RNAi as a tool to study cell biology: building the genome–phenome bridge
Introduction
In its first application as a tool, RNAi was used to help demonstrate that the C. elegans gene par-1 had been identified molecularly [1]. Soon after, but still before the crucial discovery that the active ingredient in RNAi was double-stranded RNA (dsRNA) [2], RNAi was used to study components of the wingless pathway in C. elegans and the term ‘RNA-mediated interference’ was introduced [3]. These and other early studies (for example 4., 5., 6.) established RNAi as a powerful new tool for analyzing gene function in C. elegans. It soon became apparent that a similar approach could be applied in D. melanogaster, both in vivo [7] and, importantly, in cell lines [8]. However, RNAi was not equally successful in all species or cell types tested. It was not until the discovery of short interfering RNAs (siRNAs) [9] that RNAi could be applied in mammalian cells 10., 11.. With these tools in place and the availability of complete genome sequences, it has become possible to undertake large-scale analyses to identify all the genes required in specific cellular processes in metazoans and to characterize their cellular requirements on the basis of phenotypic profiles. Initial lessons from the use of RNAi to probe gene function, including technical issues, have been previously reviewed and will not be the focus here 12., 13., 14.. Instead, this review will concentrate more on issues related to transforming large-scale RNAi-based data into models of how the genome drives cell biological processes.
Section snippets
Large-scale RNAi screens in C. elegans, D. melanogaster, and mammalian cells
Large-scale RNAi studies were first undertaken in C. elegans 15., 16., 17., 18.. These studies tested over a third of predicted genes and identified >900 genes that elicit embryonic lethality or gross developmental defects in larval or adult stages. The majority of genes identified had no previously known function. Indeed, at the time these studies were published, only 1572 genes in C. elegans (∼8% of all genes) had any biochemical or genetic information associated with them, despite several
From analog to digital representation of RNAi phenotypes
The RNAi studies touched upon above showcase both technical issues and screening philosophies. Technical choices — including the design of dsRNA reagents, delivery methods, timing and scoring protocols — can affect the range and severity of phenotypes recovered, both in vivo 12., 13. and in cell culture [14]. New tools, including RNAi libraries (e.g. those based on the ORFeome [53••]) and strains that are differentially sensitive to RNAi (e.g. the C. elegans rrf-3 strain [21]), continue to be
Conclusions
While not yet routine, genome-scale RNAi screens are now feasible for C. elegans and D. melanogaster, and are imminent for mammalian cells. The studies performed to date have shown that RNAi-based reverse genetics approaches can identify new genes that play a role in heavily studied processes like cytokinesis. Other studies have used the new technology to gain a holistic view of the phenome by describing all observable phenotypes in a systematic way. This latter approach has shown that RNAi can
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgements
We are grateful to U Eggert and B Baum for communicating results before publication. Work in the authors’ laboratory is supported by NSF - DBI-0137617 (to KCG) and NIH - R01HD046236 (to FP).
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2008, Trends in GeneticsCitation Excerpt :Therefore, it is important to develop reverse genetic tools to test the function of the gene of interest, for example, by the knockdown of endogenous gene function. With the advent of RNA interference (RNAi) and morpholino technology, these reverse genetic approaches are becoming easily accessible [45–47]. As overexpression techniques are also developed, including direct injection of mRNA molecules or proteins into tissues, viral vectors or transgenic expression assays, phenotypes of interest can be further tested and are expected to mirror the results of the downregulation experiments.