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6th ESACP Congress, Heidelberg, April 7-11, 1999 |
A015
Messenger RNA (mRNA) is transcribed and processed in the nucleus of
eucaryotic cells and then exported to the cytoplasm through nuclear pores.
It is currently not clear whether the movement of mRNA from its synthesis
site to the nuclear pore is directed or random. Employing fluorescently
labeled oligo(dT) as a hybridization tag, we studied the movement of
endogenous intranuclear poly(A) RNA in living cells using newly-developed
methods. Utilizing fluorescence correlation spectroscopy coupled with
confocal microscopy, we found that over half of intranuclear poly(A) RNA
moved with apparent diffusion coefficients in the same range as that measured
for an average-sized RNA in solution (~9 microns squared/sec). We also used
high speed digital imaging microscopy to visualize the movement and spatial
disposition of poly(A) RNA tagged with an oligo(dT) labeled with chemically
masked (caged) fluorescein. Laser spot photolysis uncaged the oligo(dT) tag
at a given intranuclear site and the resultant fluorescent signal was
tracked over time. Poly(A) RNA moved away from the uncaging spot in all
directions with a mean square displacement that varied linearly with time,
and the same apparent diffusion coefficient was measured for the movement
at both 37°C and 23°C. High resolution three-dimensional imaging of live
cells containing both Hoechst-labeled chromosomes and uncaged oligo(dT)
showed that, excluding nucleoli, the poly(A) RNA could access most, if not
all, of the non-chromosomal space in the nucleus. We conclude that poly(A)
RNA can move freely throughout the interchromatin space of the nucleus with
properties indistinguishable from classical diffusion.
TRACKING THE MOVEMENT OF NUCLEAR POLY(A)RNA IN LIVING CELLS
Politz JC, Tuft RA, SingerRH *, Pederson T
University of Massachusetts Medical School, Worcester,
MA and *Albert Einstein College of Medicine, Bronx, NY, USA