J C Politz1, R A Tuft, T Pederson, R H Singer. 1. Department of Biochemistry and Molecular Biology, Biomedical Imaging Center, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA. Joan.Politz@ummed.edu
Abstract
BACKGROUND: Messenger RNA (mRNA) is transcribed and processed in the nucleus of eucaryotic cells and then exported to the cytoplasm through nuclear pores. It is not known whether the movement of mRNA from its site of synthesis to the nuclear pore is directed or random. Directed movement would suggest that there is an energy-requiring step in addition to the step required for active transport through the pore, whereas random movement would indicate 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 chemically masked (caged) fluorescein was allowed to penetrate cells and hybridize to nuclear poly(A) RNA. Laser spot photolysis then uncaged the oligo(dT) at a given intranuclear site and the resultant fluorescent, hybridized oligo(dT) was tracked using high-speed imaging microscopy. 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 degrees C and 23 degrees C. These properties are characteristic of a random diffusive process. 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. CONCLUSIONS: Poly(A) RNA can move freely throughout the interchromatin space of the nucleus with properties characteristic of diffusion.
BACKGROUND: Messenger RNA (mRNA) is transcribed and processed in the nucleus of eucaryotic cells and then exported to the cytoplasm through nuclear pores. It is not known whether the movement of mRNA from its site of synthesis to the nuclear pore is directed or random. Directed movement would suggest that there is an energy-requiring step in addition to the step required for active transport through the pore, whereas random movement would indicate 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 chemically masked (caged) fluorescein was allowed to penetrate cells and hybridize to nuclear poly(A) RNA. Laser spot photolysis then uncaged the oligo(dT) at a given intranuclear site and the resultant fluorescent, hybridized oligo(dT) was tracked using high-speed imaging microscopy. 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 degrees C and 23 degrees C. These properties are characteristic of a random diffusive process. 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. CONCLUSIONS:Poly(A) RNA can move freely throughout the interchromatin space of the nucleus with properties characteristic of diffusion.
Authors: C Molenaar; S A Marras; J C Slats; J C Truffert; M Lemaître; A K Raap; R W Dirks; H J Tanke Journal: Nucleic Acids Res Date: 2001-09-01 Impact factor: 16.971
Authors: Jan Peter Siebrasse; Roman Veith; Akos Dobay; Heinrich Leonhardt; Bertil Daneholt; Ulrich Kubitscheck Journal: Proc Natl Acad Sci U S A Date: 2008-12-12 Impact factor: 11.205