By Tony Fitzpatrick

It's a little like finding out that Superman is actually Clark Kent.

A team of biologists at Washington University in St. Louis has discovered
that two vital cellular components, nuclear RNA Polymerases IV and V (Pol IV
and V), found only in plants, are actually specialized forms of RNA
Polymerase II, an essential enzyme of all eukaryotic organisms, including
humans.

"We've caught evolution in the act," said Craig Pikaard, Ph.D., WUSTL
professor of biology in Arts & Sciences. "We've known for decades that RNA
Polymerases I, II and III are found in all eukaryotes, but it's only over
the past several years that we've been aware that plants have two more
nuclear polymerases, Pol IV and Pol V. Now it is clear that these enzymes
evolved from Pol II over the past several hundred-million years. This is a
new snapshot into the evolution of RNA polymerases, which are the enzymes
responsible for decoding the information stored in the chromosomes."

Analyzing purified Pol IV and Pol V by a sophisticated technique known as
tandem mass spectrometry, the Pikaard lab and a team of collaborators at
Pacific Northwest National Laboratory, led by Ljiljana Paa-Toli, discovered
12 subunits in both Pol IV and Pol V that correspond one-for-one to the 12
subunits of Pol II. Some of the Pol IV and Pol V subunits are encoded by the
same genes as the corresponding Pol II subunits, but others come from
duplicated Pol II subunit genes that have changed over time. Overall, four
subunits of Pol IV are distinct from their Pol II counterparts, six subunits
of Pol IV are different from their Pol II counterparts, and four subunits
differ between Pol IV and Pol V. Yet, all of the Pol IV and Pol V subunits
are "apples that haven't fallen far from the Pol II tree."

The finding is important because it reveals more about the roles played by
RNA in complex organisms. RNA polymerases are the enzymes responsible for
making RNA from DNA templates. They are key players in determining which
genes get switched on and which get turned off. RNA Polymerase II, for
instance, is vital in the production of messenger RNAs that specify the
amino acid sequences of each of the proteins in the cell. Despite having
evolved from Pol II, Pol IV and V do not appear to be involved in protein
synthesis, or to be absolutely essential for life. Instead, they have taken
on specialized roles in gene silencing in plants.

This is important to prevent the expression of potentially harmful genes,
such as virus-derived "jumping genes" known as retrotransposons, and
invading nucleic acids, such as the genomes of replicating viruses. The
Pikaard lab has shown that Pol IV is required for the production of small
interfering RNAs (abbreviated as siRNAs) that specify the silencing of
matching DNA sequences, whereas Pol V makes longer RNAs that are thought to
pair with the siRNAs at the affected chromosomal sites.

Pikaard and his colleagues' work may have 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. The
silencing process may have parallels to the pathway in plants that makes use
of Pol IV and Pol V. Pikaard hypothesizes that Pol II accomplishes the
functions of Pol IV and Pol V in other non-plant eukaryotes.

The research revealing the subunit compositions of RNA Polymerases II, IV
and V in the plant genus Arabidopsis was published online Dec. 23, 2008 in
Molecular Cell. The work was supported by the National Institutes of Health
and the U.S. Department of Energy.

Pikaard's laboratory has been investigating the functions of Pol IV and Pol
V since playing a leading role in their discovery in 2005. The Dec. 23
Molecular Cell paper is one of three related papers published by the Pikaard
lab in rapid succession. In a paper published Nov. 14, 2008 in Cell, Pikaard
and his colleagues explain how Pol IV and Pol V work together to use the
non-coding region of DNA to prevent destructive, virus-derived genes from
being activated. Then in a paper published Dec. 4 in Molecular Cell, the
Pikaard lab announced a breakthrough in understanding the phenomenon of
nucleolar dominance, the silencing of an entire parental set of ribosomal
RNA genes in a hybrid plant or animal. That study is one of the first to
demonstrate how siRNAs can play a role in controlling the dosage of vital
genes, and not just harmful genes, and implicates the pathway in which Pol
IV and Pol V function. The research of the Nov. 14 and Dec. 4 papers has
been supported by the National Institutes of Health and by the National
Science Foundation.
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