BACKGROUND: Chemical modifications in mRNAs, tRNAs, rRNAs, and non-coding RNAs stabilize these nucleic acids and regulate their function. In addition to regulating the translation of genetic information from mRNA to proteins, it has been revealed that modifications in RNAs regulate repair processes in the genome. METHODS: Using local laser microirradiation, confocal microscopy, dot blots, and mass spectrometry we studied the role of N7-methylguanosine (m7G), which is co-transcriptionally installed in RNA. RESULTS: Here, we show that after UVC and UVA irradiation, the level of m7G RNA is increased initially in the cytoplasm, and after local laser microirradiation, m7G RNA is highly abundant in UVA-damaged chromatin. This process is poly(ADP-ribose) polymerase (PARP)-dependent, but not accompanied by changes in the level of m7G-writers, including methyltransferases RNMT, METTL1, and WBSCR22. We also observed that METTL1 deficiency does not affect the recruitment of m7G RNA to microirradiated chromatin. Analyzing the levels of mRNA, let-7e, and miR-203a in both the cytoplasm and the cell nucleus, we revealed that UVC irradiation changed the level of mRNA, and significantly increased the pool of both let-7e and miR-203a, which correlated with radiation-induced m7G RNA increase in the cytoplasm. CONCLUSIONS: Irradiation by UV light increases the m7G RNA pool in the cytoplasm and in the microirradiated genome. Thus, epigenetically modified RNAslikely contribute to DNA damage responses or m7G signals the presence of RNA damage.

• Klíčová slova
• DNA repair, RNA methylation, mRNA, miRNA, snRNA,
• Publikační typ
• časopisecké články 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.

• Klíčová slova
• DNA repair, METTL-like enzymes, RNA methylation, epigenetics, histones,
• MeSH
• adenosin analogy a deriváty   metabolismus   MeSH
• chromatin metabolismus   MeSH
• demetylace DNA účinky záření   MeSH
• fyziologický stres účinky záření   MeSH
• guanosin analogy a deriváty   metabolismus   MeSH
• metylace DNA genetika   účinky záření   MeSH
• metylace účinky záření   MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• nekódující RNA metabolismus   MeSH
• nestabilita genomu účinky záření   MeSH
• poškození DNA MeSH
• RNA metabolismus   MeSH
• ultrafialové záření * MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosin MeSH
• chromatin MeSH
• guanosin MeSH
• N-methyladenosine MeSH Prohlížeč
• N(2),N(2),7-trimethylguanosine MeSH Prohlížeč
• nekódující RNA MeSH
• RNA MeSH

Nuclear architecture plays a significant role in DNA repair mechanisms. It is evident that proteins involved in DNA repair are compartmentalized in not only spontaneously occurring DNA lesions or ionizing radiation-induced foci (IRIF), but a specific clustering of these proteins can also be observed within the whole cell nucleus. For example, 53BP1-positive and BRCA1-positive DNA repair foci decorate chromocenters and can appear close to nuclear speckles. Both 53BP1 and BRCA1 are well-described factors that play an essential role in double-strand break (DSB) repair. These proteins are members of two protein complexes: 53BP1-RIF1-PTIP and BRCA1-CtIP, which make a "decision" determining whether canonical nonhomologous end joining (NHEJ) or homology-directed repair (HDR) is activated. It is generally accepted that 53BP1 mediates the NHEJ mechanism, while HDR is activated via a BRCA1-dependent signaling pathway. Interestingly, the 53BP1 protein appears relatively quickly at DSB sites, while BRCA1 is functional at later stages of DNA repair, as soon as the Mre11-Rad50-Nbs1 complex is recruited to the DNA lesions. A function of the 53BP1 protein is also linked to a specific histone signature, including phosphorylation of histone H2AX (γH2AX) or methylation of histone H4 at the lysine 20 position (H4K20me); therefore, we also discuss an epigenetic landscape of 53BP1-positive DNA lesions.

• Klíčová slova
• 53BP1, BRCA1, DNA damage, epigenetics, histone modifications,
• MeSH
• 53BP1 genetika   metabolismus   MeSH
• buněčné jádro genetika   metabolismus   MeSH
• fosforylace MeSH
• lidé MeSH
• oprava DNA * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• 53BP1 MeSH

Methylation of histones H4 at lysine 20 position (H4K20me), which is functional in DNA repair, represents a binding site for the 53BP1 protein. Here, we show a radiation-induced increase in the level of H4K20me3 while the levels of H4K20me1 and H4K20me2 remained intact. H4K20me3 was significantly pronounced at DNA lesions in only the G1 phase of the cycle, while this histone mark was reduced in very late S and G2 phases when PCNA was recruited to locally micro-irradiated chromatin. H4K20me3 was diminished in locally irradiated Suv39h1/h2 double knockout (dn) fibroblasts, and the same phenomenon was observed for H3K9me3 and its binding partner, the HP1β protein. Immunoprecipitation showed the existence of an interaction between H3K9me3-53BP1 and H4K20me3-53BP1; however, HP1β did not interact with 53BP1. Together, H3K9me3 and H4K20me3 represent epigenetic markers that are important for the function of the 53BP1 protein in non-homologous end joining (NHEJ) repair. The very late S phase represents the cell cycle breakpoint when a DDR function of the H4K20me3-53BP1 complex is abrogated due to recruitment of the PCNA protein and other DNA repair factors of homologous recombination to DNA lesions.

• Klíčová slova
• DNA damage, H3K9me3, H4K20me1/me2/me3, Suv39h1/h2, epigenetics,
• MeSH
• 53BP1 genetika   metabolismus   MeSH
• buněčné jádro genetika   metabolismus   účinky záření   MeSH
• buněčné linie MeSH
• buněčný cyklus MeSH
• chromozomální proteiny, nehistonové metabolismus   MeSH
• epigeneze genetická * účinky záření   MeSH
• histony metabolismus   MeSH
• homolog proteinu s chromoboxem 5 MeSH
• lidé MeSH
• metylace DNA * účinky záření   MeSH
• metylace MeSH
• myši MeSH
• oprava DNA spojením konců * MeSH
• poškození DNA * MeSH
• proliferační antigen buněčného jádra metabolismus   MeSH
• restrukturace chromatinu MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 53BP1 MeSH
• CBX1 protein, human MeSH Prohlížeč
• Cbx1 protein, mouse MeSH Prohlížeč
• chromozomální proteiny, nehistonové MeSH
• histony MeSH
• homolog proteinu s chromoboxem 5 MeSH
• PCNA protein, human MeSH Prohlížeč
• proliferační antigen buněčného jádra MeSH
• TP53BP1 protein, human MeSH Prohlížeč
• Trp53bp1 protein, mouse MeSH Prohlížeč

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological approach to analyzing protein kinetics at DNA lesions over time or protein-protein interactions on locally microirradiated chromatin. We also show how to recognize individual phases of the cell cycle using the Fucci cellular system to study cell-cycle-dependent protein kinetics at DNA lesions. A methodological description of the use of two UV lasers (355 nm and 405 nm) to induce different types of DNA damage is also presented. Only the cells microirradiated by the 405-nm diode laser proceeded through mitosis normally and were devoid of cyclobutane pyrimidine dimers (CPDs). We also show how microirradiated cells can be fixed at a given time point to perform immunodetection of the endogenous proteins of interest. For the DNA repair studies, we additionally describe the use of biophysical methods including FRAP (Fluorescence Recovery After Photobleaching) and FLIM (Fluorescence Lifetime Imaging Microscopy) in cells with spontaneously occurring DNA damage foci. We also show an application of FLIM-FRET (Fluorescence Resonance Energy Transfer) in experimental studies of protein-protein interactions.

• MeSH
• geny p53 * MeSH
• interakční proteinové domény a motivy * MeSH
• kinetika MeSH
• konfokální mikroskopie metody   MeSH
• poškození DNA * MeSH
• Publikační typ
• audiovizuální média MeSH
• časopisecké články MeSH

DNA repair is a complex process that prevents genomic instability. Many proteins play fundamental roles in regulating the optimal repair of DNA lesions. Proliferating cell nuclear antigen (PCNA) is a key factor that initiates recombination-associated DNA synthesis after injury. Here, in very early S-phase, we show that the fluorescence intensity of mCherry-tagged PCNA after local micro-irradiation was less than the fluorescence intensity of non-irradiated mCherry-PCNA-positive replication foci. However, PCNA protein accumulated at locally irradiated chromatin in very late S-phase of the cell cycle, and this effect was more pronounced in the following G2 phase. In comparison to the dispersed form of PCNA, a reduced mobile fraction appeared in PCNA-positive replication foci during S-phase, and we observed similar recovery time after photobleaching at locally induced DNA lesions. This diffusion of mCherry-PCNA in micro-irradiated regions was not affected by cell cycle phases. We also studied the link between function of PCNA and A-type lamins in late S-phase. We found that the accumulation of PCNA at micro-irradiated chromatin is identical in wild-type and A-type lamin-deficient cells. Only micro-irradiation of the nuclear interior, and thus the irradiation of internal A-type lamins, caused the fluorescence intensity of mCherry-tagged PCNA to increase. In summary, we showed that PCNA begins to play a role in DNA repair in late S-phase and that PCNA function in repair is maintained during the G2 phase of the cell cycle. However, PCNA mobility is reduced after local micro-irradiation regardless of the cell cycle phase.

• Klíčová slova
• DNA repair, Lamins, Micro-irradiation, PCNA, S/G2 phases, rDNA,
• MeSH
• buněčné dělení genetika   fyziologie   MeSH
• buněčné jádro metabolismus   MeSH
• buněčný cyklus genetika   fyziologie   MeSH
• chromatin genetika   metabolismus   MeSH
• G2 fáze genetika   fyziologie   MeSH
• oprava DNA genetika   fyziologie   MeSH
• proliferační antigen buněčného jádra genetika   metabolismus   MeSH
• S fáze genetika   fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• chromatin MeSH
• proliferační antigen buněčného jádra MeSH

Cajal bodies (CBs) are important compartments containing accumulated proteins that preferentially regulate RNA-related nuclear events, including splicing. Here, we studied the nuclear distribution pattern of CBs in neurogenesis. In adult brains, coilin was present at a high density, but CB formation was absent in the nuclei of the choroid plexus of the lateral ventricles. Cells of the adult hippocampus were characterized by a crescent-like morphology of coilin protein. We additionally observed a 70 kDa splice variant of coilin in adult mouse brains, which was different to embryonic brains and mouse pluripotent embryonic stem cells (mESCs), characterized by the 80 kDa standard variant of coilin. Here, we also showed that depletion of coilin is induced during neural differentiation and HDAC1 deficiency in mESCs caused coilin accumulation inside the fibrillarin-positive region of the nucleoli. A similar distribution pattern was observed in adult brain hippocampi, characterized by lower levels of both coilin and HDAC1. In summary, we observed that neural differentiation and HDAC1 deficiency lead to coilin depletion and coilin accumulation in body-like structures inside the nucleoli.

• Publikační typ
• časopisecké články MeSH

The nucleolus is a well-organized site of ribosomal gene transcription. Moreover, many DNA repair pathway proteins, including ATM, ATR kinases, MRE11, PARP1 and Ku70/80, localize to the nucleolus (Moore et al., 2011 ). We analyzed the consequences of DNA damage in nucleoli following ultraviolet A (UVA), C (UVC), or γ-irradiation in order to test whether and how radiation-mediated genome injury affects local motion and morphology of nucleoli. Because exposure to radiation sources can induce changes in the pattern of UBF1-positive nucleolar regions, we visualized nucleoli in living cells by GFP-UBF1 expression for subsequent morphological analyses and local motion studies. UVA radiation, but not 5 Gy of γ-rays, induced apoptosis as analyzed by an advanced computational method. In non-apoptotic cells, we observed that γ-radiation caused nucleolar re-positioning over time and changed several morphological parameters, including the size of the nucleolus and the area of individual UBF1-positive foci. Radiation-induced nucleoli re-arrangement was observed particularly in G2 phase of the cell cycle, indicating repair of ribosomal genes in G2 phase and implying that nucleoli are less stable, thus sensitive to radiation, in G2 phase.

• Klíčová slova
• DNA damage, UBF1, live cells, nucleolus, nuncleoli tracking,
• MeSH
• apoptóza účinky záření   MeSH
• buněčné jadérko účinky záření   MeSH
• buněčné linie MeSH
• buněčný cyklus účinky záření   MeSH
• G2 fáze účinky záření   MeSH
• genetická transkripce MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• poškození DNA účinky záření   MeSH
• transkripční iniciační komplex Pol1 - proteiny genetika   metabolismus   MeSH
• ultrafialové záření MeSH
• výpočetní biologie MeSH
• záření gama škodlivé účinky   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• transcription factor UBF MeSH Prohlížeč
• transkripční iniciační komplex Pol1 - proteiny MeSH