www.czu.cz ; www.usab-tm.ro; www.raupp.info

Biologists at Washington University in St. Louis have discovered an entirely
new cellular "machine" in plants that plays a significant role in plant
flowering and DNA methylation, a key chemical process essential for an
organism's development, February 2005 by Tony Fitzpatrick.

A team headed by Craig Pikaard, Ph, D., Washington University professor of
biology in Arts & Sciences, has discovered a fourth kind of RNA polymerase
found only in plants and speculated to have been a plant feature for more
than 200 million years.

RNA polymerase is an enzyme, or protein machine, essential for carrying out
functions of cells and for expression of biological traits. It does its job
by copying a template of DNA genetic information in order to make RNAs that
encode proteins or that function directly in the cell.

Biologists have studied three kinds of RNA polymerase for decades in
organisms ranging from brewer's yeast to humans. In all eukaryotes, the RNA
polymerases Pol I, II, and III perform the same distinct , though separate,
functions in different species.

But then along came Pol IV. Pikaard first noticed the evidence for a fourth
polymerase when analyzing gene sequences after Arabidopsis thaliana , the
"laboratory rat" of the plant world, was sequenced in 2001. It originally
looked to him like an alternative form of either Polymerase I (Pol I), which
makes the largest of the ribosomal RNAs, Pol II which makes RNAs for
protein-coding genes, or Pol III, a specialist in making the shortest of the
ribosomal RNAs and tRNAs.


The big 'subunit'
He and his colleagues looked specifically at two polypeptides that would be
the key subunits if the fourth polymerase were functional, namely the
largest and second largest subunits, what Pikaard refers to as the
catalytic, or "business end" of any known polymerase.

"So, we took a reverse-genetics approach" said Pikaard. " We thought: 'What
happens if we knock these genes out?' So, we knocked out the genes
responsible for these subunits and there were no huge consequences. The
plants survived, but there were slight delays in flowering and some strange
floral defects. The plants were having trouble with organ identity ? stamens
tried to turn into petals, for instance. Our first hypothesis was that the
fourth polymerase was involved with what are known as micro RNAs, which are
known to regulate flower development, but that proved wrong."

In a series of genetic and biochemical tests , Pikaard and his collaborators
discovered that Pol IV does not share in the duties of Pol I, II or III. But
when the Pol IV subunits are knocked out, the most tightly packed DNA in the
nucleus becomes less condensed, small RNAs called siRNAs corresponding to
highly repeated 5S rRNA genes and retrotransposons (jumping genes) are
completely eliminated and DNA methylation at 5S genes and retrotransposons
is lost.

Methylation is a vital process involving a chemical modification in
cytosine, one of the four chemical subunits of DNA. Without proper DNA
methylation, higher organisms from plants to humans have a host of
developmental problems, from dwarfing in plants to tumor development in
humans to certain death in mice.

Pikaard thinks that Pol IV helps make siRNAs that then direct DNA
methylation to sequences matching the siRNAs.

The results were published in Cell online, Feb. 10, 2005 and will appear in
the March, 2005 print version of the journal.

"Pol IV is somehow involved in maintaining the integrity of the Arabidopsis
genome, principally in keeping the silent DNA silent," Pikaard said. "Plants
can get by without Pol IV, whereas they can't do without the other three. We
don't see anything obviously like Pol IV in any other genome, but it's
possible it might have been overlooked."

While Pikaard and his collaborators have indirect evidence that Pol IV is a
distinct RNA polymerase, they still have many aspects of Pol IV to unravel.

"We know what happens when its gone, but not how it behaves, at this point,"
he said. "We don't know its template, or what kind of RNA ? long or short ?
it makes. Presumably, because it is inherently different from the other RNA
polymerases, the rules of activity are different for Pol IV."

Pikaard said the Pol IV has a perfect match in rice, the only other plant
genome to be sequenced, despite rice being a monocotyledon and Arabidopsis a
dicotyledon.

"These two plants diverged 200 million years ago, and there is some
speculation that this form of polymerase might extend twice as far back in
evolution,' Pikaard said.

Pikaard said that it is strange that so far this kind of polymerase has been
found only in plants.

"Why would plants only have these?" he questioned. "It is a bit of a mystery
how other organisms that use small RNAs and that also do methylation get by
without a Pol IV. It might be possible that they have something equivalent,
and maybe we haven't looked hard enough. "

[www.eurekalert.org]

------------------------------------------
Posted to Phorum via PhorumMail