Recent advances in genomics, such as the sequencing of entire genomes and the discovery of RNA-interference as a means of testing the effects of gene loss, have opened up the possibility to systematically analyze the function of all known and predicted genes in an organism. Until now, this type of functional genomics approach has not been applied to the study of very complex cells, such as the brain's neurons, on a full-genome scale.
Dr. Katharine Sepp and her fellow researchers took fresh neuronal cells extracted from embryos of the fruit fly genus Drosophila and screened them using RNA interference techniques. The team tested all genes, one by one in a rapid manner, for their potential role in neuronal development. The team then validated the method in mice.
A combination of live-cell imaging and quantitative analysis allowed Sepp et al to characterize neurons' morphological phenotypes in response to RNAi-mediated gene knockdown. The researchers focused on 104 evolutionary conserved genes that, when downregulated by RNAi, have morphological defects. The team developed algorithms to help streamline the analysis of the thousands of images created in the process.
The analysis revealed unexpected, essential roles in neurite outgrowth for genes representing a wide range of functional categories including signalling molecules, enzymes, channels, receptors, and cytoskeletal proteins. Results also determined that genes known to be involved in protein and vesicle trafficking show similar RNAi phenotypes.
The researchers believe that this study provides an effective method for future studies of a large variety of genes, including those with important functions in the nervous system.
CITATION: Sepp KJ, Hong P, Lizarraga SB, Liu JS, Mejia LA, et al. (2008) Identification of Neural Outgrowth Genes using Genome-Wide RNAi. PLoS Genet4(7): e1000111. doi:10.1371/journal.pgen.1000111
plosgenetics/doi/pgen.1000111
The researchers found that their man-made RNA strand bound to the RNA transcript, which then recruited certain proteins to form an RNA-protein complex. The whole complex then bound to the promoter region, an action that could then either activate or inhibit gene expression.
"Involvement of RNA at a gene promoter is a new concept, potentially a big new concept," Dr. Janowski said. "Interactions at gene promoters are critical for understanding disease, and our results bring a new dimension to understanding how genes can be regulated."
Until recently, many scientists believed that proteins alone control gene expression at promoters, but Drs. Corey and Janowski's results suggest that this assumption is not necessarily true.
"By demonstrating how small RNAs can be used to recruit proteins to gene promoters, we have provided further evidence that this phenomenon should be in the mainstream of science," Dr. Corey said.
Although using synthetic RNA to regulate gene expression and possibly treat disease in humans is still in the future, Dr. Corey noted that the type of man-made RNA molecules employed by the UT Southwestern team are already being used in human clinical trials, so progress toward the development of gene-regulating drugs could move quickly.
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