Patrick J. Paddison

Stable suppression of gene expression by RNAi in mammalian cells

In a diverse group of organisms including plants, Caenorhabditis elegans, Drosophila, and trypanosomes, double-stranded RNA (dsRNA) is a potent trigger of gene silencing. In several model systems, this natural response has been developed into a powerful tool for the investigation of gene function. Use of RNA interference (RNAi) as a genetic tool has recently been extended to mammalian cells, being inducible by treatment with small, ≈22-nt RNAs that mimic those produced in the first step of the silencing process. Here, we show that some cultured murine cells specifically silence gene expression upon treatment with long dsRNAs (≈500 nt). This response shows hallmarks of conventional RNAi including silencing at the posttranscriptional level and the endogenous production of ≈22-nt small RNAs. Furthermore, enforced expression of long, hairpin dsRNAs induced stable gene silencing. The ability to create stable “knock-down” cell lines expands the utility of RNAi in mammalian cells by enabling examination of phenotypes that develop over long time periods and lays the groundwork for by using RNAi in phenotype-based, forward genetic selections.

A resource for large-scale RNA-interference-based screens in mammals

Gene silencing by RNA interference (RNAi) in mammalian cells using small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) has become a valuable genetic tool. Here, we report the construction and application of a shRNA expression library targeting 9,610 human and 5,563 mouse genes. This library is presently composed of about 28,000 sequence-verified shRNA expression cassettes contained within multi-functional vectors, which permit shRNA cassettes to be packaged in retroviruses, tracked in mixed cell populations by means of DNA ‘bar codes’, and shuttled to customized vectors by bacterial mating. In order to validate the library, we used a genetic screen designed to report defects in human proteasome function. 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.

Second-generation shRNA libraries covering the mouse and human genomes

Loss-of-function phenotypes often hold the key to understanding the connections and biological functions of biochemical pathways. We and others previously constructed libraries of short hairpin RNAs that allow systematic analysis of RNA interference–induced phenotypes in mammalian cells. Here we report the construction and validation of second-generation short hairpin RNA expression libraries designed using an increased knowledge of RNA interference biochemistry. These constructs include silencing triggers designed to mimic a natural microRNA primary transcript, and each target sequence was selected on the basis of thermodynamic criteria for optimal small RNA performance. Biochemical and phenotypic assays indicate that the new libraries are substantially improved over first-generation reagents. We generated large-scale-arrayed, sequence-verified libraries comprising more than 140,000 second-generation short hairpin RNA expression plasmids, covering a substantial fraction of all predicted genes in the human and mouse genomes. These libraries are available to the scientific community.

Synthetic shRNAs as potent RNAi triggers

Designing potent silencing triggers is key to the successful application of RNA interference (RNAi) in mammals. Recent studies suggest that the assembly of RNAi effector complexes is coupled to Dicer cleavage. Here we examine whether transfection of optimized Dicer substrates results in an improved RNAi response. Dicer cleavage of chemically synthesized short hairpin RNAs (shRNAs) with 29-base-pair stems and 2-nucleotide 3′ overhangs produced predictable homogeneous small RNAs comprising the 22 bases at the 3′ end of the stem. Consequently, direct comparisons of synthetic small interfering RNAs and shRNAs that yield the same small RNA became possible. We found synthetic 29-mer shRNAs to be more potent inducers of RNAi than small interfering RNAs. 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.

Wnt and TGF-beta signaling are required for the induction of an in vitro model of primitive streak formation using embryonic stem cells.

The establishment of the primitive streak and its derivative germ layers, mesoderm and endoderm, are prerequisite steps in the formation of many tissues. To model these developmental stages in vitro, an ES cell line was established that expresses CD4 from the foxa2 locus in addition to GFP from the brachyury locus. A GFP-Bry+ population expressing variable levels of CD4-Foxa2 developed upon differentiation of this ES cell line. Analysis of gene-expression patterns and developmental potential revealed that the CD4-Foxa2hiGFP-Bry+ population displays characteristics of the anterior primitive streak, whereas the CD4-Foxa2loGFP-Bry+ cells resemble the posterior streak. Using this model, we were able to demonstrate that Wnt and TGF-β/nodal/activin signaling simultaneously were required for the generation of the CD4-Foxa2+GFP-Bry+ population. Wnt or low levels of activin-induced a posterior primitive streak population, whereas high levels of activin resulted in an anterior streak fate. Finally, sustained activin signaling was found to stimulate endoderm commitment from the CD4-Foxa2+GFP-Bry+ ES cell population. These findings demonstrate that the early developmental events involved in germ-layer induction in the embryo are recapitulated in the ES cell model and uncover insights into the signaling pathways involved in the establishment of mesoderm and endoderm.

Genome-wide RNA-mediated interference screen identifies miR-19 targets in Notch-induced T-cell acute lymphoblastic leukaemia

MicroRNAs (miRNAs) have emerged as novel cancer genes. In particular, the miR-17–92 cluster, containing six individual miRNAs, is highly expressed in haematopoietic cancers and promotes lymphomagenesis in vivo. Clinical use of these findings hinges on isolating the oncogenic activity within the 17–92 cluster and defining its relevant target genes. Here we show that miR-19 is sufficient to promote leukaemogenesis in Notch1-induced T-cell acute lymphoblastic leukaemia (T-ALL) in vivo. In concord with the pathogenic importance of this interaction in T-ALL, we report a novel translocation that targets the 17–92 cluster and coincides with a second rearrangement that activates Notch1. To identify the miR-19 targets responsible for its oncogenic action, we conducted a large-scale short hairpin RNA screen for genes whose knockdown can phenocopy miR-19. Strikingly, the results of this screen were enriched for miR-19 target genes, and include Bim (Bcl2L11), AMP-activated kinase (Prkaa1) and the phosphatases Pten and PP2A (Ppp2r5e). Hence, an unbiased, functional genomics approach reveals a coordinate clampdown on several regulators of phosphatidylinositol-3-OH kinase-related survival signals by the leukaemogenic miR-19.

RNA interference: the new somatic cell genetics?

RNAi is evolving into a powerful tool for manipulating gene expression in mammalian cells with potential utility for investigating gene function, for high-throughput, function-based genetic screens and potentially for development as a therapeutic tool.

Cloning of short hairpin RNAs for gene knockdown in mammalian cells.

. We and others have chosen to explore the shRNAs for several reasons: first, the considerable cost of chemically synthesized siRNAs; second, the possibility of enforceable and stable expression of shRNAs; and third, the availability of applications of expression constructs in primary cell types (for example, using retroviruses) and in whole organisms (for example, in mouse). We have developed a system to drive expression of shRNAs by placing them under the control of the human RNA polymerase III U6 small nuclear RNA (snRNA) promoter, which normally controls expression of small RNAs in cells. This system has now been demonstrated to be effective both in

Exit from Pluripotency Is Gated by Intracellular Redistribution of the bHLH Transcription Factor Tfe3

Summary Factors that sustain self-renewal of mouse embryonic stem cells (ESCs) are well described. In contrast, the machinery regulating exit from pluripotency is ill defined. In a large-scale small interfering RNA (siRNA) screen, we found that knockdown of the tumor suppressors Folliculin (Flcn) and Tsc2 prevent ESC commitment. Tsc2 lies upstream of mammalian target of rapamycin (mTOR), whereas Flcn acts downstream and in parallel. Flcn with its interaction partners Fnip1 and Fnip2 drives differentiation by restricting nuclear localization and activity of the bHLH transcription factor Tfe3. Conversely, enforced nuclear Tfe3 enables ESCs to withstand differentiation conditions. Genome-wide location and functional analyses showed that Tfe3 directly integrates into the pluripotency circuitry through transcriptional regulation of Esrrb. These findings identify a cell-intrinsic rheostat for destabilizing ground-state pluripotency to allow lineage commitment. Congruently, stage-specific subcellular relocalization of Tfe3 suggests that Flcn-Fnip1/2 contributes to developmental progression of the pluripotent epiblast in vivo.

Production of complex nucleic acid libraries using highly parallel in situ oligonucleotide synthesis

Generation of complex libraries of defined nucleic acid sequences can greatly aid the functional analysis of protein and gene function. Previously, such studies relied either on individually synthesized oligonucleotides or on cellular nucleic acids as the starting material. As each method has disadvantages, we have developed a rapid and cost-effective alternative for construction of small-fragment DNA libraries of defined sequences. This approach uses in situ microarray DNA synthesis for generation of complex oligonucleotide populations. These populations can be recovered and either used directly or immortalized by cloning. From a single microarray, a library containing thousands of unique sequences can be generated. As an example of the potential applications of this technology, we have tested the approach for the production of plasmids encoding short hairpin RNAs (shRNAs) targeting numerous human and mouse genes. We achieved high-fidelity clone retrieval with a uniform representation of intended library sequences.