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RNAi: Mechanism, Biology, and Application to Cancer

Research Summary

Greg Hannon's lab works primarily on mechanisms and applications of RNA interference. The lab is also interested in cancer biology, in particular in developing tools to understand cancer initiation and progression.

In most eukaryotes that have been examined to date, exposure to double-stranded RNA (dsRNA) activates a biological response known as RNA silencing or RNA interference (RNAi). This process was first noted more than a dozen years ago when transgenesis experiments in Petunia resulted in loss of expression for both the transgene and its endogenous homolog. Known at that time as transgene cosuppression, this mysterious phenomenon implied communication between unlinked loci through an unidentified but diffusible intermediary. Subsequent work in Caenorhabditis elegans led to the realization that sequence-specific silencing events could be triggered by dsRNA. Mechanistic studies in a variety of systems led to the realization that transgene cosuppression is really just one example of an evolutionary conserved pathway for dsRNA-mediated gene regulation.

My group focuses on three areas. First, we take biochemical approaches in a variety of experimental systems to probe the underlying mechanism of RNAi. Second, we use mouse genetics to investigate the biological roles of this pathway in mammals. Third, we combine the knowledge gained from our biochemical and biological studies of RNAi to build tools for exploiting this pathway as an experimental tool in mammals.

Following observations by Richard Carthew (Northwestern University) and colleagues that an RNAi response could be triggered in Drosophila embryos by injection of dsRNA, we sought to use cultured Drosophila cells as a model system for probing RNAi at a biochemical level. My laboratory showed that an RNAi response could be initiated by transfection of S2 cells with long dsRNA triggers, and we developed in vitro systems from these cells in which both the initiation of silencing and the effector steps of the process could be studied effectively. Use of these systems led to the discovery of Dicer as the enzyme that starts the interference process by cleaving dsRNA into small interfering RNAs (siRNAs), discrete RNA products of ~22 nucleotides in length. We also isolated the effector complex of RNAi, the RNA-induced silencing complex (RISC), and showed that it contained these small RNAs, which serve as guides for the enzyme to identify its targets. These experiments paralleled very closely those carried out by Phillip Zamore (HHMI, University of Massachusetts), Thomas Tuschl (HHMI, Rockefeller University), Phillip Sharp (Massachusetts Institute of Technology), and David Bartel (HHMI, Massachusetts Institute of Technology) and led to the establishment of a basic scaffold of the RNAi pathway.

Genetic lesions in core RNAi components, for example in C. elegans and human cells, led to a link between the RNAi machinery and small endogenous noncoding RNAs that are now known collectively as microRNAs. These represent a large class of genes that function through the RNAi pathway to regulate the expression of protein-coding genes, generally at the post-transcriptional level. Given the ever-expanding breadth of the microRNA class, however, the biological range of these small RNAs may yet expand to other processes. Indeed, genetic analyses of loss-of-function mutants for Dicer and Argonaute proteins in mice indicate a surprising range of biological impacts for the RNAi machinery as a whole and by implication the small RNAs that act as its specificity determinants.

Studies in many systems have suggested that RISC, loaded with a small RNA, can control target genes through a number of different mechanisms. RISC can direct target cleavage. It can repress in a cleavage-independent but still post-transcriptional fashion, or it can affect chromatin structure. My laboratory continues to pursue a detailed biochemical understanding of each of these repression mechanisms. With Leemor Joshua-Tor's group (HHMI, Cold Spring Harbor Laboratory), we found that Argonaute proteins themselves are the heart of RISC and provide it with its catalytic activity. With Roy Parker (HHMI, University of Arizona ), we have recently provided a possible model for cleavage-independent repression, with the observation that microRNAs can bring their targets to processing bodies (P-bodies). These cytoplasmic locales are the site of translationally inert RNAs and concentrate the cellular RNA degradation machinery. Precisely how microRNAs signal this localization is a topic of intensive investigation.

Our analyses of the phenotypes of Argonaute mutants in animals and of small RNAs associated with Argonaute-family proteins led to the discovery of piRNAs, a new class of small RNAs largely specific to germ cells. In mammals and in flies, these RNAs act to protect germ cell genomes against the unwanted activity of parasitic, mobile genetic elements. piRNAs form an elegant and elaborate small RNA–based immune system with both genetically encoded and adaptive components. Major generative loci for piRNAs, called piRNA clusters, represent areas of high transposon density in both fly and mammalian genomes, essentially forming an evolutionary record of the elements with which an organism has adapted to coexist. Small RNAs and cluster transcripts interact with mRNAs derived from active transposons in the adaptive phase of the response, called the ping-pong cycle. This provides an amplification loop that both consumes transposon mRNAs and in so doing also generates new piRNAs corresponding to active elements. Recently, we have shown that piRNAs in flies are transmitted from mothers to progeny and that these maternally inherited species are important for successfully mounting a piRNA-based response to active elements. Thus, small RNAs can serve as vectors for transmitting epigenetic information. Overall, our studies contribute to an understanding of how transposons (nonself) are discriminated from endogenous genes (self), with the former being selectively silenced.

The identification of Dicer as an initiator of the process and the isolation of Argonaute proteins as core components of RISC provided a strong indication that the RNAi machinery is evolutionarily conserved, even in mammals. This sparked an interest in exploiting the RNAi pathway as a tool for probing gene function in mammals. Our basic approach has been to exploit the microRNA pathway for this purpose. The underlying idea is that genes might be silenced experimentally by redirecting a microRNA from its normal target to a gene of interest. This could be accomplished by reconfiguring the microRNA precursor so that the mature RNA it generates would precisely match the desired target gene. We called these synthetic microRNAs, short hairpin RNAs (shRNAs). Beginning with rather simple models for microRNAs, we have now evolved to the point where we express our experimental small RNA within the context of a natural microRNA precursor. This is processed through the normal microRNA maturation pathway to generate a small RNA of our design in RISC.

With Scott Lowe (HHMI, Cold Spring Harbor Laboratory) and Stephen Elledge (HHMI, Brigham and Women's Hospital, Boston), we have been devising methods to exploit shRNAs not only in cells but also in animals. We have demonstrated that regulated expression of shRNAs can be used to toggle gene expression on and off in a regulated and tissue-specific manner in mosaic animals with effects as predicted by similar analyses of animals with conventional genetic lesions.

Given the potential power of these tools, we have set out with the Elledge lab to construct comprehensive collections of synthetic shRNAs that cover all of the genes in the human, mouse, and rat genomes. This effort, now in progress, has generated silencing triggers for more than 30,000 human and 28,000 mouse genes. These materials are being made available to the academic community and are being used in-house to probe oncogene and tumor-suppression mechanisms through forward genetic screening strategies. Toward this end, we take advantage of high-throughput approaches that examine the consequences of suppression one clone at a time and of genetic selections that operate on pools of shRNAs. We are refining strategies that combine the advantages of both approaches, using molecular barcodes to track the properties of shRNAs in complex populations.

Finally, we have examined not only the impact of artificial microRNAs on tumor formation and progression but also the possibility that endogenous microRNAs might act as oncogenes and tumor suppressors themselves. One particular microRNA cluster, miR17–92, was found as a chromosome 13 amplicon in B cell lymphomas. Subsequent studies with Scott Hammond's group (University of North Carolina) showed that the microRNA components of this cluster are substantially overexpressed in a variety of human tumors, but particularly so in B cell lymphomas. Using a mouse model of this disease, we showed that the miR17–92 is a bona fide oncogene and could collaborate with another oncogenic lesion, overexpression of c-myc, to produce an accelerated and aggressive disease. We are probing the mechanisms by which this cluster contributes to tumorigenesis and using mouse genetics to pursue its normal biological functions. We are pursuing similar studies of the miR-34 family, which we recently showed acts within the p53 tumor-suppressor network.

Overall, my laboratory probes the biology of small RNAs at many different levels and in many systems. Ours has been a field that has expanded at an astounding rate over the past several years. All indications are that we have yet to see the full range of the biological impact of these remarkable pathways.

As of April 13, 2009

Scientist Profile

Investigator
Cold Spring Harbor Laboratory
Biochemistry, Genetics