Building mammalian signalling pathways with RNAi screens

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Abstract

Technological advances in mammalian systems are providing new tools to identify the molecular components of signalling pathways. Foremost among these tools is the ability to knock down gene function through the use of RNA interference (RNAi). The fact that RNAi can be scaled up for use in high-throughput techniques has motivated the creation of genome-wide RNAi reagents. We are now at the brink of being able to harness the power of RNAi for large-scale functional discovery in mammalian cells.

Key Points

  • Functional annotation of human genes has been hampered by the lack of tools to probe gene function systematically and quickly. RNA interference (RNAi) offers the cell biologist an approach to perturb gene function that can be applied in a high-throughput fashion on the cell or organism scale.

  • Various approaches to RNAi-mediated gene knockdown have been engineered for screening gene function in mammalian cells, including small interfering RNAs (siRNAs), plasmid-based short hairpin RNAs (shRNAs), plasmid-based shRNAs in a microRNA context (shRNA-mirs), virus-based shRNAs (retroviral and lentiviral), enzymatically prepared siRNAs (esiRNAs) and others. This review surveys several of the screens that were undertaken using these approaches to show their applicability.

  • A number of different formats for high-throughput mammalian RNAi screens have been developed and are discussed, including well-based arrayed screening, pooled screening and cell microarrays.

  • Methods for undertaking a mammalian RNAi screen focus on component discovery, component validation, component classification and systems-type analysis. Ideas for navigating through each of these screening steps are discussed.

  • A road map for RNAi screening using a hypothetical example to look for regulators of cell growth and proliferation is presented as an example of how the architecture of a signalling pathway could quickly be constructed using RNAi gene-knockdown technology.

  • Further development of RNAi technology will provide greater control over gene expression as well as advancing organ and organismal studies.

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Figure 1: Identification of components in a biological process using high-throughput RNA interference.
Figure 2: Formats for high-throughput mammalian RNAi screens.
Figure 3: Probing binary genetic interactions with RNA interference.
Figure 4: A hypothetical screen for regulators of growth and proliferation.

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Acknowledgements

We thank K. Ottina, J. Reiling, C. Thoreen and D. Guertin for helpful comments and critical reading of the manuscript and all laboratory members for valuable discussions. We apologize to authors whose primary works have not been cited due to space constraints. The work of the authors is supported by a Natural Sciences and Engineering Research Council of Canada postdoctoral fellowship (J.M.) and grants from the National Institutes of Health, Keck Foundation and Stewart Trust (D.M.S.). The authors would also like to acknowledge members of the RNAi Consortium (http://www.broad.mit.edu/rnai_platform/) for their continued support.

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Correspondence to David M. Sabatini.

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Glossary

Small interfering RNA

(siRNA). A class of 19?22-nucleotide-long RNA molecules that interfere with the expression of genes by eliciting the RNAi response. siRNAs are short double-stranded RNA molecules with 2-nucleotide overhangs on either end, including a 5′ phosphate group and a 3′ hydroxyl group. They can be artificially introduced into cells to bring about the knockdown of a particular gene.

Interferon response

A primitive antiviral response to dsRNAs of >30 base pairs, which triggers the sequence-nonspecific degradation of mRNA and the downregulation of cellular protein synthesis.

Short hairpin RNA

(shRNA). A short RNA that contains sense and antisense sequences from a target gene that are connected by a hairpin loop. shRNAs can be expressed from a pol-III-type promoter or in the context of a microRNA by pol II promoters. Following processing of the shRNAs, the resulting siRNAs can decrease the expression of a gene that has complementary sequences by RNAi.

MicroRNA

(miRNA). A small non-coding RNA of 19?25 nucleotides in length that regulates the expression of genes at the stage of protein synthesis.

Reverse transfection

A process whereby cells are transfected with features (for example, DNA or RNA) that are immobilized on glass slides or in multi-well plates.

RNA polymerase III promoter

A promoter that uses RNA pol III to drive the production of 5S RNA, tRNA and other small RNAs. U6 and H1 pol III promoters have all the elements that are required for the initiation of transcription upstream of a defined start site and the termination of transcription at four or more Ts.

Primary polyadenylated RNAs

(pri-miRNA). A long primary polyadenylated miRNA that is transcribed by RNA pol II. The miRNA sequence and its reverse complement base pair to form a dsRNA hairpin loop, which forms the primary RNA structure.

Microprocessor complex

A small protein complex consisting of Drosha and DGCR8 that is necessary and sufficient for mediating the genesis of miRNAs from the primary miRNA transcript.

Pre-miRNA

A miRNA precursor that is converted from the pri-miRNA in the nucleus by the Microprocessor complex and exported to the cytoplasm by a mechanism that is mediated by exportin-5. The Dicer enzyme then cuts 20?25 nucleotides from the base of the hairpin to release the mature miRNA.

High-content image-based screen

(HCS). A method that uses high-resolution images as the readout for a screen. This type of screening is typically carried out using automated microscopy to acquire images. The images are analysed by eye or by automated image analysis, which is sometimes referred to as HCA (high-content analysis).

Epistasis

The masking of a phenotype that is caused by a mutation in one gene, by a mutation in another gene. Epistasis analysis can therefore be used to dissect the order in which genes function in a genetic pathway.

Perturbagen

A reagent (for example, a chemical or siRNA) or condition that disrupts or modifies the function of a specific gene or signalling pathway.

Cell microarray

A method for studying cells that take up perturbagens and that have been printed in an arrayed format on the surface of glass slides.

Reverse genetics

Genetic analysis that proceeds from genotype to phenotype through gene-manipulation techniques.

Blastocyst

The stage of the developing embryo when the number of cells reaches 40?150, a central fluid-filled cavity called the blastocoel forms and the zona pellucida begins to degenerate. This stage lasts approximately until implantation into the uterus.

Tetraploid aggregation

A method that is used to generate embryos that are completely derived from embryonic stem cells. The approach provides a quick evaluation of phenotype in embryonic development; for example, one can observe a knockout phenotype by aggregating mouse embryos with gene-knockout or transgenic RNAi embryonic stem cells.

Perivitelline space

Region between the surface of the oocyte or more specifically the oolemma and the zona pellucida, an extracellular matrix synthesized by the oocyte. The perivitelline space has contents that change during development and that appear to have various roles before, during and after fertilization.

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