Nucleocytoplasmic transport of fluorescent mRNA in living mammalian cells: nuclear mRNA export is coupled to ongoing gene transcription.
In eukaryotic cells, export of mRNA from the nucleus to the cytoplasm is one of the essential steps in gene expression. To examine mechanisms involved in the nucleocytoplasmic transport of mRNA, we microinjected fluorescently labeled fushi tarazu (ftz) pre-mRNA into the nuclei of HeLa cells. The injected intron-containing ftz pre-mRNA was distributed to the SC35 speckles and exported to the cytoplasm after splicing by an energy-requiring active process. In contrast, the injected intron-less ftz mRNA was diffusely distributed in the nucleus and then presumably degraded. Interestingly, export of the ftz pre-mRNA was inhibited by treatment with transcriptional inhibitors (actinomycin D, alpha-amanitin or DRB). Cells treated with transcriptional inhibitor showed foci enriched with the injected mRNA, which localize side by side with SC35 speckles. Those nuclear foci, referred to as TIDRs (transcriptional-inactivation dependent RNA domain), do not overlap with paraspeckles. In addition, in situ hybridization analysis revealed that the export of endogenous poly(A)+ mRNA is also affected by transcriptional inactivation. These results suggest that nuclear mRNA export is coupled to ongoing gene transcription in mammalian cells.
Department of Biological Science, Faculty of Science, Kumamoto University, Kumamoto 860-8555, Japan.
Kazuaki
K
Tokunaga
Toshiharu
T
Shibuya
Yo
Y
Ishihama
Hisashi
H
Tadakuma
Miyuki
M
Ide
Minoru
M
Yoshida
Takashi
T
Funatsu
Yasumi
Y
Ohshima
Tokio
T
Tani
D021581:Active Transport, Cell Nucleus; D002467:Cell Nucleus; D003593:Cytoplasm; D003609:Dactinomycin / Q000494:pharmacology; D005456:Fluorescent Dyes; D051717:Fushi Tarazu Transcription Factors / Q000235:genetics / Q000378:metabolism*; D005914:Globins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D017403:In Situ Hybridization; D007438:Introns; D008845:Microinjections; D009687:Nuclear Proteins / Q000378:metabolism; D011061:Poly A; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012322:RNA Precursors / Q000378:metabolism*; D015347:RNA Probes; D012323:RNA Processing, Post-Transcriptional; D012326:RNA Splicing; D012333:RNA, Messenger / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000378:metabolism; D000068103:Serine-Arginine Splicing Factors; D014158:Transcription, Genetic
D005456:Fluorescent Dyes; D051717:Fushi Tarazu Transcription Factors; D009687:Nuclear Proteins; D011500:Protein Synthesis Inhibitors; D012322:RNA Precursors; D015347:RNA Probes; D012333:RNA, Messenger; D012261:Ribonucleoproteins; C073288:SRSF2 protein, human; D000068103:Serine-Arginine Splicing Factors; D003609:Dactinomycin; D011061:Poly A; D005914:Globins