I’m going to talk today about C. elegans and the role of RNAi in C elegans development. This animal is aptly named for its elegant simplicity (Figure 1). Only one millimeter in length and yet capable of producing 300 progeny in three days by self fertilization. One of the most beautiful things about C. elegans, immediately apparent upon viewing it in the microscope, is its transparency. Sydney Brenner recognized the importance of this attribute when deciding what organism to work on. As animals go, C. elegans is relatively simple, having only about a thousand cells in the adult organism. Indeed, the origin and fate of every cell, both in the embryo and adult, has been determined - an amazing accomplishment. At any stage of Mello Nobel Lecture 2 development, you can look at a cell and know where that cell came from, tracing its origin back in time to the first division of the embryo.

If one looks carefully, the complexity of living things becomes strikingly clear. Consider for example the natural environment of C. elegans. Figure 3 is an electron micrograph taken by George Barron, who works on nematophagous fungi. The unfortunate worm shown here has become ensnared in a trap set by a fungus that preys on nematodes. It really is a jungle out there for these poor little animals; they struggle to survive, just like the rest of us. The soil is filled with hundreds of different species of these fungi that prey on worms as they’re swimming around in the soil. These fungi can sense the motion or contact of a worm and, after the worm has entered its lariat, the fungus inflates it to constrict the snare around the animal, trapping it. The fungus can then send hyphae into the worm to digest it. So, imagine that these poor elegant little animals are actually struggling to survive out there. Nature is filled with complexity that we don’t appreciate. This is so tiny, that you would walk over millions, if not billions, of these little creatures in the soil every day on your way to work, never realizing the things that are happening there.

Public broadcasting has the luxury of an audience that tends to have a bit more patience, and they came up with a 15 minute segment and another strategy, “the cop”, to explain RNAi (Figure 5). They describe a little policeman who looks out for viruses and other misbehavior in the cell. When he sees double-stranded RNA he realizes something is not right. He then goes on to use “enzymatic kung fu” to destroy not only the dsRNA with that sequence, but all RNAs with that sequence that he encounters in the cell.

A very exciting possibility occurred to us after we cloned rde-1. To explain this possibility, I first have to describe some fundamental facts about genes and how the amazingly successful genome-sequencing projects around the world have impacted biological research. Genes are composed of long sequences of nucleotides that specify the protein-coding potential and/or other functions of their gene products. The relationships between genes can be inferred by looking at the nucleotide sequence of the gene. For example, by using the nucleotide sequence to infer the protein-coding potential of all the known genes related to rde-1, it was possible to build what is called a phylogenetic tree (Figure 7), in which the most similar members of the gene family (often referred to as homologs) are closest to each other on the tree. Interestingly, it turns out that rde-1 is a member of a large gene family, with 26 related genes in C. elegans. Similarly, there are multiple Argonaute genes in almost every organism. The organisms from which each gene in the tree is derived are indicated by a prefix as follows: C. elegans (Ce), humans (Hs), the plant A. thaliana (At), fruit fly (Dm), and fission yeast (Sp). The genes named in black represent the most highly conserved branch of the family, with members in plants, animals and fungi. The green branch, often referred to as the Piwi family after its founding member, has family members in all animals (but not in plants or fungi). Finally, the red branch of the tree represents a C. elegans-specific subfamily of genes that are equally divergent from both the black and green families. This remarkable diversity of Argonaute genes raised the exciting possibility that different members of the family have become specialized in each organism to perform distinct functions. For example, RDE-1 according to our genetic studies is required for gene silencing in response to foreign dsRNA. Perhaps other members of the C. elegans Argonaute gene family mediate transposon and transgene silencing. Still others may function in developmental pathways related to RNAi. An outstanding graduate student, Alla Grishok (Figure 8), took on the task of trying to test these ideas.

This previous work goes back to 1993 when, after several years of trying, Victor Ambros’ lab succeeded in cloning the lin-4 gene [11]. One of the reasons lin-4 had been so hard to clone was that the gene was tiny and did not encode a protein. Instead, the lin-4 gene appeared to encode two RNA products: an ~70 nucleotide-long RNA capable of forming a double-stranded RNA molecule with a hairpin-like structure, and a single-stranded 22 nucleotide RNA that appeared to be derived from this longer RNA (Figure 9). This short RNA was capable of binding directly to sites in the transcript of the lin-14 gene, a gene that is negatively regulated by lin-4 during the normal course of worm development.

With these findings, the first link was established between RNA interference and a natural developmental mechanism for regulating gene expression. This was extremely exciting, and we envisioned a model (Figure 11), in which the RNAi and microRNA pathways utilized different members of the RDE-1 family and converged on Dicer. Downstream of Dicer these pathways appeared to diverge again, through the action of unknown effectors that direct different types of silencing, including mRNA destruction, transcriptional silencing and inhibition of translation. And yet we still had not identified the RDE-1 family member involved in transposon and transgene silencing.

The mechanism of silencing mediated by these downstream Argonautes remains unknown. It could be through mRNA destruction, but comparison of members of this group of Argonautes to RDE-1 and other members of the family suggest that these downstream Argonautes are not likely to have an intact RNA-cleaving nuclease domain. Our studies of these proteins indicate that they also function in endogenous silencing pathways (Figure 13), including pathways likely to have a role in silencing transposons, transgenes and other genes at the chromatin level [21].

It’s a beautiful system. In fact, the researchers who work in C. elegans have their own lineage. Almost all of us can trace ourselves back to Sydney Brenner, who pioneered the modern genetic analysis of this organism. My particular ancestors, if you will, in the lineage of researchers are shown in Figure 2. I owe a tremendous amount of thanks to Dan Stinchcomb, for teaching me molecular biology and really being a fantastic mentor during my initial years in graduate school; Victor Ambros who, along with Dan, provided a wonderful joint laboratory at Harvard University where I did graduate work; and then Jim Priess, who taught me genetics, and was a tremendous mentor and a great friend out in Seattle where I conducted my postdoctoral research at the Fred Hutchinson Cancer Research Center. I owe a tremendous amount to these individuals. I’ll show you more pictures of people I will need to thank as I go along.

But, one of the problems with a discovery like this one, of DNA, is that we tend to become overconfident in the explanatory power of the discovery. Does the DNA sequence information control all of the events in the cell? Cells are constantly responding to their environment and to surrounding cells, and these external influences can alter the cell in heritable ways that do not require changes in the primary sequence information in the DNA. Consider the early C. elegans embryo. During these early divisions, maternal mRNA and protein products that are stored in the egg direct numerous cell-cell signaling and differentiation events that give rise to the multicellular organism. These are exemplified by the distribution of the PIE-1 protein (Figure 4). PIE-1 tracks with, and is essential for, germline specification. As shown in this image from a movie, PIE-1 - in this case tagged with a glowing jellyfish protein - becomes localized after each division to the germ-line cell. In this two-cell embryo PIE-1 protein is localized exclusively to the posterior cell where it is concentrated in the nucleus. This occurs through a fundamental developmental process called asymmetric (unequal) cell division. As a result of this process, the two daughter cells differ with respect to their content of maternally provided products, like PIE-1. These products, in turn, can direct the subsequent development of these cells such that, once differentiated in this way, these cells remain committed to their specific tasks in the animal through numerous rounds of cell division. These remarkably stable differentation events can be maintained for the entire life of an organism without any underlying changes in the DNA sequence. The germline cells, which in C. elegans inherit PIE-1 protein, are the only cells that retain the potential to launch the developmental program again in the next generation.

The inheritance properties and systemic nature of RNAi, along with its remarkable potency in C. elegans, all pointed toward an active organismal response to the double-stranded RNA. What we wanted to do immediately, upon realizing that there was an active response in the organism, was to find the genes in the animal that encode that response. Therefore, we set out to use the powerful genetics of C. elegans to look for mutant strains defective in RNAi. We imagined that these mutants would define genes required for the recognition of the foreign double-stranded RNA, genes required for the transport of the silencing signal from cell to cell, genes required for the amplification of silencing, and genes required for the silencing apparatus itself. Hiroaki Tabara (Figure 6), was the first person doing RNAi genetics in the world. He was a courageous postdoc who came to my lab to study development, but was willing to tackle something as unusual and as odd as RNAi. The screen that he did was very simple. Basically, he mutagenized animals, let them grow for two generations until mutations that had been induced would become homozygous and then, using a trick first developed by Lisa Timmons in Andy’s lab [5], he fed the worms E. coli expressing double-stranded RNA targeting an essential worm gene. According to this strategy, if the animals have an intact RNAi response the RNAi would knock out the activity of the essential gene, causing lethality. Now, if by chance a mutant exists in the population that lacks an RNAi response, then RNAi would not occur, and the corresponding animal and its progeny would be viable. Hiroaki used this very powerful genetic selection to identify mutants defective in RNAi, and his screen worked very, very well.

A very exciting possibility occurred to us after we cloned rde-1. To explain this possibility, I first have to describe some fundamental facts about genes and how the amazingly successful genome-sequencing projects around the world have impacted biological research. Genes are composed of long sequences of nucleotides that specify the protein-coding potential and/or other functions of their gene products. The relationships between genes can be inferred by looking at the nucleotide sequence of the gene. For example, by using the nucleotide sequence to infer the protein-coding potential of all the known genes related to rde-1, it was possible to build what is called a phylogenetic tree (Figure 7), in which the most similar members of the gene family (often referred to as homologs) are closest to each other on the tree. Interestingly, it turns out that rde-1 is a member of a large gene family, with 26 related genes in C. elegans. Similarly, there are multiple Argonaute genes in almost every organism. The organisms from which each gene in the tree is derived are indicated by a prefix as follows: C. elegans (Ce), humans (Hs), the plant A. thaliana (At), fruit fly (Dm), and fission yeast (Sp). The genes named in black represent the most highly conserved branch of the family, with members in plants, animals and fungi. The green branch, often referred to as the Piwi family after its founding member, has family members in all animals (but not in plants or fungi). Finally, the red branch of the tree represents a C. elegans-specific subfamily of genes that are equally divergent from both the black and green families. This remarkable diversity of Argonaute genes raised the exciting possibility that different members of the family have become specialized in each organism to perform distinct functions. For example, RDE-1 according to our genetic studies is required for gene silencing in response to foreign dsRNA. Perhaps other members of the C. elegans Argonaute gene family mediate transposon and transgene silencing. Still others may function in developmental pathways related to RNAi. An outstanding graduate student, Alla Grishok (Figure 8), took on the task of trying to test these ideas.

Alla’s work provided an answer. When Alla knocked out alg-1 and alg-2, she observed a phenotype that was very similar to that observed when you knock out let-7. To confirm this connection we collaborated with Gary Ruvkun and Amy Pasquinelli who had recently developed probes for following the processing of the lin-4 and let-7 precursor RNAs into their mature 21 nucleotide RNAs. In wild-type animals the precursor forms are barely detectable. However, they found that, after inactivation of alg-1 and -2, this precursor accumulates to high levels while the product, the mature twenty-one nucleotide RNA, is greatly diminished [17] (Figure 10).

We were surprised to learn that RDE-1 was probably the slicer enzyme because our genetic studies had placed RDE-1 activity at an upstream step in the pathway. However, we realized that this observation could be explained if Argonautes function more than once during RNAi in C. elegans. For example (Figure 12), we imagined that RDE-1 could function along with small RNAs derived from processing of the trigger dsRNA in an initial round of target mRNA cleavage. The cleaved target mRNA could then serve as a template for an RNA-dependent RNA polymerase that produces new siRNAs that could, in turn, interact with other Argonautes to mediate efficient silencing of the gene.

To explain how RNAi could regulate DNA directly, I have to tell you a little bit about the physiological nature of DNA inside cells. Your DNA isn’t just lying around by itself. The unit of packaging for DNA is a protein structure called the nucleosome. The DNA wraps around the nucleosome twice, and the nucleosomes are in turn wrapped up and packaged into even thicker fibers. Chromosomes are composed of these protein/DNA fibers, also referred to as chromatin. Partly, what’s achieved by packaging the DNA into chromatin is a silencing effect. Structural studies of the nucleosome core suggest that short protein tails stick out past the DNA in such a way that they are readily accessible for modification [25]. The modification of these tails, and the resulting regulatory effects on gene expression, is turning out to be a fascinating subject - one that I’m sure this committee will need to consider in the future. Interestingly, mechanisms are now emerging that explain how small RNAs can guide the modification of these chromatin tails [24]. I will illustrate these mechanism with a model that could not only explain chromatin-based silencing mediated by RNAi but also provide a mechanism for the RNA-mediated evolution concept mentioned earlier (Figure 14).