RNAi Discovery
Larry H. Bernstein, MD, FCAP, Curator
LPBI
RETURN TO THE RNAi WORLD: RETHINKING GENE EXPRESSION AND EVOLUTION
Nobel Lecture, December 8, 2006 by Craig C. Mello
Abstracted
It’s wonderful to be here today, I would like to start with the most important part, by saying thank you. First of all, I want to thank Andy Fire for being such a tremendous colleague, friend and collaborator going back over the years. Without Andy I definitely wouldn’t be here today. I need to thank the University of Massachusetts for providing for my laboratory, for believing in me and for giving me not only a place and money, but great colleagues with whom to pursue my research.
I want to get down to the theme of my talk today, which really is, in part, about how we continually underestimate the complexity of life. It’s the correction of these underestimations that is quite often what this prize is really recognizing. As science progresses, our knowledge expands, we think we understand, and too often we become overconfident. The fact is, I think we almost always underestimate the complexity of life and of nature. Today has been a true celebration of that beauty and complexity.
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….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.
In Roger Kornberg’s talk, we heard about an RNA polymerase that can transcribe the DNA to produce RNA copies of the genetic information. These copies provide templates for the polymerization of the proteins through another elaborate and really beautiful process, called translation.
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 cellcell 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…..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.
How do developing cells, all with the same DNA content, lock in different programs of gene expression that are stable through so many rounds of cell division? One possibility, as I will discuss below, is that mechanisms related to those that mediate RNA interference have a role in this process. It has been suggested that the origin of life on Earth may have begun with selfreplicating nucleic acid polymers that were more similar chemically to RNA than to DNA, a classic hypothesis referred to as the “RNA World” hypothesis.
….primordial germ cells in the common metazoan (probably worm-like) ancestor of worms and humans, and going even farther back are direct descendants of the hypothetical self-replicating RNA molecules that gave rise to all life on Earth some 3.5 billion years ago….. RNAi itself is at least one billion years old. Biological mechanisms are far more constant than the positions of continents on our planet. That fact and the implicit concept of deep time are among the most profound discoveries of science.
1) there is a particle, containing siRNAs, for some traits; 2) these siRNAs can grow and multiply independent of cell division; 3) both the nucleus and the cytoplasm can contain the siRNAs; 4) a given siRNA may be represented by many replicas; and 5) that during cell division the daughter cells may receive different kinds and numbers of siRNAs through unequal cell division.
…here’s what CBS Evening News came up with (Figure 5). In the movie, the double stranded RNA flies onto the scene then opens at one end and begins to open and close as though it’s chewing. Defective genes, shaped like colored cheese puffs, then begin to fly into the mouth of the RNA from the left. The RNA is literally eating the DNA for lunch. Now, Andy and I knew that RNA interference was something incredible when we started working on it, but we really didn’t have any idea that it was this incredible.
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 6). 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.
I like both of these movies because they illustrate a really important concept; that is, that RNAi is an active process, that there is an organismal response to the dsRNA [4]. We realized this at an early stage, because, first of all, as Andy mentioned, the silencing was heritable. RNA injected into an animal resulted in silencing that was transmitted to progeny and even transmitted through crosses for multiple generations via the egg or the sperm. So, the interference mechanism can be initiated in one generation and then transmitted in the germline.
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 was able to identify numerous mutants. Some of these lacked the RNAi response and had no other obvious phenotypes, like rde-1 and rde-4. However, some of his mutants had additional defects, including a very striking phenotype in which the transposons, which are selfreplicating DNA elements present in the genomes of all organisms, became hyperactive, causing mutations by jumping from place to place in the genome. In addition, these same mutants had a reduced tendency to silence transgenes in the germline (transgenes are genes that are experimentally introduced into the organism). In normal worms, transgenes have the vexing property of becoming silent after introduction into the animal experimentally. The same mutants with activated transposons also exhibited activation of transgenes in the germline. These observations suggested that the normal physiological function of RNAi might be to defend cells against the potentially damaging effects of transposons and other foreign genetic elements (perhaps including viruses)…..The rde- 1 and rde-4 mutants, as I indicated earlier, had no other phenotypes. They Figure 7. Hiroaki Tabara 249 were strongly deficient in RNAi in response to double-stranded RNA, but the transposon silencing and the transgene silencing mechanisms were still functioning in these mutant strains. These observations indicated to us, even at that very early stage of our analysis, the existence of some additional, very interesting complexity.
Hiroaki cloned the rde-1 gene and showed that it encodes a highly conserved protein that we now refer to as an Argonaute protein [6]. RDE-1 was an interesting protein for a couple of reasons. It had highly conserved domains found in related genes in organisms as diverse as plants and humans, and yet nothing was known about the enzymatic activities or the biological functions of these domains. This was a very exciting time in the laboratory. We at last had a gene that we knew was involved in the mechanism. Furthermore, previous work on one gene closely related to RDE-1 from Drosophila had linked this gene family to an epigenetic silencing pathway in the fruit fly [7, 8], and work in plants had linked a member of the family to the control of development [9]. Very shortly after our paper was published, Carlo Cogoni and Giuseppe Macino [10] published a very nice paper implicating an RDE-1 family member in silencing triggered by the introduction of a transgene in the fungus Neurospora. So from these findings in other organisms, and from Hiroaki’s genetics, we hypothesized that there may be other types of triggers that initiate related silencing pathways either through natural developmental mechanisms or in response to transposons and transgenes.
…, 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 10). 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.
Even before we identified RDE-1, we were interested in the possibility of a relationship between the RNAi pathway and the lin-4 pathway. Indeed, Hiroaki had raised the concern that RNAi-defective mutants could be hard to recover as viable strains since they might also cause disruption of the lin-4 pathway. Making all of these possibilities even more exciting – while we were conducting genetic screens for RNAi deficient strains, beautiful work was published by Hamilton and Baulcombe [12], linking small RNAs of ~21 nucleotides to viral gene silencing in plants, and by Gary Ruvkun’s lab, identifying a second lin-4- like worm gene, let-7 [13]. Whereas lin-4 was a worm-specific gene, it turned out that the let-7 gene had homologs in every animal, including humans. Remarkably, every single nucleotide in the twenty-one nucleotide mature let-7 RNA products from the worm and human were identical to each other. The conservation of let-7 initiated a gold rush to find small RNA encoding genes, now referred to as micro-RNA genes, in the genomes of numerous organisms.
….activities present in Drosophila cells could process double-stranded RNA into tiny RNAs approximately 21 nucleotides long. Tom Tuschl and colleagues were the first to show that these small RNAs could silence gene expression in vertebrate cells [16]. Thus genetic studies in worms had identified small RNAs as silencing agents beginning in 1993, experimental studies of virus infections in plants identified small RNAs accumulating in infected plants, biochemical studies in fly extracts identified small RNAs in extracts, and finally experimental studies identified silencing activity in cellular assays with vertebrate cells. But were these small RNA molecules only similar in size, or did their similarity extend to mechanism?
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, we 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 11).
We also looked at the involvement of Dicer in this process. Dicer was identified by Greg Hannon’s lab as a nuclease required for processing long double-stranded RNA into approximately 21-nucleotide fragments in Drosophila cells. We were able to show, as did several other groups [18, 19], that when you knock out Dicer you also see defects in the processing of these microRNAs (Figure 11). 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 12), 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.
At that time we thought of the RDE-1 family members (also known as Argonaute proteins) as initiators of the silencing pathways. Genetic studies had placed RDE-1 at an upstream step in the pathway and, as I showed you, ALG-1 and -2 are required for processing the microRNA precursors. However, there was mounting evidence that these proteins might also function downstream in the silencing step. Definitive support for this idea came from Greg Hannon’s group through a collaboration with Ji-Joon Song and Leemor Joshua-Tor at Cold Spring Harbor [20]. They showed that Argonaute proteins have structural similarity to an enzyme domain that can cut RNA, and they presented a model for how Argonaute proteins can bind the ends of the short RNAs and utilize the sequence information to find and destroy target mRNAs in the cell. These studies demonstrated that Argonaute proteins represent the long sought “slicer” activity (or the cop) that lies at the heart of the RNA-induced silencing pathway. 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 13), 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.
The last concept I want to discuss relates to the question of how RNAi can interact with chromatin to silence genes, and the potential importance of this mechanism for gene regulation during both development and evolution. As indicated earlier in my talk, many of the genes involved in RNAi are also required for the silencing of transgenes in the germline.
….Beautiful direct evidence for a link between RNAi and chromatin silencing has more recently come from work in the fission yeast S. pombe, where a complex containing an Argonaute protein and known chromatin interacting factors has been shown to interact directly with silenced genes in the nucleus [24]. 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].
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