Alla Grishok in the lab knocked out these two genes and then analyzed the phenotype. To give you an idea of how exciting her discovery was, I have to tell you a little bit about Victor Ambros’ work. In 1993 Victor published a paper describing a little gene lin-4. The lin-4 gene had this very interesting nature in that it folds into a hairpin-like structure. And Victor described two forms of the lin-4 product. One is a 70 nucleotide precursor form that’s in this hairpin shape and the other is a 21 nucleotide RNA. So this is a naturally occurring worm gene, identified as a gene that regulates another worm gene called lin-14 that was analyzed by Gary Ruvkun’s group. Together Victor and Gary’s groups, together they worked out that lin-4 is a negative regulator of a gene, lin-14 that has complementary sites in its three prime UTR. This was the first gene which we now refer to as microRNAs, this was the first microRNA. In 1993 it was just another one of these weird things worms do. And in fact in 1994 I think Victor was denied tenure at Harvard, despite making this really exciting discovery just the year before. Everyone was very surprised. But the fact is that there was a strong bias that this was just an unusual thing that worms did and maybe it’s not relevant to humans. But something happened between then and 2000. In the year 2000 Gary Ruvkun’s group cloned the let-7 gene. And what had happened between ’93 and 2000 is we had enough human DNA sequence by that time, that you could look—Gary’s group looked and he found a perfect homologue of let-7 in the human genome sequence. So the entire 21 nucleotides in the human is identical to the 21 nucleotides in the worm. And in fact what they found is that every metazoan animal has a let-7 gene. And again let-7 seems to regulate targets by interacting with the three prime UTR. But the thing that was missing still, despite the fact that these are about the same size as siRNAs, the question remained. How are these pathways really related. Is the RNAi pathway related to the microRNA pathway or not. And Alla’s result suggested for the first time that the two pathways are related. And what she showed is that mutations in Dicer and mutations in those conserved argonautes have defects in the processing. Here’s the wild type, but in the absence of Dicer or this argonaute there’s a defect in the processing of this precursor and there’s less of this mature form accumulating in the animal. So the RNA interference mechanism which is involved in silencing genes - we use it experimentally, but it has a role also in transposon silencing and so on - has a conserved role in gene regulation, in which the worm makes double-stranded RNA by encoding it with just regular genes that then are processed into these siRNAs and can go on to regulate their targets. That was extremely exciting. And now it’s even getting more exciting in that these microRNAs are turning out to have really important roles developmentally, including some very important links to cancer biology in humans, including links in which the microRNAs are involved in preventing cell division and in suppressing cancer, sort of a tumor suppressor role for the microRNA genes or are chimiric genes, genes that promote cell division. This is a figure from Carlo Croce’s lab. It turns out that these are different patient samples and these are a set of about 100 microRNAs from the human. What they’re doing here is looking at the gene expression profile in different patient samples on this axis. You can see that certain microRNAs are up regulated in some tumors and down regulated in others. By looking at these profiles it’s actually turning out to be possible to make predictions about how a particular tumor will respond to treatment. In addition some of the microRNAs that are identified as correlated with an oncogenic state are turning out to be potential targets for suppressing tumor growth. Here’s where there’s even more. And I think that this is where the lab is still working. I’m just going to show a few slides from the work of Wai Fong who has been a new post doc in the lab. What you see here is a gel where these are tRNAs. We’re looking at RNAs. And if you look way down on the bottom of the gel there are RNA species that are here that everyone always thought was just junk. But it turns out that this is where the microRNAs run, right down here, there’s a band here that Wai Fong noticed was missing in these mutants. These are mutants that are deficient in RNAi. This is Dicer here which is required for the microRNA pathway. And you can see this band here is present in Dicer, it’s present in wild type but it’s missing in these mutants here. These are all mutations in a gene called DRH-3, Dicer-related helicase 3. Now it turns out these siRNAs are triphosphorylated, whereas Dicer, when it cleaves it, makes a monophosphate at the end because of its enzymatic action. Triphosphates would be put on by a polymerase and we believe these siRNAs are the products of a polymerase. This species here, the microRNAs are here but there’s also some other species of small RNA that’s got a 3 prime modification of some kind and we’re still analyzing those. Here’s an example of one of those, these are what we call natural endogenous small RNAs. They’re different from microRNAs in that they’re not encoded by a hairpin-like structure. This is a gene called K02. In wild type the messenger RNA for this gene is present at a low level and there’s a lot of siRNA present in the wild type animal. In the mutants that Wai Fong analyzed, the messenger RNA is now expressed at a higher level and the siRNAs are gone. So, these are naturally occurring silencing RNAs that are targeting one of the worms own genes and we don’t know why. So Wai Fong devised a strategy for sequencing these. This is just an example of how these siRNAs map from some of Wai Fong’s sequencing data. We’re just starting to sequence on a very high throughput level. Small RNAs from throughout the worm’s genome to try to find out what genes are targeted by these natural silencing RNAs. You can see this particular gene which has this long name here, has hundreds probably— or thousands of siRNAs in every cell that target the whole gene. And we don’t know how these are generated or why they exist and how they function in the animal. But we do know that out of some 6000 siRNAs that we’ve analyzed of this type we’ve identified already 3000 different genes. So most genes have just a few siRNAs targeting them in the samples that we’ve analyzed so far. So we think that this may turn out to be a very important regulatory mechanism for genes in the worm. So is it just another worm thing or not. Well, this is a mouse and this is based—we just did this experiment ourselves in our lab. But last year there were three or four papers on something called the piRNAs. These are extremely abundant in the mouse and the piRNAs are actually siRNAs that are 30 nucleotides, they’re bigger than the 21. They are 30 nucleotides long. They are so abundant that they are a blazing signal on ethidium stain gel. And it’s amazing that no one ever noticed these before. But they turn out to be interacting with the Pee-wee class, argonautes. That’s why they call them the piRNAs. And their functions are still being worked out. They appear to have a role in transposon suppression but they may have a more general role in gene regulation as well. You can probably barely see it, but here’s the band that Wai Fong has been analyzing. And there are bands here in the mouse that are similar in intensity. So there’s a lot out there still to be worked out in terms of how these small RNAs are regulating development. I’m going to come back now at the end of my talk to this concept of how the RNAs interact with the DNA. Here’s the double stranded DNA. The reason I show that slide is with the discovery of DNA I think molecular biologists sort of became overconfident. We thought we had figured out how life works, we thought we understood how information is stored inside of ourselves. The DNA explains a lot, it can explain Mendelian segregation of traits. And it can explain how the genetic material is replicated and how mutations could arise. So it’s a very powerful paradigm but I think it sort of made us overconfident. And I think RNAi is sort of bringing us back to reality a little bit. DNA doesn’t really look like that in the cell, it’s wrapped up and bundled into these higher order structures. This is a 30–nm fiber, these are the nucleosomes and here’s a crystal structure model showing the histone proteins inside and the DNA wrapping around. This is another picture here where the DNA goes around just about two times. And these little tails stick out from the histones and these tails, as you may know, are targets for multiple types of regulation. They can have a role in activating or repressing the transcription of the DNA that’s wrapped around those nucleosomes. And this information that’s put here by proteins that modify these tails is very interesting to me because it seems that siRNAs can guide these modifications by bringing to the chromatin remodeling complexes and proteins that recognize these histone tail modifications. So with that type of interaction between the chromatin and the RNA you can imagine that the DNA is more like the hardware in a computer. And that the proteins and the RNA that regulate, that interact with these histone tails are like the software. The analogy I like to make is that we think of ourselves as differentiated, right. We all have the same genome in every cell but the cells do different things. Well, why do they do different things. They do different things because the DNA, different genes are on and different genes are off and they’re on and they’re off very stably. And that’s how we remain stably differentiated creatures. The thing that I’m very interested in right now is a concept that we’re just trying to figure out how to test in the lab, that the germ line itself can differentiate and that that differentiation process allows the germ line to evolve without any changes in the underlying nucleotide sequence. So you could imagine heritable changes at this level that are maintained through the interactions with siRNAs over multiple generations. If you look back at the history of thinking on evolution prior to DNA—and this is actually going back to the turn of the century— Weismann’s theory of inheritance, he named the genes biophores. Unfortunately we all like jargon a little too much, so his word for genes was biophores. But he envisioned genes that could be replicated many, many times per cell division, not just once. And they could be segregated unequally between cells. He thought this could explain differentiation and development. And he came up with this concept of biophores which was cast aside after Mendel’s discoveries. But when you read Weismann’s theory, if you put instead of biophore the word siRNA, everywhere he used the word biophore. His theory works beautifully. So, I think we have to go back and think about evolution again and development. Darwin went even further than Weismann and came up with a word gemmules. And these were able to exit the somatic cells and enter the germ line and cause heritable changes that were… I see Hans standing up, that’s a bad sign. So I’m going to cut it short. But RNA could be, RNAi, maybe we should rename it RNA information because the RNA can guide the regulation of the DNA. Now, here’s Hans. I’m asking him to call me any time. He’s the one who makes the phone call by the way. So be nice to him. Here’s Andy and his wife Rachel. This is the class of 2006, there’s John Mather and George Smoot over there, Orhan Pamuk, Rodger Kornberg, Ed Phelps, Muhammad Yunus and a representative from the Grameen Bank. This is just a family photo with my daughter Melisa and Victoria who are here at the meeting. Here’s my wife getting some advice on economics at the banquet. And here she’s giving some advice. During the banquet Ed called my wife a communist—she grew up in Hungary. And here’s George explaining the Big Bang to me. And you can see I have this glazed look on my face because you know I think it’s a nice story but believe me there’s a lot of mysteries still in that story. And here is Victoria collecting some gold medals, these are very nice gold medals because they have chocolate inside. And here is Vickie dancing with me, it was a wonderful time there. This is the danger of one of these prizes, you can see I’m signing a chair at the Nobel Museum and you can see what's happened to my body, my head is huge and my body has gotten very small. So that’s one of the dangers. But, and I’ll tell you there is a real antidote for that and I’ll just end with this slide, this is Tara Bean, in 1998, this is her picture. She was diagnosed with a brain tumor because of vision problems that she developed in third grade. She grew up in my home town, we know her family and she was seen at the hospital where I work. And she unfortunately lost her battle with cancer. And since her death we’ve learned a lot about the basic mechanisms of cancer. We have a lot more to learn. There’s a lot of tremendous exciting work going on and a day doesn’t go by now, even here at this meeting when I don’t get an email of some kind from somebody who has got a sick loved one, someone who thinks that maybe RNAi could help them. And unfortunately the answer is still that those cures are perhaps years away and it’s very hard. So we got to get back to work, as soon as the meeting is over everybody get back to work and try to come up with some new cures. And I think I’ll end there. Sorry, I always go long. Thank you very much Craig, it was a wonderful lecture and we now learn also about the future. I felt like a bad boy walking up and interrupting you. But since we said we should have some questions and further extension, I thought we should do so. So, now this lecture is open to questions. I saw your hand first. I was promised that we have a microphone. Yes, she has one. My name is … (inaudible 50.05). I’m a medical doctor from Pakistan. You mentioned that RNAi can cause chromosomal elimination of DNA. So, is it therapeutically possible by injecting bacteria or viruses expressing double-stranded RNA to silence genes involved in the pathophysiology of cells in tumors or cancers in humans or mouse models. Well, potentially. But those silencing approaches that are being attempted now in the human are not designed to eliminate the DNA corresponding to the target gene. You can achieve knock down reduction in gene expression of a gene in a human cell using RNAi. And that is being developed as a therapeutic strategy now in multiple different companies around the world. And it’s even in human trials right now for several different indications. So it looks like it’s going to be a viable approach, whether or not you’ll ever be able to actually target the elimination of a gene effectively in a patient, I don’t know, and you may not even want to do that in most cases. So that’s probably not possible. Questions. I saw another hand very closed by and one hand over here. This is really amazing and I’m really sensitized. But in the beginning of your lecture you mentioned the importance of politics in science and I want to agree with you absolutely. I also noticed that this is a long journey what you are presenting today. And what I’d love you to mention is how much this costs, because I’m really interested in economy and this must have cost a lot of a fortune. And I know that the Bush administration has put a lot of limitation on Africa research because he felt we should not waste money on research, we should face poverty. And some of us felt research is another way out of poverty. From your experience, what would you say to this comment. Well, I’m not sure I understood your question completely. But let me just say that the kinds of drugs that you can make with RNAi, using RNAi as a drug, are going to be so expensive and so tailored to individuals that they have a lot of potential. But I really doubt they’ll penetrate to more than a few percent of the world’s population. The easy to deliver drugs are pills. And if you can develop orally available small molecule, then you can really get drugs inexpensively into patients. RNAi is being used to help discover pathways and so on, to find new druggable targets. But RNAi as a drug I think is going to be very hard to get into the Third World as a therapy because it’s an injectable. So it’s somewhat more difficult. I think that’s actually one of the things that we really need to fix. We can spend a lot of time trying to cure diseases that have never been cured. But in a lot of the world the diseases—it’s just malnutrition and diarrhea that are killing most of the people, or malaria or some well known disease that’s treatable. And the medicines are not there because of political instability or infrastructure problems. These are the kinds of things that I think require an interdisciplinary effort to try to solve medicine. We can continue to go along these paths towards more and more complex, more and more specialized medicine without benefiting mankind. Nearly as much as we could if we invested some time in helping to bring—really to reinvent economies that work for the Third World and we’re not doing that. So I think that sort of answers your question. Thank you. And then it was you, yes. Hi, I’m Shawn from … (inaudible 54.16) UK. So, I had a question, looking at the system biology aspect. I always used to think about this problem, like how the cell in terms of system biology decides which part for small RNA you should take. Like if you have viral response you have mRNAs or you have saRNA mechanism. So in terms of the wall cell concept, how would cell choose the mechanism for RNA defense. I have no idea. It’s a very interesting question. But you know, the regulation of these mechanisms is something that really just hasn’t been worked out yet. We don’t know how microRNAs really work and how they regulate their targets. We don’t even know how they—probably because they have multiple ways of silencing—we don’t know which one they—how they choose, how to either cleave the gene or just silence it or send it to a storage place. You know there are a lot of possibilities. One of the things though that's very exciting about RNAi is for the eukaryotic cell. I think it provides a mechanism for linking transcription and translation so that the cell can, while its transcribing a message, mark that message for storage and use later. A concept that would allow the cell, despite the existence of the nuclear envelope to export a message that’s been marked for use later. And that’s a really interesting concept. So there are all kinds of interesting possibilities about regulation. I think it’s going to take years to sort it all out. Any more questions. I saw your hand and then yours. I don’t think Hans can see further than … Well, thank you.(laughter) My name is Matthew Albert from INSERM in France. I’m curious to know about double-stranded RNA and single-stranded RNA with 5-prime-triphosphates. They’re known as immune adjuvants, signaling through pattern recognition molecules like toll-like receptors and intercellular helicases. I was curious to know whether or not worms have a similar mechanism for immune activation. And how you imagine sort of cell versus viral RNA could be recognized in different ways that would avoid aberrant immune responses. That’s a great question. And in fact the Dicer-related helicase that I was referring to is a homologue of RIG-I, MDA-5 and LGP-2, which all are members of the Dicer family of helicases. That's our name for it, Dicer-related helicases. But the fascinating thing about that is that those are all involved in the human innate immune response in anti-viral response. And recently the Dicer-related helicase, human versions have been linked to recognition of triphosphorylated, not siRNAs but viral products that are triphosphorylated. So there’s a homology really between the anti-viral mechanism in humans and the RNAi mechanism in worms. The worms have retained a very potent sequence specific response. The human has either lost that response or it’s much less important or it’s only in certain cell types, we don’t know which. The human cell when it recognizes a viral process will commit suicide and these same receptors are involved in recognizing the viral replication products and then activating an apoptotic pathway. So, a very efficient way to get rid of a virus if you have 10 trillion cells but if you’ve only got 1000 you better not kill the cells. So the worms may be very good at the sequence specific aspect of anti-viral or silencing. So the homology and the similarities between those mechanisms are fascinating and in fact it turns out that those LGP-2 of the three human members of this family interacts with Dicer and with another protein that we’ve identified in the worm called pure 1. It’s a phosphatase that recognizes triphosphates and removes two to make a monophosphate. There is a complex in the human that appears to have at least those three proteins. So we think that the human cell may have this mechanism as well. And we’re still trying to sort out how the human does the sequence specific silencing. Then I promised you. And after that we go further away. My name is . (inaudible 59.16). I’m from India. What I wanted to ask you was if you’re using RNA interference in the cure of cancer or the battle against cancer. Is there a mechanism or is there a method to make RNA cell-specific to suppress mitosis in the cancer cells. Is there a specific way to just target the cells in there. Because the mitotic mechanisms are the same in normal cells as well as cancerous cells. Right, cancer is a very challenging target for an RNAi therapeutic. And getting it delivered to the tumor specifically would obviously be a very important aspect because if you’re trying to interfere, say with mitosis, you’d kill the healthy cells as well. The same problem with many cancer therapies. So, it’s a problem again that hasn’t been solved. But one that people are working on. There are possible solutions but I don’t have any for you at the moment. Could we possibly get the microphone over here. Worms are extraordinarily sensitive to RNAi. I mean, they’re sensitive to it in their food. What do you think the evolutionary advantages of evolving such a sensitivity would have been. I mean, considering you don’t have this in other organisms. Why are they so sensitive that they can actually transmit it in their food. It’s very interesting. You know, these worms are hermaphroditic, they’re also capable of fertilization, cross fertilization by males. But they colonize food in the soil, quite often an individual worm will find the food and have to populate the food source without a mate. So they’ll self fertilize and make thousands and millions of progeny. When they begin to starve they do something very interesting. These mothers are very altruistic, they actually hold on to their eggs when they’re starving and they allow the eggs to hatch internally. That way the progeny hatch in the presence of an abundant food source, the mother. And then they consume the mother, they reach an age that’s old enough to become a dower larva which is a very resistant form and then they can go off in the soil to find new sources of food. The interesting thing there though is that if an animal is starving and allows the progeny to eat itself, it might have developed immunity against viruses during its lifetime that could then be transmitted to the progeny via feeding. That’s one explanation that I’ve just sort of back of an envelope idea. But I think it’s possible that—because of the way they live, they want to be able to transmit these silencing activities to their progeny. The sort of embarrassing aspect of this is that C. elegans researchers So we don’t know of any viruses that we can use to sort of test this model. There are no viral, existing viral examples from nematodes. Maybe they’re so good at silencing them. Probably it’s just not a stable interaction in the laboratory. So we lose them very quickly. Thank you very much. I’ve just thought, I should say we stop. But a final question to you. You can shout it out. I’ll hear it. Concerning the use of siRNA and therapy. Isn’t there a problem between the high specificity of siRNA and the allele variation of defective genes. For instance by using shRNA to fight against AIDS, there are a lot of HIV mutants. So, how would you imagine. Well, you could target cellular genes which are less apt to mutate, that’s one strategy. And of course you can design an siRNA against the region of the genome that for some reason can’t mutate very often. And those strategies are being tried. I think there’s an HIV trial now using gene therapy to deliver the silencing RNA. That’s either approved or will be shortly in the US. So they’re trying that approach even for HIV. But normally there’s not that much allele variation so you can choose a region. That’s not for normal genes. For viral genes—yes, there are. But for normal cellular genes there is less variability. So you can choose an siRNA that will target a conserved region. Interestingly, in the case of a dominant mutation for example that’s causing a phenotype, you can even design your siRNA so that it will only target the mutant allele. And that’s with some degree of selectivity, you can get silencing specifically of the mutant allele by designing the siRNA. So that it won’t cleave the wild type allele but only the mutant. So there’s a lot of possibilities still for using RNAi as a therapeutic. Thank you very much.

Craig Mello (2007)

RNAi and development in C. elegans

Craig Mello (2007)

RNAi and development in C. elegans

Abstract

Argonaute proteins interact with small RNAs to mediate gene silencing. C. elegans contains 27 Argonaute homologs, raising the question of what roles these genes play in RNAi and related gene-silencing pathways. Through our collaborator, Dr. Shohei Mitani, we have obtained a set of 30 deletion alleles representing all of the previously uncharacterized Argonaute genes. Analysis of single- and multiple-Argonaute mutant strains reveals essential functions in several pathways including: (i) chromosome segregation, (ii) fertility, and (iii) at least two separate steps in the RNAi pathway. We show that RDE-1 interacts with trigger-derived sense and antisense RNAs to initiate RNAi, while several other Argonaute proteins interact with amplified antisense siRNAs to mediate downstream silencing. Overexpression of downstream Argonautes enhances silencing, suggesting that these proteins are limiting for RNAi.

These downstream Argonautes also function in endogenous RNAi (endo-RNAi) pathways of unknown function. A distinct Argonaute, ERGO-1, appears to function in a manner analogous to RDE-1 at an upstream step in the endo-RNAi pathway. The ERGO-1 and RDE-1 mediated pathways appear to compete for the downstream secondary Argonautes which lack key residues required for mRNA cleavage. Thus our findings support a two-step model for RNAi, in which Argonaute proteins function sequentially, and downstream silencing is mediated by a set of Argonautes unlikely to harbor catalytic-slicer activity.

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