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

N4-acetylcytidine (ac4C) is a post-transcriptional RNA modification that plays a crucial role in the epitranscriptome, influencing gene expression and cellular function. This modification occurs at the cytosine base, where an acetyl group is installed to the nitrogen at the 4th position (N4). This co-transcription modification affects RNA stability, RNA structure, and translation efficiency. Recent studies have uncovered a potential link between RNA modifications and DNA repair mechanisms, suggesting that ac4C-modified or methylated RNAs may interact with factors involved in DNA repair pathways; thus, influencing the cellular response to DNA damage. Dysregulation of modified RNAs, including ac4C RNA, has been implicated in cancer development, where aberrant levels of these RNAs may contribute to oncogenic transformation by altering genome stability and the expression of key genes regulating cell proliferation, cell cycle progression, and apoptosis. Understanding the dynamics of modified RNAs offers promising insights into the role of epitranscriptome in DNA repair processes and cancer treatment.

• Keywords
• DNA damage repair, N-acetylcytidine, NAT10, RNA modifications, epigenetics, epitranscriptomics,
• MeSH
• Cytidine * analogs & derivatives   metabolism   MeSH
• Epigenesis, Genetic * MeSH
• Humans MeSH
• Neoplasms * genetics   metabolism   MeSH
• DNA Repair * MeSH
• RNA Processing, Post-Transcriptional * MeSH
• Gene Expression Regulation, Neoplastic MeSH
• RNA * metabolism   genetics   MeSH
• Transcriptome * MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Cytidine * MeSH
• N-acetylcytidine MeSH Browser
• RNA * MeSH

METTL16 methyltransferase is responsible for the methylation of N6-adenosine (m6A) in several RNAs. In mouse cells, we showed that the nuclear distribution of METTL16 is cell cycle-specific. In the G1/S phases, METTL16 accumulates to the nucleolus, while in the G2 phase, the level of METTL16 increases in the nucleoplasm. In metaphase and anaphase, there is a very low pool of the METTL16 protein, but in telophase, residual METTL16 appears to be associated with the newly formed nuclear lamina. In A-type lamin-depleted cells, we observed a reduction of METTL16 when compared with the wild-type counterpart. However, METTL16 does not interact with A-type and B-type lamins, but interacts with Lamin B Receptor (LBR) and Lap2α. Additionally, Lap2α depletion caused METTL16 downregulation in the nuclear pool. Furthermore, METTL16 interacted with DDB2, a key protein of the nucleotide excision repair (NER), and also with nucleolar proteins, including TCOF, NOLC1, and UBF1/2, but not fibrillarin. From this view, the METTL16 protein may also regulate the transcription of ribosomal genes because we observed that the high level of m6A in 18S rRNA appeared in cells with upregulated METTL16.

• Keywords
• METTL16, cell cycle, epitranscriptome, nucleolus, rDNA,
• Publication type
• Journal Article MeSH

Cellular senescence, induced by genotoxic or replication stress, is accompanied by defects in nuclear morphology and nuclear membrane-heterochromatin disruption. In this work, we analyzed cytological and molecular changes in the linker of nucleoskeleton and cytoskeleton (LINC) complex proteins in senescence triggered by γ-irradiation. We used human mammary carcinoma and osteosarcoma cell lines, both original and shRNA knockdown clones targeting lamin B receptor (LBR) and leading to LBR and lamin B (LB1) reduction. The expression status and integrity of LINC complex proteins (nesprin-1, SUN1, SUN2), lamin A/C, and emerin were analyzed by immunodetection using confocal microscopy and Western blot. The results show frequent mislocalization of these proteins from the nuclear membrane to cytoplasm and micronuclei and, in some cases, their fragmentation and amplification. The timing of these changes clearly preceded the onset of senescence. The LBR deficiency triggered neither senescence nor changes in the LINC protein distribution before irradiation. However, the cytological changes following irradiation were more pronounced in shRNA knockdown cells compared to original cell lines. We conclude that mislocalization of LINC complex proteins is a significant characteristic of cellular senescence phenotypes and may influence complex events at the nuclear membrane, including trafficking and heterochromatin attachment.

• Keywords
• LINC complex proteins, SUN1/2, heterochromatin-nuclear membrane disconnection, lamin B receptor, nesprin-1, γ-irradiation,
• MeSH
• Spatio-Temporal Analysis MeSH
• Nuclear Envelope metabolism   MeSH
• Humans MeSH
• Membrane Proteins metabolism   MeSH
• Lamin B Receptor MeSH
• Receptors, Cytoplasmic and Nuclear genetics   MeSH
• Cellular Senescence genetics   MeSH
• Gamma Rays therapeutic use   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Membrane Proteins MeSH
• Receptors, Cytoplasmic and Nuclear MeSH

The DNA damage response is mediated by both DNA repair proteins and epigenetic markers. Here, we observe that N6-methyladenosine (m6A), a mark of the epitranscriptome, was common in RNAs accumulated at UV-damaged chromatin; however, inhibitors of RNA polymerases I and II did not affect the m6A RNA level at the irradiated genomic regions. After genome injury, m6A RNAs either diffused to the damaged chromatin or appeared at the lesions enzymatically. DNA damage did not change the levels of METTL3 and METTL14 methyltransferases. In a subset of irradiated cells, only the METTL16 enzyme, responsible for m6A in non-coding RNAs as well as for splicing regulation, was recruited to microirradiated sites. Importantly, the levels of the studied splicing factors were not changed by UVA light. Overall, if the appearance of m6A RNAs at DNA lesions is regulated enzymatically, this process must be mediated via the coregulatory function of METTL-like enzymes. This event is additionally accompanied by radiation-induced depletion of 2,2,7-methylguanosine (m3G/TMG) in RNA. Moreover, UV-irradiation also decreases the global cellular level of N1-methyladenosine (m1A) in RNAs. Based on these results, we prefer a model in which m6A RNAs rapidly respond to radiation-induced stress and diffuse to the damaged sites. The level of both (m1A) RNAs and m3G/TMG in RNAs is reduced as a consequence of DNA damage, recognized by the nucleotide excision repair mechanism.

• Keywords
• DNA repair, METTL-like enzymes, RNA methylation, epigenetics, histones,
• MeSH
• Adenosine analogs & derivatives   metabolism   MeSH
• Chromatin metabolism   MeSH
• DNA Demethylation radiation effects   MeSH
• Stress, Physiological radiation effects   MeSH
• Guanosine analogs & derivatives   metabolism   MeSH
• DNA Methylation genetics   radiation effects   MeSH
• Methylation radiation effects   MeSH
• Mice MeSH
• Cell Line, Tumor MeSH
• RNA, Untranslated metabolism   MeSH
• Genomic Instability radiation effects   MeSH
• DNA Damage MeSH
• RNA metabolism   MeSH
• Ultraviolet Rays * MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenosine MeSH
• Chromatin MeSH
• Guanosine MeSH
• N-methyladenosine MeSH Browser
• N(2),N(2),7-trimethylguanosine MeSH Browser
• RNA, Untranslated MeSH
• RNA MeSH