In this study, we investigated the behavior of rDNA loci in senescent MCF-7 mammary cancer cells induced by gamma irradiation. To analyze changes in nucleolar structure we used rDNA-FISH and immunohistochemical staining with fibrillarin and UBF transcription factor. The expression levels of rDNAs and nucleolar proteins were determined by RNA-seq of total and poly-A libraries. The cytological and molecular parameters of nucleoli were monitored throughout the 7-day interval following irradiation. Senescent cells exhibited a higher proportion of smaller nucleoli as compared to cycling cells, indicating nucleolar fragmentation. The rDNA copy number and expression of rDNA variants remained stable in cycling and senescent cells. However, the levels of polyadenylated rRNA species derived from external (5'ETS) and internal (ITS1) rDNA spacers tend to increase (c.2 fold) following irradiation. At the protein level, senescent cells showed decreased levels of fibrillarin and UBF transcription factor while localization of both proteins in the nucleolus was not impaired. We conclude that withdrawal from cell cycle does not change expression patterns of rDNA variants. However, defects in rRNA processing may lead to fragmentation of nucleoli in senescent cells.

• Keywords
• Cancer cell, Irradiation, Nucleolus, Senescence, rDNA,
• Publication type
• Journal Article MeSH

In human cells, the intergenic spacers (IGS), which separate ribosomal genes, are complex approximately 30 kb-long loci. Recent studies indicate that all, or almost all, parts of IGS may be transcribed, and that at least some of them are involved in the regulation of the ribosomal DNA (rDNA) transcription, maintenance of the nucleolar architecture, and response of the cell nucleus to stress. However, since each cell contains hundreds not quite identical copies of IGS, the structure and functions of this locus remain poorly understood, and the dynamics of its products has not been specially studied. In this work, we used quantitative PCR to measure the expression levels of various rDNA regions at different times after inhibition of the transcription by Actinomycin D applied in high doses. This approach allowed us to measure real or extrapolated half-life times of some IGS loci. Our study reveals characteristic dynamic patterns suggestive of various pathways of RNA utilization and decay.

• Keywords
• Intergenic spacer, Processing, RNA decay, lncRNAs, rDNA,
• MeSH
• HeLa Cells MeSH
• Humans MeSH
• DNA, Ribosomal Spacer chemistry   genetics   metabolism   MeSH
• RNA analysis   biosynthesis   genetics   isolation & purification   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA, Ribosomal Spacer MeSH
• RNA 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

BACKGROUND: The repair of spontaneous and induced DNA lesions is a multistep process. Depending on the type of injury, damaged DNA is recognized by many proteins specifically involved in distinct DNA repair pathways. RESULTS: We analyzed the DNA-damage response after ultraviolet A (UVA) and γ irradiation of mouse embryonic fibroblasts and focused on upstream binding factor 1 (UBF1), a key protein in the regulation of ribosomal gene transcription. We found that UBF1, but not nucleolar proteins RPA194, TCOF, or fibrillarin, was recruited to UVA-irradiated chromatin concurrently with an increase in heterochromatin protein 1β (HP1β) level. Moreover, Förster Resonance Energy Transfer (FRET) confirmed interaction between UBF1 and HP1β that was dependent on a functional chromo shadow domain of HP1β. Thus, overexpression of HP1β with a deleted chromo shadow domain had a dominant-negative effect on UBF1 recruitment to UVA-damaged chromatin. Transcription factor UBF1 also interacted directly with DNA inside the nucleolus but no interaction of UBF1 and DNA was confirmed outside the nucleolus, where UBF1 recruitment to DNA lesions appeared simultaneously with cyclobutane pyrimidine dimers; this occurrence was cell-cycle-independent. CONCLUSIONS: We propose that the simultaneous presence and interaction of UBF1 and HP1β at DNA lesions is activated by the presence of cyclobutane pyrimidine dimers and mediated by the chromo shadow domain of HP1β. This might have functional significance for nucleotide excision repair.

• Keywords
• DNA repair, DNA-damage response, Irradiation, Live-cell studies, Nucleolus, UBF1,
• Publication type
• Journal Article MeSH