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Living View in Animals Shows How Cells Decide To Make Proteins

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Duke Health News 919-660-1306

DURHAM, N.C. -- Scientists at Duke University Medical Center
have visualized in a living animal how cells use a critical
biological process to dice and splice genetic material to
create unique and varied proteins.

The scientists say the findings, made in mice, help explain
a key wonder of human biology: how the same genes found in
every cell of an individual's body can produce different
proteins in different tissues and organs. These varied
proteins, in turn, dictate the function of each tissue or
organ.

The findings also may offer insight into a number of
diseases, including cancer, in which the genetic process --
called alternative splicing -- goes awry and produces the wrong
proteins, the scientists said.

The scientists published the findings in the Dec. 1, 2006,
issue of the journal RNA. The study was funded by the National
Institutes of Health.

Scientists previously have examined alternative splicing in
cells and tissues in test tubes, but this study marks its first
successful visualization in a living mammal, said senior
investigator Mariano Garcia-Blanco, M.D., Ph.D., a professor of
molecular genetics and microbiology.

"We were able to watch alternative splicing as it occurred
in different tissues," he said. "It's an excellent example of
how experiments in living organisms provide a much more
complete picture of how genes and proteins behave than do
experiments using cells in culture."

Until 20 years ago, scientists believed that a single gene
made a single protein. With the discovery of alternative
splicing, it became clear that one gene can produce multiple
proteins.

In alternative splicing, microscopic "scissors" in a gene
chop the genetic material RNA into bits called "exons" and then
reassemble the bits in a different order to form a new RNA
molecule. In the process, some of the exons are retained while
others are excluded. The exons that are retained in the final
RNA determine which proteins the RNA produces within the
cell.

The scissors that do the genetic chopping are, in most
cells, proteins called splicing silencers and splicing
enhancers.

In the current study, Garcia-Blanco's team sought to
identify which silencers chop out an important segment of RNA
in a gene called fibroblast growth receptor 2 (FGFR2). This
gene plays a critical role in normal mouse and human
development, and the order in which its RNA is assembled can
alter an animal's development.

As a model system to study, the scientists genetically
created a "glowing" mouse. The mouse carried in its FGFR2 gene
a green fluorescent tag that would glow when a common type of
silencer, called an "intronic silencer," chopped out a specific
exon, called IIIb.

In this way, the scientists could track whether intronic
silencers were chopping out the IIIb exon -- and if so, in
which tissues and organs -- or whether other types of silencers
or helper proteins were involved.

By tracking the green glow, the team found that cells in
most tissues made the same decision to silence exon IIIb, but
the cells used a variety of silencers and helper proteins to
accomplish this task, said Vivian I. Bonano, a graduate student
in the University Program in Genetics and Genomics and lead
author of the journal report.

"Identifying which silencers are active in a given tissue or
organ will ultimately help scientists understand how exons are
erroneously included or excluded in various disease processes,"
Bonano said.

For example, a cell's decision to include exon IIIb is
critical because the exon's presence or absence determines
which variant of the FGFR2 protein is produced, she said. Such
subtle variations in proteins can alter the cell's behavior,
just as switching ingredients in a favorite recipe can change
the food's flavor, according to the scientists.

"Viewing these decisions is most relevant in a living
animal, because cells behave differently in their natural
environment versus an artificially created environment such as
a laboratory tissue culture," Garcia-Blanco said. "The
complexity of alternative splicing necessitates its
visualization as the decisions are occurring, because taking a
cell out of its context shows only its current status and not
how it arrived at that place."

For instance, the splicing process can change even from day
to day as an animal develops, he said, adding that extracting
cells and watching them in a culture cannot convey all of these
transient changes.

Moreover, different cell types within the brain or other
organs can exhibit different splicing decisions, Garcia-Blanco
said. For example, neurons reside next to glial cells in the
brain, yet they express different proteins in different
amounts, and detecting such differences in cell cultures can be
exceedingly difficult, he said.

"This is a powerful tool to apply to mouse genetics to learn
when and where in the animals' bodies alternative splicing
decisions are made and, eventually, to learn what factors are
critical in making these decisions," Garcia-Blanco said.

"Given the importance of alternative splicing in health and
disease," he added, "this anatomic mapping of splicing
decisions may give us considerable insight into the many human
diseases associated with improper regulation of splicing."

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