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.

• Klíčová slova
• PARP inhibitor, RNA pol II, SC-35, splicing,
• 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
• Názvy látek
• laminy MeSH
• PARP inhibitory MeSH
• PARP1 protein, human MeSH Prohlížeč
• poly(ADP-ribosa)polymerasa 1 MeSH
• RNA-polymerasa II MeSH
• sestřihové faktory MeSH

53BP1 is a very well-known protein that is recruited to DNA lesions. The focal accumulation of p53 binding protein, 53BP1, is a main feature indicating the repair of spontaneous or irradiation-induced foci (IRIF). Thus, here, we addressed the question of whether mutations in the TP53 gene, which often affect the level of p53 protein, can change the recruitment of 53BP1 to γ- or UVA-irradiated chromatin. In various TP53 mutants, we observed a distinct accumulation of 53BP1 protein to UV-induced DNA lesions: in R273C mutants, 53BP1 appeared transiently at DNA lesions, during 10-30 min after irradiation; the mutation R282W was responsible for accumulation of 53BP1 immediately after UVA-damage; and in L194F mutants, the first appearance of 53BP1 protein at the lesions occurred during 60-70 min. These results showed that specific mutations in the TP53 gene stand behind not only different levels of p53 protein, but also affect the localized kinetics of 53BP1 protein in UVA-damaged chromatin. However, after γ-irradiation, only G245S mutation in TP53 gene was associated with surprisingly decreased level of 53BP1 protein. In other mutant cell lines, levels of 53BP1 were not affected by γ-rays. To these effects, we conversely found a distinct number of 53BP1-positive irradiation-induced foci in various TP53 mutants. The R280K, G245S, L194F mutations, or TP53 deletion were also characterized by radiation-induced depletion in MDC1 protein. Moreover, in mutant cells, an interaction between MDC1 and 53BP1 proteins was abrogated when compared with wild-type counterpart. Together, the kinetics of 53BP1 accumulation at UV-induced DNA lesions is different in various TP53 mutant cells. After γ-irradiation, despite changes in a number and a volume of 53BP1-positive foci, levels of 53BP1 protein were relatively stable. Here, we showed a link between the status of MDC1 protein and TP53 gene, which specific mutations caused radiation-induced MDC1 down-regulation. This observation is significant, especially with regard to radiotherapy of tumors with abrogated function of TP53 gene.

• Klíčová slova
• 53BP1 protein, DNA repair, Histone γH2AX, MDC1 protein, TP53 gene,
• MeSH
• 53BP1 metabolismus   MeSH
• adaptorové proteiny signální transdukční MeSH
• down regulace MeSH
• jaderné proteiny nedostatek   metabolismus   MeSH
• lidé MeSH
• mutace * MeSH
• nádorové buňky kultivované MeSH
• nádorový supresorový protein p53 nedostatek   genetika   metabolismus   MeSH
• poškození DNA * MeSH
• proteiny buněčného cyklu MeSH
• trans-aktivátory nedostatek   metabolismus   MeSH
• ultrafialové záření * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• 53BP1 MeSH
• adaptorové proteiny signální transdukční MeSH
• jaderné proteiny MeSH
• MDC1 protein, human MeSH Prohlížeč
• nádorový supresorový protein p53 MeSH
• proteiny buněčného cyklu MeSH
• TP53 protein, human MeSH Prohlížeč
• TP53BP1 protein, human MeSH Prohlížeč
• trans-aktivátory MeSH