Movement of nuclear poly(A) RNA throughout the interchromatin space in living cells

Background: Messenger RNA (mRNA) is transcribed and processed in the nucleu s of eucaryotic cells and then exported to the cytoplasm through nuclear po res. It is not known whether the movement of mRNA from its site of synthesi s to the nuclear pore is directed or random. Directed movement would sugges t that there is an energy-requiring step in addition to the step required f or active transport through the pore, whereas random movement would indicat e that mRNAs can make their way to the nuclear envelope by diffusion. Results: We devised a method to visualize movement of endogenous polymerase II transcripts in the nuclei of living cells. Oligo(dT) labeled with chemi cally masked (caged) fluorescein was allowed to penetrate cells and hybridi ze to nuclear poly(A) RNA. Laser spot photolysis then uncaged the oligo(dT) at a given intranuclear site and the resultant fluorescent, hybridized oli go(dT) was tracked using high-speed imaging microscopy. Poly(A) RNA moved a way from the uncaging spot in all directions with a mean square displacemen t that varied linearly with time, and the same apparent diffusion coefficie nt was measured for the movement at both 37 degrees C and 23 degrees C. The se properties are characteristic of a random diffusive process. High resolu tion three-dimensional imaging of live cells containing both Hoechst-labele d chromosomes and uncaged oligo(dT) showed that, excluding nucleoli, the po ly(A) RNA could access most, if not all, of the non-chromosomal space in th e nucleus. Conclusions: Poly(A) RNA can move freely throughout the interchromatin spac e of the nucleus with properties characteristic of diffusion.