St. Louis, Missouri
November 14, 2008
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., WUSTL 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 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
controlling 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.
"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 and his colleagues study 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.
"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," says Pikaard.
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
Using Arabidopsis thaliana, the "laboratory rat" of the plant
world, Pikaard and his colleagues carried out a series of
genetic tests to pinpoint where in the genome Pol V was getting
down to business.
Following a hunch, postdoctoral scholar Andrzej Wierzbicki
decided to take a closer look at the stretches of DNA that lie
between genes, the so-called intergenic regions. Biologists have
long been baffled by this alleged "junk DNA" because
transcription seems to occur here, and just about everywhere,
genes and junk alike.
"This is a hot topic in genetics right now," says Pikaard. "It
looks like not just the genes are getting transcribed, but
pretty much all of the DNA is getting transcribed. And why?
What's the purpose of all this transcription?"
Wierzbicki's instincts paid off. Using high levels of PCR
amplification, he determined that Pol V was indeed hard at work
within the intergenic region. In this space between genes, Pol V
makes noncoding RNA transcripts that he and Pikaard think bind
with the siRNAs generated by Pol IV. By acting as a scaffold for
these siRNAs, the Pol V transcripts enable silencing of
adjacent, virus-derived genes such as retrotransposons (jumping
genes) that can be detrimental if activated. Pikaard and
colleagues were able to confirm that both Pol IV and Pol V are
necessary for silencing by examining mutations that knock out,
or disable, the different genes coding for the polymerases.
'Junk' no more
This research adds to a growing body of evidence suggesting that
"junk DNA" is in fact a functional part of the genome, since
transcription of the intergenic regions is necessary to keep
potentially harmful genes turned off. In addition, Pikaard and
his colleagues have resolved a paradox that has recently puzzled
geneticists: the need for transcription in order to
transcriptionally silence the same region. In the case of
plants, this paradox is resolved by the interactive effects of
Pol IV and Pol V. The combination of Pol IV and Pol V products
modify the DNA such that RNA Pol I, II, and III are prevented
from transcribing potentially deleterious genes.
By Rachel Shulman |
|
