Having helped to identify many miRNAs, Christopher Burge and his colleagues at the Massachusetts Institute of Technology are one of many teams now tackling an even bigger job — to find out which genes are regulated by the known miRNAs, and how they fit into physiological pathways.

James Carrington: taking a systems look at miRNA.

Finding targets begins computationally, using the TargetScan algorithms developed by Benjamin Lewis working with Burge and with David Bartel at the Whitehead Institute for Biomedical Research in Cambridge, Massachusetts. These algorithms “rely on evolutionary conservation of segments complementary to the microRNA ‘seed’ region in the 3′ untranslated regions of orthologous genes from multiple vertebrate organisms”, says Burge. The seed region, six or seven bases at the 5′ end of the miRNA, is thought to be key to specifying which genes an miRNA will regulate. Targets have been verified in Bartel's lab using a dual luciferase reporter system, which measures the effect of predicted miRNA interaction sites on protein production in cultured human cells. In a computational analysis published earlier this year, Lewis, Burge and Bartel estimated that more than a third of our genes might be regulated by miRNAs.

The task will be complicated by the fact that an miRNA may regulate as many as 200 genes, according to a computational study by Nikolaus Rajewsky and his colleagues at New York University and Rockefeller University, using their PicTar algorithm to identify miRNA targets. Other software for miRNA target prediction includes miRANDA from Anton Enright and his colleagues at the Memorial Sloan-Kettering Cancer Center in New York and DIANA-microT from Artemis Hatzigorgeou and Axel Bernal at the University of Pennsylvania, Philadelphia.

Frank Slack's team at Yale University uses in situ hybridization, northern blots and fluorescent protein fusions to find when and where miRNAs and their targets are expressed. “We use genetics and RNA interference to reduce the expression of potential targets to see if we suppress the effects of a mutation in the corresponding miRNA, and use reporter gene assays to test if the miRNA-complementary sites function in gene regulation,” he says.

“The classic tools of developmental biology and physiology are needed to correlate miRNA expression and targeting to biological function,” agrees James Carrington at Oregon State University, Corvallis, who is looking at pathways regulated by miRNAs in Arabidopsis. “miRNA sensors involving miRNA target sites within gene constructs expressing a fluorescent protein are quite useful in understanding spatial and temporal miRNA expression and activity patterns,” he says. But to address the question of how miRNAs integrate with cellular pathways, “the more quantitative approaches using the tools of systems biology and computational analysis are the trend in this lab”, he says.

Caitlin Smith