The affiliation number 1, "Institute of Biophysics of the Czech Academy of Sciences, Královopolská 135, 612 65 Brno, Czech Republic" changed its zip code to 612 00 [...].
- Publikační typ
- tisková chyba MeSH
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.
- Publikační typ
- časopisecké články MeSH
RNA modifications have been known for many years, but their function has not been fully elucidated yet. For instance, the regulatory role of acetylation on N4-cytidine (ac4C) in RNA can be explored not only in terms of RNA stability and mRNA translation but also in DNA repair. Here, we observe a high level of ac4C RNA at DNA lesions in interphase cells and irradiated cells in telophase. Ac4C RNA appears in the damaged genome from 2 to 45 min after microirradiation. However, RNA cytidine acetyltransferase NAT10 did not accumulate to damaged sites, and NAT10 depletion did not affect the pronounced recruitment of ac4C RNA to DNA lesions. This process was not dependent on the G1, S, and G2 cell cycle phases. In addition, we observed that the PARP inhibitor, olaparib, prevents the recruitment of ac4C RNA to damaged chromatin. Our data imply that the acetylation of N4-cytidine, especially in small RNAs, has an important role in mediating DNA damage repair. Ac4C RNA likely causes de-condensation of chromatin in the vicinity of DNA lesions, making it accessible for other DNA repair factors involved in the DNA damage response. Alternatively, RNA modifications, including ac4C, could be direct markers of damaged RNAs.
- MeSH
- acetylace MeSH
- chromatin MeSH
- cytidin * genetika metabolismus MeSH
- PARP inhibitory MeSH
- RNA * metabolismus MeSH
- Publikační typ
- časopisecké články 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.
- Publikační typ
- časopisecké články MeSH
The essential components of splicing are the splicing factors accumulated in nuclear speckles; thus, we studied how DNA damaging agents and A-type lamin depletion affect the properties of these regions, positive on the SC-35 protein. We observed that inhibitor of PARP (poly (ADP-ribose) polymerase), and more pronouncedly inhibitors of RNA polymerases, caused DNA damage and increased the SC35 protein level. Interestingly, nuclear blebs, induced by PARP inhibitor and observed in A-type lamin-depleted or senescent cells, were positive on both the SC-35 protein and another component of the spliceosome, SRRM2. In the interphase cell nuclei, SC-35 interacted with the phosphorylated form of RNAP II, which was A-type lamin-dependent. In mitotic cells, especially in telophase, the SC35 protein formed a well-visible ring in the cytoplasmic fraction and colocalized with β-catenin, associated with the plasma membrane. The antibody against the SRRM2 protein showed that nuclear speckles are already established in the cytoplasm of the late telophase and at the stage of early cytokinesis. In addition, we observed the occurrence of splicing factors in the nuclear blebs and micronuclei, which are also sites of both transcription and splicing. This conclusion supports the fact that splicing proceeds transcriptionally. According to our data, this process is A-type lamin-dependent. Lamin depletion also reduces the interaction between SC35 and β-catenin in mitotic cells.
- MeSH
- HeLa buňky MeSH
- laminy metabolismus MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- PARP inhibitory terapeutické užití MeSH
- poly(ADP-ribosa)polymerasa 1 MeSH
- RNA-polymerasa II metabolismus MeSH
- sestřihové faktory metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem 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.
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- 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
Repair of ribosomal DNA (rDNA) is a very important nuclear process due to the most active transcription of ribosomal genes. Proper repair of rDNA is required for physiological biogenesis of ribosomes. Here, we analyzed the epigenetics of the DNA damage response in a nucleolar compartment, thus in the ribosomal genes studied in nonirradiated and UVA-irradiated mouse embryonic fibroblasts (MEFs). We found that the promoter of ribosomal genes is not abundant on H4K20me2, but it is densely occupied by H4K20me3. Ribosomal genes, regulated via UBF1/2 proteins, were characterized by an interaction between UBF1/2 and H4K20me2/me3. This interaction was strengthened by UVA irradiation that additionally causes a focal accumulation of H4K20me3 in the nucleolus. No interaction has been found between UBF1/2 and H3K9me3. Interestingly, UVA irradiation decreases the levels of H3K9me3 and H4K20me3 at 28S rDNA. Altogether, the UVA light affects the epigenetic status of ribosomal genes at 28S rDNA and strengthens an interaction between UBF1/2 proteins and H4K20me2/me3.
- MeSH
- buněčné jadérko metabolismus MeSH
- buněčné jádro metabolismus MeSH
- chromatinová imunoprecipitace MeSH
- DNA vazebné proteiny MeSH
- epigeneze genetická účinky záření MeSH
- fluorescenční protilátková technika MeSH
- histony metabolismus MeSH
- metylace MeSH
- myši MeSH
- promotorové oblasti (genetika) MeSH
- regulace genové exprese účinky záření MeSH
- ribozomální DNA genetika MeSH
- transkripční iniciační komplex Pol1 - proteiny metabolismus MeSH
- ultrafialové záření * MeSH
- vazba proteinů MeSH
- vysoce účinné nukleotidové sekvenování MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem 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.
- 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
We examined the levels and distribution of post-translationally modified histones and protamines in human sperm. Using western blot immunoassay, immunofluorescence, mass spectrometry (MS), and FLIM-FRET approaches, we analyzed the status of histone modifications and the protamine P2. Among individual samples, we observed variability in the levels of H3K9me1, H3K9me2, H3K27me3, H3K36me3, and H3K79me1, but the level of acetylated (ac) histones H4 was relatively stable in the sperm head fractions, as demonstrated by western blot analysis. Sperm heads with lower levels of P2 exhibited lower levels of H3K9ac, H3K9me1, H3K27me3, H3K36me3, and H3K79me1. A very strong correlation was observed between the levels of P2 and H3K9me2. FLIM-FRET analysis additionally revealed that acetylated histones H4 are not only parts of sperm chromatin but also appear in a non-integrated form. Intriguingly, H4ac and H3K27me3 were detected in sperm tail fractions via western blot analysis. An appearance of specific histone H3 and H4 acetylation and H3 methylation in sperm tail fractions was also confirmed by both LC-MS/MS and MALDI-TOF MS analysis. Taken together, these data indicate that particular post-translational modifications of histones are uniquely distributed in human sperm, and this distribution varies among individuals and among the sperm of a single individual.
- MeSH
- acetylace MeSH
- chromatin genetika MeSH
- histonlysin-N-methyltransferasa biosyntéza genetika MeSH
- histony genetika metabolismus MeSH
- lidé MeSH
- metylace MeSH
- posttranslační úpravy proteinů genetika MeSH
- sekvence aminokyselin MeSH
- spermie růst a vývoj metabolismus MeSH
- tandemová hmotnostní spektrometrie MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Although physiological steroid levels are often pulsatile (ultradian), the genomic effects of this pulsatility are poorly understood. By utilizing glucocorticoid receptor (GR) signaling as a model system, we uncovered striking spatiotemporal relationships between receptor loading, lifetimes of the DNase I hypersensitivity sites (DHSs), long-range interactions, and gene regulation. We found that hormone-induced DHSs were enriched within ± 50 kb of GR-responsive genes and displayed a broad spectrum of lifetimes upon hormone withdrawal. These lifetimes dictate the strength of the DHS interactions with gene targets and contribute to gene regulation from a distance. Our results demonstrate that pulsatile and constant hormone stimulations induce unique, treatment-specific patterns of gene and regulatory element activation. These modes of activation have implications for corticosteroid function in vivo and for steroid therapies in various clinical settings.
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- buněčné linie MeSH
- chromatin genetika metabolismus MeSH
- chromatinová imunoprecipitace MeSH
- deoxyribonukleasa I genetika metabolismus MeSH
- glukokortikoidy farmakologie MeSH
- membránové proteiny genetika metabolismus MeSH
- myši MeSH
- receptory glukokortikoidů genetika metabolismus MeSH
- regulace genové exprese MeSH
- responzivní elementy * MeSH
- sekvenční analýza DNA MeSH
- transportní proteiny genetika metabolismus MeSH
- vazba proteinů MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- Research Support, N.I.H., Intramural MeSH
