By Mehdi Banan, Ph.D.
Science Content/Business Development Analyst

Almost five years ago Fire and Mello discovered that double stranded RNAs (dsRNAs) mediate gene-specific silencing in worms--a phenomenon they referred to as RNA interference or RNAi (1). Since then, loss-of-function phenotypes created by RNAi have been used extensively for functional genome studies in worms. Now, with the construction of a comprehensive C. elegans RNAi library, these studies will undoubtedly take a major step forward (2). In the 16 January 2003 issue of Nature, Julie Ahringer and colleagues report on the construction of a bacterial RNAi library that encompasses ~86% of the 19,427 C. elegans open reading frames (ORFs). (One way of introducing dsRNAs into worms is by feeding them bacteria that express complementary transcripts from plasmids, which then form into dsRNAs. This strategy was used to create the above library.)

The C. elegans RNAi library was put to use quickly. In the same issue of Nature, Gary Ruvkun and colleagues used the library to do a genome-wide analysis of fat regulatory genes in worms (3). The authors identified 305 gene 'knockdowns' that caused reduced fat storage and another 112 that led to increased fat storage. Interestingly, a large number of these genes turned out to have mammalian orthologues, suggesting that some of these genes may play a role in obesity in humans.

Visualizing fat storage

In order to visualize the fat storage droplets, Ruvkun and colleagues added a lipid-associating dye (Nile Red) to the worms' bacterial diet. The dyes were then consumed, incorporated into fat droplets, and visualized by microscopy. The pattern of incorporation of Nile Red dye in the 16,757 RNAi 'knockdown' worms was subsequently compared to that of wild-type worms.

Using this approach, the authors identified 305 gene knockdowns (1.8% of the genes) that cause reduced fat storage. These include genes that are involved in lipid biosynthesis, fatty acid elongation, and fatty acid binding proteins. Interestingly, the list also includes olfactory receptors, presumably required for a healthy appetite.

Conversely, RNAi inactivation of 112 other genes (0.7% of the genes) led to increased fat storage. The list includes the hepatocyte nuclear factor 4-a, several cytochrome P450 enzymes, and an orthologue of the transcription factor Nhlh-2, which causes obesity when 'knocked out' in mice.

Discussion

The findings by Ruvkun and colleagues point towards the enormous utility of comprehensive RNAi libraries. The authors were able to identify 'fat storage' genes in worms and numerous mammalian orthologues, some of which could be involved in obesity in humans. Interestingly, many of these human orthologues happen to encode for proteins with known small molecule regulators (such as kinases and hormone receptors), suggesting that they could also serve as potential drug targets.

Similar RNAi libraries could also be created for mammalian cells. While long dsRNAs are used to silence genes in worms, 20-25 nt small interfering RNAs (siRNAs) are deployed to silence genes in mammalian cells. Before long, siRNA libraries to all mammalian genes should be available. Such libraries will greatly enhance the ability to screen for loss-of-function phenotypes in mammalian cells and, as expressed by Thomas Tuschl, "with the development of phenotypic read-outs based on cell biology, the hunt will begin." (4)

References

1. Fire A, Xu SQ, Montgomery MK, Kostas SA, Driver SE, and Mello CC. (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391:806-811.
2. Kamath RS, Fraser AG, Dong Y, Poulin G, Durbin R, Gotta M, Kanapin A, Le Bot N, Moreno S, Sohrmann M, Welchman DP, Zipperlen P, and Ahringer J. (2003). Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature 421:231-237.
3. Ashrafi K, Chang FY, Watts JL, Fraser AG, Kamath RS, Ahringer J, and Ruvkun G. (2003). Genome-wide RNAi analysis of Caenorhabditis elegans fat regulatory genes. Nature 421:268-272.
4. Tuschl T. (2003). RNA sets the standard. Nature 421:220-221.