Separation of replication and transcription domains in nucleoli

Separation of replication and transcription domains in nucleoli

In mammalian cells, active ribosomal genes produce the 18S, 5.8S and 28S RNAs of ribosomal particles. Transcription levels of these genes are very high throughout interphase, and the cell needs a special strategy to avoid collision of the DNA polymerase and RNA polymerase machineries. To investigate...

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Bibliographic Details
Published in:Journal of structural biology Vol. 188; no. 3; pp. 259 - 266
Main Authors: Smirnov, E., Borkovec, J., Kováčik, L., Svidenská, S., Schröfel, A., Skalníková, M., Švindrych, Z., Křížek, P., Ovesný, M., Hagen, G.M., Juda, P., Michalová, K., Cardoso, M.C., Cmarko, D., Raška, I.
Format: Journal Article
Language:English
Published: United States Elsevier Inc 01-12-2014
Subjects:
Cell Line
Cell Nucleolus - genetics
Cell Nucleolus - metabolism
DNA Replication
HeLa Cells
Humans
Nucleolus
rDNA
Replication
Single-molecule localization microscopy
Super-resolution
Transcription
Transcription, Genetic
rDNA
Replication
Super-resolution
Single-molecule localization microscopy
Nucleolus
Transcription
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Summary:In mammalian cells, active ribosomal genes produce the 18S, 5.8S and 28S RNAs of ribosomal particles. Transcription levels of these genes are very high throughout interphase, and the cell needs a special strategy to avoid collision of the DNA polymerase and RNA polymerase machineries. To investigate this problem, we measured the correlation of various replication and transcription signals in the nucleoli of HeLa, HT-1080 and NIH 3T3 cells using a specially devised software for analysis of confocal images. Additionally, to follow the relationship between nucleolar replication and transcription in living cells, we produced a stable cell line expressing GFP-RPA43 (subunit of RNA polymerase I, pol I) and RFP-PCNA (the sliding clamp protein) based on human fibrosarcoma HT-1080 cells. We found that replication and transcription signals are more efficiently separated in nucleoli than in the nucleoplasm. In the course of S phase, separation of PCNA and pol I signals gradually increased. During the same period, separation of pol I and incorporated Cy5-dUTP signals decreased. Analysis of single molecule localization microscopy (SMLM) images indicated that transcriptionally active FC/DFC units (i.e. fibrillar centers with adjacent dense fibrillar components) did not incorporate DNA nucleotides. Taken together, our data show that replication of the ribosomal genes is spatially separated from their transcription, and FC/DFC units may provide a structural basis for that separation.
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ISSN:1047-8477
1095-8657
DOI:10.1016/j.jsb.2014.10.001