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Kinetics of HCMV immediate early mRNA expression in stably transfected fibroblasts

Department of Molecular Cell Biology, Laboratory for Cytochemistry and Cytometry, Leiden University Medical Center, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands

Corresponding author (e-mail: R.W.Dirks{at}lumc.nl)

Accepted October 12, 2001

Compelling evidence supports an intimate link in time and space between eukaryotic pre-mRNA synthesis and processing and nucleocytoplasmic transport of mature mRNA. In this study, we analyzed the kinetic behavior of these processes in a quantitative manner. We used FISH and confocal scanning laser microscopy to detect transcripts produced by an inducible human cytomegalovirus immediate early (HCMV-IE) expression system. Upon induction, a large amount of pre-mRNA accumulated in nuclear foci at or near their transcription sites and, at later time, throughout the nucleoplasm. Inhibition of RNA polymerase II activity resulted in a rapid decrease in the number of transcripts in the nuclear RNA foci (half time two minutes), indicating that accumulated transcripts were rapidly spliced and then released. The dispersed nucleoplasmic transcripts exited the nucleus with a half time of 10 minutes. Both processes were temperature dependent, suggesting that mRNA export is an active process. RNA polymerase II activation revealed that production of mature HCMV IE mRNAs required less than five minutes. Transcripts radiated from the gene at an average speed of 0.13 µm²/sec from this time on. Thus, it appears that these processes are tightly linked in time and space, with the splicing reaction as a rate-limiting factor.

Key words: Fluorescent in situ hybridization, Confocal scanning laser microscopy, RNA polymerase II, Human cytomegalovirus

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