Imaging of single mRNA molecules moving within a living cell nucleus

Abstract

In eukaryotic cells, pre-mRNAs are transcribed in the nucleus, processed by 5′ capping, 3′-polyadenylation, and splicing, and exported to the cytoplasm for translation. To examine the nuclear mRNA transport mechanism, intron-deficient mRNAs of truncated β-globin and EGFP were synthesized, fluorescently labeled in vitro, and injected into the nucleus of living Xenopus A6 cells. The trajectories of single mRNA molecules in the nucleus were visualized using video-rate confocal microscopy. Approximately half the mRNAs moved by Brownian motion in the nucleoplasm, except the nucleoli, with an apparent diffusion coefficient of 0.2 μm 2/s, about 1/150 of that in water. The slow diffusion could not be explained by simple diffusion obeying the Stokes–Einstein equation, suggesting interactions of the mRNAs with nuclear components. The remaining mRNAs were stationary with an average residence time of about 30 s, comparable to the time required for mRNA diffusion from the site of synthesis to nuclear pores.