August 5, 2002
New technique shows how cells interpret genetic
By Tim Stephens
A surprising amount of the DNA sequence in the genes of humans and
other higher organisms ends up on the cutting-room floor, so to speak,
spliced out by the cellular machinery that turns genetic code into functional
DNA microarrays ("gene chips") enable researchers
to monitor the RNA splicing process for all the genes in a cell
simultaneously. The colored spots on this microarray show the
results of an experiment comparing normal yeast with a yeast strain
that carries a mutation in an essential RNA splicing factor.
Photo: Tyson Clark
Differences in the editing of genetic information may, in fact, be
a significant source of genetic variability. UCSC researchers have now
taken a big step toward understanding how this editing process (known
as splicing) is regulated.
Using DNA microarrays (also called "gene chips"), the researchers
are able to analyze the editing of all the genes in a cell simultaneously.
This enables them to study how mutations or environmental perturbations
affect the editing process.
"In the past, you had to analyze one gene at a time, but now we
can look at all of the genes in parallel," said Manuel Ares, professor
of molecular, cell, and developmental biology.
Master copies of all of an organism's genes are found on the chromosomes
in every cell. When a gene is "expressed" or activated, its
DNA sequence is copied into a "messenger RNA" molecule. The
messenger RNA directs the production of a protein molecule, but not
before a bit of cutting and splicing of the RNA code.
In the genes of higher organisms (as opposed to bacteria), the protein-coding
information is broken up into fragments called "exons" that
are separated by long sections of noncoding sequence called "introns."
The splicing of pre-messenger RNA involves cutting out the introns and
joining together the exons. The most striking thing about this process
is that there are often several and sometimes many different ways to
splice together the exons of a particular gene.
"It's estimated that more than 50 percent of human genes produce
alternatively spliced messenger RNAs," Ares said. "To study
this, we started by looking at yeast, because yeast genes have very
few introns. The yeast genome is sort of a toy version of the bigger
genomes of more complex organisms like humans."
Applying the microarray technique to yeast, Ares and his coworkers
obtained the first genome-wide view of RNA splicing for any organism.
They published their findings in the May 3 issue of the journal Science.
Tyson Clark, a graduate student in molecular, cell, and developmental
biology, is lead author of the paper. Coauthor Charles Sugnet is a graduate
student in computational biology.
Ares's lab is also looking at RNA splicing in other organisms, including
For example, graduate student Valerie Welch has developed a set of
microarrays to analyze changes in splicing as human cells in tissue
culture progress from normal to cancerous states, and as cells are infected
The idea that every gene acts by directing the production of a specific
protein--known as the "one gene, one protein" hypothesis--was
a Nobel Prize-winning milestone in the development of modern biology.
But alternative splicing means that genes are actually much more complicated
than that, and some genes can produce many proteins through alternative
splicing. Most cases of alternative splicing involve just a few different
versions, but one gene in the fruit fly apparently generates 38,000
different versions of its messenger RNA.
In humans, a good example is the gene for tropomyosin, a structural
Alternative splicing gives rise to five different versions of tropomyosin,
which are produced in five different tissues in the body: skeletal muscle,
smooth muscle, fibroblast, liver, and brain. Cells in each type of tissue
splice the 11 exons of the tropomyosin gene differently to produce the
different forms of the protein.
"The coding sequences of our genes are all broken up and spread
out, and there is a whole cellular machinery involved in patching it
together so that the code makes sense. This splicing process gives the
cell the ability to try new combinatorial arrangements of information,"
Ares said. "You have all this information in the genome, but then
the cell can interpret it in different ways. Our microarray technology
allows us to access all that information in the cell at one time."
The technique depends on the recognition of the unique sequence created
when two exons are spliced together. The initial step of identifying
these splice-junction sequences involves comparing the genome sequence
with the "expressed" sequence. To do this, Ares works with
bioinformatics researchers led by David Haussler, professor of computer
science and director of the Center for Biomolecular Science and Engineering.
Sugnet performed this analysis for the yeast genome.
"He was able to identify the splicing events and, in each case,
give us the coding sequence that spans the junction between the two
exons. Then we created a synthetic DNA molecule that can recognize that
sequence and spotted it onto the microarray," Ares said.
The microarrays for the yeast study consisted of glass slides with
tiny spots of synthetic DNA laid out in a grid pattern. For every intron-containing
gene in the yeast genome, the microarray included three synthetic DNA
probes: splice-junction probes to detect spliced messenger RNA; intron
probes to detect unspliced RNA; and exon probes to detect both spliced
and unspliced RNA. RNA extracted from yeast cells and tagged with fluorescent
labels was then placed on the microarray, allowing the RNA molecules
present in the cells to bind to the specific DNA probes.
The intensity of the fluorescence at each spot serves as an indicator
of the relative abundance of the corresponding RNAs. The microarrays
are analyzed with an instrument that measures the fluorescence at each
The researchers compared splicing patterns in normal and mutant strains
of yeast. The mutations affected various factors known to be involved
in the processing of messenger RNA molecules. In general, the results
showed that the effects of RNA processing factors on splicing events
depend on the particular intron involved.
"Each splicing event has its own character. When you alter the
cell in some way, it's hard to predict the consequences for any particular
intron," Ares said. "We hope to use our data to figure out
what the rules are, why some splicing events depend more on certain
parts of the RNA processing machinery than others."
Ares is already extending this work to human genes, starting with genes
involved in cancer (tumor suppressor genes and oncogenes). Other researchers
have reported that the splicing of some cancer genes changes as the
Ultimately, Ares wants to understand the mechanisms that regulate RNA
splicing in all cells. RNA splicing is basically an interpretive process--the
genetic information contained in the genome is interpreted differently
in different contexts.
"That interpretive process, and how it varies within a population,
has really not been explored in any organism, including humans,"
Ares said. "I'd like to look at how much polymorphism [i.e., genetic
variability] there is in the human population as far as how genes are
This could be an important factor in understanding genetic diseases.
The severity of certain genetic diseases, such as cystic fibrosis, varies
tremendously depending on the patient's genetic background. In other
words, the severity of the disease in two people with the same genetic
defect might be quite different because of differences in other genes.
Ares said differences in RNA splicing may have a lot to do with that.
"We know of cases where the way the disease gene is handled by
the splicing machinery is different in different people," he said.
"We have a lot to learn about this kind of variation."
Return to Front Page