Numerous studies show that various genes in all kinds of organisms are transcribed discontinuously, i.e. in short bursts or pulses with periods of inactivity between them. But it remains unclear whether ribosomal DNA (rDNA), represented by multiple copies in every cell, is also expressed in such manner. In this work, we synchronized the pol I activity in the populations of tumour derived as well as normal human cells by cold block and release. Our experiments with 5-fluorouridine (FU) and BrUTP confirmed that the nucleolar transcription can be efficiently and reversibly arrested at +4°C. Then using special software for analysis of the microscopic images, we measured the intensity of transcription signal (incorporated FU) in the nucleoli at different time points after the release. We found that the ribosomal genes in the human cells are transcribed discontinuously with periods ranging from 45 min to 75 min. Our data indicate that the dynamics of rDNA transcription follows the undulating pattern, in which the bursts are alternated by periods of rare transcription events.

• MeSH
• Cell Nucleolus genetics   MeSH
• Epithelial Cells metabolism   MeSH
• Transcription, Genetic * MeSH
• HeLa Cells MeSH
• Kinetics MeSH
• Middle Aged MeSH
• Humans MeSH
• Limbus Corneae cytology   MeSH
• Cadaver MeSH
• Cold Temperature MeSH
• DNA, Ribosomal genetics   MeSH
• Ribosomes genetics   MeSH
• RNA, Ribosomal genetics   MeSH
• Aged MeSH
• Software MeSH
• Transfection MeSH
• Uridine analogs & derivatives   immunology   metabolism   MeSH
• Uridine Triphosphate analogs & derivatives   immunology   metabolism   MeSH
• Check Tag
• Middle Aged MeSH
• Humans MeSH
• Aged MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 5-bromouridine triphosphate MeSH Browser
• 5-fluorouridine MeSH Browser
• DNA, Ribosomal MeSH
• RNA, Ribosomal MeSH
• Uridine MeSH
• Uridine Triphosphate MeSH

Nucleoli are formed on the basis of ribosomal DNA (rDNA) clusters called Nucleolus Organizer Regions (NORs). Each NOR contains multiple genes coding for RNAs of the ribosomal particles. The prominent components of the nucleolar ultrastructure, fibrillar centers (FC) and dense fibrillar components (DFC), together compose FC/DFC units. These units are centers of rDNA transcription by RNA polymerase I (pol I), as well as the early processing events, in which an essential role belongs to fibrillarin. Each FC/DFC unit probably corresponds to a single transcriptionally active gene. In this work, we transfected human-derived cells with GFP-RPA43 (subunit of pol I) and RFP-fibrillarin. Following changes of the fluorescent signals in individual FC/DFC units, we found two kinds of kinetics: 1) the rapid fluctuations with periods of 2-3 min, when the pol I and fibrillarin signals oscillated in anti-phase manner, and the intensities of pol I in the neighboring FC/DFC units did not correlate. 2) fluctuations with periods of 10 to 60 min, in which pol I and fibrillarin signals measured in the same unit did not correlate, but pol I signals in the units belonging to different nucleoli were synchronized. Our data indicate that a complex pulsing activity of transcription as well as early processing is common for ribosomal genes.

• Keywords
• fibrillarin, fluctuation, pol I, rDNA, transcription pulsing,
• MeSH
• Cell Nucleolus chemistry   enzymology   MeSH
• Chromosomal Proteins, Non-Histone chemistry   metabolism   MeSH
• DNA-Directed RNA Polymerases chemistry   metabolism   MeSH
• HeLa Cells MeSH
• Immunohistochemistry MeSH
• Microscopy, Confocal MeSH
• Humans MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Chromosomal Proteins, Non-Histone MeSH
• DNA-Directed RNA Polymerases MeSH
• fibrillarin MeSH Browser

DNA damage response (DDR) in ribosomal genes and mechanisms of DNA repair in embryonic stem cells (ESCs) are less explored nuclear events. DDR in ESCs should be unique due to their high proliferation rate, expression of pluripotency factors, and specific chromatin signature. Given short population doubling time and fast progress through G1 phase, ESCs require a sustained production of rRNA, which leads to the formation of large and prominent nucleoli. Although transcription of rRNA in the nucleolus is relatively well understood, little is known about DDR in this nuclear compartment. Here, we directed formation of double-strand breaks in rRNA genes with I- PpoI endonuclease, and we studied nucleolar morphology, DDR, and chromatin modifications. We observed a pronounced formation of I- PpoI-induced nucleolar caps, positive on BRCA1, NBS1, MDC1, γH2AX, and UBF1 proteins. We showed interaction of nucleolar protein TCOF1 with HDAC1 and TCOF1 with CARM1 after DNA injury. Moreover, H3R17me2a modification mediated by CARM1 was found in I- PpoI-induced nucleolar caps. Finally, we report that heterochromatin protein 1 is not involved in DNA repair of nucleolar caps.

• Keywords
• CARM1, DNA repair, HDAC1, NBS1, PpoI, chromatin, nucleolus,
• MeSH
• Acetylation MeSH
• Arginine metabolism   MeSH
• Cell Nucleolus genetics   ultrastructure   MeSH
• Cell Line MeSH
• DNA Breaks, Double-Stranded * MeSH
• Embryonic Stem Cells metabolism   ultrastructure   MeSH
• Phosphoproteins metabolism   MeSH
• Genes, rRNA MeSH
• Histone Deacetylase 1 metabolism   MeSH
• Histones metabolism   MeSH
• Intracellular Signaling Peptides and Proteins MeSH
• Nuclear Proteins metabolism   MeSH
• Methylation MeSH
• Mice MeSH
• DNA Repair MeSH
• Protein-Arginine N-Methyltransferases metabolism   MeSH
• RNA, Ribosomal genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Arginine MeSH
• coactivator-associated arginine methyltransferase 1 MeSH Browser
• Phosphoproteins MeSH
• Hdac1 protein, mouse MeSH Browser
• Histone Deacetylase 1 MeSH
• Histones MeSH
• Intracellular Signaling Peptides and Proteins MeSH
• Nuclear Proteins MeSH
• Protein-Arginine N-Methyltransferases MeSH
• RNA, Ribosomal MeSH
• Tcof1 protein, mouse MeSH Browser

The essential structural components of the nucleoli, Fibrillar Centers (FC) and Dense Fibrillar Components (DFC), together compose FC/DFC units, loci of rDNA transcription and early RNA processing. In the present study we followed cell cycle related changes of these units in 2 human sarcoma derived cell lines with stable expression of RFP-PCNA (the sliding clamp protein) and GFP-RPA43 (a subunit of RNA polymerase I, pol I) or GFP-fibrillarin. Correlative light and electron microscopy analysis showed that the pol I and fibrillarin positive nucleolar beads correspond to individual FC/DFC units. In vivo observations showed that at early S phase, when transcriptionally active ribosomal genes were replicated, the number of the units in each cell increased by 60-80%. During that period the units transiently lost pol I, but not fibrillarin. Then, until the end of interphase, number of the units did not change, and their duplication was completed only after the cell division, by mid G1 phase. This peculiar mode of reproduction suggests that a considerable subset of ribosomal genes remain transcriptionally silent from mid S phase to mitosis, but become again active in the postmitotic daughter cells.

• Keywords
• FC/DFC units, cell cycle, nucleolus, rDNA, replication,
• MeSH
• Cell Nucleolus metabolism   MeSH
• HeLa Cells MeSH
• Humans MeSH
• S Phase MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH