6th ESACP Congress, Heidelberg, April 7-11, 1999

A015
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

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.