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Poly(A)⁺ RNAs roam the cell nucleus and pass through speckle domains in transcriptionally active and inactive cells

Department of Molecular Cell Biology, Leiden University Medical Center, 2333 AL Leiden, Netherlands

Address correspondence to R.W. Dirks, Dept. of Molecular Cell Biology, Leiden University Medical Center, Wassenaarseweg 72, 2333 AL Leiden, Netherlands. Tel.: 31-71-5276026. Fax.: 31-71-5276180. email: r.w.dirks{at}lumc.nl

Many of the protein factors that play a role in nuclear export of mRNAs have been identified, but still little is known about how mRNAs are transported through the cell nucleus and which nuclear compartments are involved in mRNA transport. Using fluorescent 2'O-methyl oligoribonucleotide probes, we investigated the mobility of poly(A)⁺ RNA in the nucleoplasm and in nuclear speckles of U2OS cells. Quantitative analysis of diffusion using photobleaching techniques revealed that the majority of poly(A)⁺ RNA move throughout the nucleus, including in and out of speckles (also called SC-35 domains), which are enriched for splicing factors. Interestingly, in the presence of the transcription inhibitor 5,6-dichloro-1-ß-D-ribofuranosylbenzimidazole, the association of poly(A)⁺ RNA with speckles remained dynamic. Our results show that RNA movement is energy dependent and that the proportion of nuclear poly(A)⁺ RNA that resides in speckles is a dynamic population that transiently interacts with speckles independent of the transcriptional status of the cell. Rather than the poly(A)⁺ RNA within speckles serving a stable structural role, our findings support the suggestion of a more active role of these regions in nuclear RNA metabolism and/or transport.

Key Words: RNA transport; mRNA; 2'O-methyl RNA; FRAP; live cell imaging

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