RNAi as a tool to study cell biology: building the genome–phenome bridge

doi:10.1016/j.ceb.2004.12.008

Current Opinion in Cell Biology

Volume 17, Issue 1, February 2005, Pages 3-8

https://doi.org/10.1016/j.ceb.2004.12.008 Get rights and content

In the few short years since its discovery, RNA interference (RNAi) has revolutionized the functional analysis of genomes: both technical and conceptual approaches to the investigation of gene function are being transformed as a result of this new technology. Genome-scale RNAi analyses have already been performed in the model organisms Caenorhabditis elegans (in vivo) and Drosophila melanogaster (in cell lines), ushering in a new era of RNAi-based approaches to probing the inner workings of the cell. The transformation of complex phenotypic data into mineable ‘digitized’ formats is fostering the emergence of a new area of bioinformatics related to the phenome.

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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RNAi as a tool to study cell biology: building the genome–phenome bridge

Introduction

Section snippets

Large-scale RNAi screens in C. elegans, D. melanogaster, and mammalian cells

From analog to digital representation of RNAi phenotypes

Conclusions

References and recommended reading

Acknowledgements

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