Gregory Hannon

..Harnessed as an experimental tool, RNAi has revolutionized approaches to decoding gene function. It also has the potential to be exploited therapeutically, and clinical trials to test this possibility are already being planned...

..Our findings indicate a function for pseudogenes in regulating gene expression by means of the RNA interference pathway and may, in part, explain the evolutionary pressure to conserve argonaute-mediated catalysis in mammals...

..Purified RISC complexes contain short interfering RNAs and endogenously expressed miRNAs and will be useful for studying many aspects of the RNAi machinery...

..Thus, our evidence supports a model in which Argonaute contributes "Slicer" activity to RISC, providing the catalytic engine for RNAi...

..Truncation of the regulatory carboxy-terminal domain (CTD) of RNA pol II disrupts transcriptional silencing, indicating that, like other RNA processing events, RNAi-directed chromatin modification is coupled to transcription...

..We find that specific members of the Dicer and Argonaute families have distinct biochemical activities, which provides insight into their roles within RNA silencing pathways in Arabidopsis...

..In this review, we discuss the recent developments in our understanding of plant RNAi mechanisms from a biochemical perspective...

..In practice, this primary microRNA-based short hairpin RNA vector system is markedly similar to cDNA overexpression systems and is a powerful tool for studying gene function in cells and animals...

..These libraries are available to the scientific community...

..An emerging theme is that miRNAs, and siRNAs to some extent, target mRNAs to the general eukaryotic machinery for mRNA degradation and translation control...

..Second, AGO4 catalytic activity can be crucial for the generation of secondary siRNAs that reinforce its repressive effects...

..Our studies identify Dicer as central to a regulatory network that controls oocyte gene expression programs and that promotes genomic integrity in a cell type notoriously susceptible to aneuploidy...

..Our findings suggest that the miRNA pathway and some siRNA pathways are ancient mechanisms of gene regulation that evolved prior to the emergence of multicellularity...

..These studies establish a practical platform for genome-scale screening of complex phenotypes in mammalian cells and demonstrate that RNAi can be used to expose genotype-specific sensitivities...

..Such efforts are complementary to the Cancer Genome Atlas and provide an alternative functional view of cancer cells...

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..This suggests that specialization of AGO complexes might involve remodeling the 5' end-binding pocket to accept certain small RNA sequences, perhaps explaining the evolutionary drive for miRNAs to initiate with uridine...

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..The possibility that dFXR, and potentially FMRP, use, at least in part, an RNAi-related mechanism for target recognition suggests a potentially important link between RNAi and human disease...

..In this review, some of the key features of RNAi in mammalian systems are discussed...

..We also show the therapeutic potential of this technique by demonstrating effective targeting of a sequence from hepatitis C virus by RNA interference in vivo...

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..We describe methods for the construction and transfer of stable shRNA expressing vectors suitable for generating loss of function alleles in mammalian cells in vitro or in vivo...

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..Recently, a related RNAse III enzyme, Drosha, has surfaced as another component of the RNAi pathway. In addition to biochemistry, protein structures have proven to be helpful in deciphering the enzymology of RNase III molecules...

..Thus, Bcl2L12 contributes to the classical tumor biological features of GBM such as intense apoptosis resistance and florid necrosis, and may provide a target for enhanced therapeutic responsiveness of this lethal cancer...

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..The p53 network suppresses tumour formation through the coordinated activation of multiple transcriptional targets, and miR-34 may act in concert with other effectors to inhibit inappropriate cell proliferation...

..A likely function of RNAi throughout that history is to protect the genome from both pathogenic and parasitic invaders...

..This methodology provides an adaptable route toward rapid and efficient resequencing of any sizeable, non-repeat portion of the human genome...

..This implicates piRNAs as specificity determinants of DNA methylation in germ cells...

..RNAi has been cultivated as a means to manipulate gene expression experimentally and to probe gene function on a whole-genome scale...

..Together, our studies demonstrate essential roles for Dicer in cardiac contraction and indicate that miRNAs play critical roles in normal cardiac function and under pathological conditions...

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..Platypus and echidna testes contain a robust Piwi-interacting (piRNA) system, which appears to be participating in ongoing transposon defense...

..These observations expand the repertoire of small RNAs in Drosophila, adding a class that blurs distinctions based on known biogenesis mechanisms and functional roles...

..These results highlight the utility of pooled shRNA screens for identifying genetic determinants of chemotherapy response and suggest strategies for improving the effectiveness of topoisomerase poisons in the clinic...

..Library cassettes can be easily shuttled into vectors that contain different promoters and/or that provide different modes of viral delivery...

..These results reveal critical roles for Dicer in the skin and implicate miRNAs in key aspects of epidermal and hair-follicle development and function...

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..We have validated this approach by targeting genes involved in cytokinesis and proteasome-mediated proteolysis...

..We also provide methods that use shRNAs to permit different levels of gene expression. Additionally, we discuss some aspects important for constructing an information pipeline to support development of a large shRNA library...

..These results suggest that Aurora-A and p160ROCK act in a common genetic pathway that promotes and monitors progression through G(2)/M...

..quot; The architecture of the molecule and the placement of the PAZ and PIWI domains define a groove for substrate binding and suggest a mechanism for siRNA-guided mRNA cleavage...

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..Maximal inhibition of target genes was achieved at lower concentrations and silencing at 24 h was often greater. These studies provide the basis for an improved approach to triggering experimental silencing via the RNAi pathway...

..These studies demonstrate that Argonaute proteins catalyze mRNA cleavage within RISC and provide a source of recombinant enzyme for detailed biochemical studies of the RNAi effector complex...

..These results provide a link between miRNA function and mammalian P-bodies and suggest that translation repression by RISC delivers mRNAs to P-bodies, either as a cause or as a consequence of inhibiting protein synthesis...

..These results implicate REST as a human tumor suppressor and provide a novel approach to identifying candidate genes that suppress the development of human cancer...

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..Tudor-SN is the first RISC subunit to be identified that contains a recognizable nuclease domain, and could therefore contribute to the RNA degradation observed in RNAi...

..These studies provide a conditional model of RNAi deficiency in mammals that will permit the dissection of the biological roles of the RNAi machinery in cultured mammalian cells...

..The high penetrance of these vectors will facilitate genomewide loss-of-function screens and is an important step toward using bar-coding strategies to follow loss of specific sequences in complex populations...

..In addition, intrinsic differences between individual shRNA expression vectors targeting the same gene can be used to create an 'epi-allelic series' for dissecting gene function in vivo...

..Thus, our results support a functional link between cytoplasmic P-bodies and the ability of a microRNA to repress expression of a target mRNA...

..Our results suggest that our large-scale RNAi library can be used in specific, genetic applications in mammals, and will become a valuable resource for gene analysis and discovery...

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..The findings from this Program have the potential both to inform the effective application of current therapies and to identify proteins and networks that may become targets for the development of new therapeutic agents. ..

..In this application, we outline our approach toward elucidating the mechanisms underlying dsRNA-induced gene silencing through a combination of in vitro and in vivo studies. ..

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..The development of this technology will allow not only allow a search for anti-cancer targets but will also undoubtedly find application to a broad range of biological problems. ..

..They serve as tools for understanding normal and aberrant biological processes and will perhaps transform clinical practice by serving as the basis for a new generation of therapeutic agents. ..