Our
work is focused on the expression and travels of RNA within the cell:
from the site of its birth to its ultimate biological destiny in the
cytoplasm where it makes proteins in specific locations. Our new technology,
based on in situ hybridization allows us to visualize specific nucleic
acid sequences within individual cells. Synthetic nucleic acid probes
are labeled with fluorochromes. Subsequently these molecules are hybridized
to the cell and detected using high resolution digital imaging microscopy.
We have developed imaging methodologies and algorithms capable of detecting
a single RNA molecule within a cell. This enables the detection of specific
nucleic acid molecules for comparison between normal or cancer cells.
This method of molecular diagnosis is the clinical application of the
technology. As an additional result of this approach, we have found
specific RNA sequences located in particular cellular compartments.
An example is the messenger RNA for beta-actin, which is located in
the periphery of the cell where actin protein is needed for cell motility.
These transcripts are not free to diffuse, and appear to be associated
with a cellular matrix or skeleton from the moment of their synthesis
through translation. We are investigating how this spatial information
is encoded within the gene and how the RNA transcript is processed within
the nucleus and then transported to its correct compartment in the cytoplasm,
resulting in asymmetric protein distribution. We have recently discovered
that RNA localization also occurs in yeast. During budding, a nuclear
factor represses mating type switching asymmetrically, only in the daughter
cell. This is because the factor is synthesized only in the bud because
the mRNA was transported there by a motor, myosin. This discovery has
provided a model by which to investigate the mechanisms responsible
for moving RNA within the cell. For example, we have constructed genetically
altered yeast and vertebrate cells in order to elucidate the sequences
responsible for mRNA localization. A reporter gene can be "delivered"
to a variety of cellular compartments by using specific sequences, or
"zipcodes" from the mRNAs found in those compartments. These
"zipcodes" consist of short sequences in the 3' untranslated
region of the mRNA. We have isolated and cloned proteins which bind
to the zipcode and decode this information. Recently we have developed
technology that allows us to visualize RNA movement in living cells.
Currently our efforts are to develop imaging methods to see fast movements
in order to characterize how the motors connect with and drive the RNA.
