Biologists at Washington University in St. Louis have made major headway in explaining a mechanism by which plant cells silence potentially harmful genes.
Differential gene expression profoundly influences the way in which organisms grow and develop. For instance, although every cell in the human body has the same genetic information, different subsets of the DNA get activated to make an eye different from a toe. RNA polymerases, the enzymes responsible for making RNA from DNA templates, are key players in determining which genes get switched on and which get left off.
A team led by Craig Pikaard, Ph.D., Washington University professor of biology in Arts & Sciences, has been investigating the role of two plant-specific RNA polymerases since playing a leading role in their discovery in 2005. In a paper published Nov. 14, 2008 in Cell , Pikaard and his colleagues explain how these RNA polymerases work together to use the non-coding region of DNA to prevent destructive, virus-derived genes from being activated.
"There's a lot of interest in harnessing this sort of silencing on purpose to be able to silence the genes that you care about," says Pikaard. Understanding the cellular machinery responsible for gene silencing has major implications for gene therapy, where RNA-centric approaches are showing real promise for control of diseases such as cancer and HIV.
Pikaard and his colleagues' work may have important implications for applied medical research. For instance, gene therapy procedures sometimes use retroviral vectors as a way of introducing a foreign gene to replace a function impaired by disease. Often this foreign gene, called a transgene, restores the missing function for a while and then unexpectedly goes silent. Pikaard explains, "It gets inactivated and it's probably the same sort of RNA-directed silencing mechanism." he explains. " If you could prevent the silencing of the transgene or if you could purposefully silence something that you wanted inactivated, that could be a good thing."
Pikaard studies what's known as transcriptional gene silencing. This phenomenon is often regulated by short interfering RNAs, or siRNAs, which University of Cambridge scientist David Baulcombe has called "the dark matter of genetics". By bringing about changes in DNA that interfere with transcription -- the copying of DNA to RNA -- siRNAs can effectively extinguish gene expression at its earliest stage. Pikaard explains, "From yeast to plants to humans these small RNAs can specify the modification of DNA somehow in a way that prevents transcription in the first place." According to Pikaard, most eukaryotes use the same two-pronged method for silencing genes at the transcriptional level: DNA methylation, or adding chemical flags to genes, and modification of proteins called histones that act as spools for DNA.
All eukaryotes share three essential RNA polymerases: Pol I, II, and III. These polymerases are indispensable for expressing biological traits and play a critical role in maintaining basic metabolic functions necessary for survival. "If you're mutated for any of those, you die," says Pikaard. "However, Pol IV and Pol V -- which only plants have -- you don't need them to stay alive but they turn out to be really important for this whole RNA-directed silencing phenomenon."
Since discovering these plant-specific RNA polymerases a few years ago, Pikaard's lab has been on a hunt to figure out what Pol IV and Pol V are making. In 2005, Pikaard and his collaborators published research showing that the major function of Pol IV is to generate siRNAs, thereby singling out this RNA polymerase as a potential player in gene silencing. However, when subsequent genetic tests suggested that Pol V is also needed for gene silencing, but not siRNA production, Pikaard and his colleagues suspected that Pol V and Pol IV cooperate, but work independently.
The Space between Genes