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

Cajal bodies are important nuclear structures containing proteins that preferentially regulate RNA-related metabolism. We investigated the cell-type specific nuclear distribution of Cajal bodies and the level of coilin, a protein of Cajal bodies, in non-irradiated and irradiated human tumor cell lines and embryonic stem (ES) cells. Cajal bodies were localized in different nuclear compartments, including DAPI-poor regions, in the proximity of chromocenters, and adjacent to nucleoli. The number of Cajal bodies per nucleus was cell cycle-dependent, with higher numbers occurring during G2 phase. Human ES cells contained a high coilin level in the nucleoplasm, but coilin-positive Cajal bodies were also identified in nuclei of mouse and human ES cells. Coilin, but not SMN, recognized UVA-induced DNA lesions, which was cell cycle-independent. Treatment with γ-radiation reduced the localized movement of Cajal bodies in many cell types and GFP-coilin fluorescence recovery after photobleaching was very fast in nucleoplasm in comparison with GFP-coilin recovery in DNA lesions. By contrast, nucleolus-localized coilin displayed very slow fluorescence recovery after photobleaching, which indicates very slow rates of protein diffusion, especially in nucleoli of mouse ES cells.

• Klíčová slova
• Cajal bodies, DNA repair, chromatin, coilin, nucleolus, nucleus,
• MeSH
• buněčné jádro genetika   metabolismus   účinky záření   MeSH
• buněčné linie MeSH
• buňky K562 MeSH
• Cajalova tělíska genetika   metabolismus   účinky záření   MeSH
• DNA genetika   účinky záření   MeSH
• G2 fáze genetika   MeSH
• HeLa buňky MeSH
• jaderné proteiny genetika   metabolismus   MeSH
• lidé MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• rekombinantní fúzní proteiny genetika   metabolismus   MeSH
• ultrafialové záření škodlivé účinky   MeSH
• záření gama škodlivé účinky   MeSH
• zelené fluorescenční proteiny genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA MeSH
• jaderné proteiny MeSH
• p80-coilin MeSH Prohlížeč
• rekombinantní fúzní proteiny MeSH
• zelené fluorescenční proteiny MeSH

The U4/U6·U5 tri-small nuclear ribonucleoprotein particle (tri-snRNP) is an essential pre-mRNA splicing factor, which is assembled in a stepwise manner before each round of splicing. It was previously shown that the tri-snRNP is formed in Cajal bodies (CBs), but little is known about the dynamics of this process. Here we created a mathematical model of tri-snRNP assembly in CBs and used it to fit kinetics of individual snRNPs monitored by fluorescence recovery after photobleaching. A global fitting of all kinetic data determined key reaction constants of tri-snRNP assembly. Our model predicts that the rates of di-snRNP and tri-snRNP assemblies are similar and that ∼230 tri-snRNPs are assembled in one CB per minute. Our analysis further indicates that tri-snRNP assembly is approximately 10-fold faster in CBs than in the surrounding nucleoplasm, which is fully consistent with the importance of CBs for snRNP formation in rapidly developing biological systems. Finally, the model predicted binding between SART3 and a CB component. We tested this prediction by Förster resonance energy transfer and revealed an interaction between SART3 and coilin in CBs.

• MeSH
• antigeny nádorové genetika   metabolismus   MeSH
• buněčné jádro genetika   metabolismus   MeSH
• Cajalova tělíska genetika   metabolismus   MeSH
• HeLa buňky MeSH
• jaderné proteiny metabolismus   MeSH
• kinetika MeSH
• lidé MeSH
• malý jaderný ribonukleoprotein U4-U6 genetika   metabolismus   MeSH
• malý jaderný ribonukleoprotein U5 genetika   metabolismus   MeSH
• molekulární modely * MeSH
• nádorové buněčné linie MeSH
• prekurzory RNA genetika   metabolismus   MeSH
• proteiny vázající RNA genetika   metabolismus   MeSH
• ribonukleoproteiny malé jaderné genetika   metabolismus   MeSH
• RNA-helikasy genetika   metabolismus   MeSH
• sestřih RNA genetika   MeSH
• spliceozomy genetika   metabolismus   MeSH
• vazba proteinů genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny nádorové MeSH
• DHX15 protein, human MeSH Prohlížeč
• jaderné proteiny MeSH
• malý jaderný ribonukleoprotein U4-U6 MeSH
• malý jaderný ribonukleoprotein U5 MeSH
• p80-coilin MeSH Prohlížeč
• prekurzory RNA MeSH
• proteiny vázající RNA MeSH
• ribonukleoproteiny malé jaderné MeSH
• RNA-helikasy MeSH
• SART3 protein, human MeSH Prohlížeč
• SNRNP200 protein, human MeSH Prohlížeč

The Cajal body (CB) is a nuclear structure closely associated with import and biogenesis of small nuclear ribonucleoprotein particles (snRNPs). Here, we tested whether CBs also contain mature snRNPs and whether CB integrity depends on the ongoing snRNP splicing cycle. Sm proteins tagged with photoactivatable and color-maturing variants of fluorescent proteins were used to monitor snRNP behavior in living cells over time; mature snRNPs accumulated in CBs, traveled from one CB to another, and they were not preferentially replaced by newly imported snRNPs. To test whether CB integrity depends on the snRNP splicing cycle, two human orthologues of yeast proteins involved in distinct steps in spliceosome disassembly after splicing, hPrp22 and hNtr1, were depleted by small interfering RNA treatment. Surprisingly, depletion of either protein led to the accumulation of U4/U6 snRNPs in CBs, suggesting that reassembly of the U4/U6.U5 tri-snRNP was delayed. Accordingly, a relative decrease in U5 snRNPs compared with U4/U6 snRNPs was observed in CBs, as well as in nuclear extracts of treated cells. Together, the data show that particular phases of the spliceosome cycle are compartmentalized in living cells, with reassembly of the tri-snRNP occurring in CBs.

• MeSH
• biologické markery metabolismus   MeSH
• Cajalova tělíska metabolismus   MeSH
• HeLa buňky MeSH
• lidé MeSH
• malá interferující RNA metabolismus   MeSH
• malý jaderný ribonukleoprotein U4-U6 metabolismus   MeSH
• malý jaderný ribonukleoprotein U5 metabolismus   MeSH
• protein přežití motorických neuronů 1 metabolismus   MeSH
• proteiny vázající RNA MeSH
• ribonukleoproteiny malé jaderné metabolismus   MeSH
• spliceozomy metabolismus   MeSH
• transportní proteiny metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• biologické markery MeSH
• malá interferující RNA MeSH
• malý jaderný ribonukleoprotein U4-U6 MeSH
• malý jaderný ribonukleoprotein U5 MeSH
• protein přežití motorických neuronů 1 MeSH
• proteiny vázající RNA MeSH
• PRPF8 protein, human MeSH Prohlížeč
• ribonukleoproteiny malé jaderné MeSH
• transportní proteiny MeSH

Spliceosomal small nuclear ribonucleoprotein particles (snRNPs) are essential pre-mRNA splicing factors that consist of small nuclear RNAs (snRNAs) complexed with specific sets of proteins. A considerable body of evidence has established that snRNP assembly is accomplished after snRNA synthesis in the nucleus through a series of steps involving cytoplasmic and nuclear phases. Recent work indicates that snRNPs transiently localize to the Cajal body (CB), a nonmembrane-bound inclusion present in the nuclei of most cells, for the final steps in snRNP maturation, including snRNA base modification, U4/U6 snRNA annealing, and snRNA-protein assembly. Here, we review these findings that suggest a crucial role for CBs in the spliceosome cycle in which production of new snRNPs--and perhaps regenerated snRNPs after splicing--is promoted by the concentration of substrates in this previously mysterious subnuclear organelle. These insights allow us to speculate on the role of nuclear bodies in regulating the dynamics of RNP assembly to maintain a functional pool of factors available for key steps in gene expression.

• MeSH
• biologické modely MeSH
• Cajalova tělíska genetika   metabolismus   MeSH
• histony genetika   metabolismus   MeSH
• jaderné proteiny nedostatek   genetika   metabolismus   MeSH
• lidé MeSH
• posttranskripční úpravy RNA MeSH
• ribonukleoproteiny malé jaderné chemie   genetika   metabolismus   MeSH
• RNA malá jaderná chemie   genetika   metabolismus   MeSH
• sestřih RNA MeSH
• spliceozomy genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• histony MeSH
• jaderné proteiny MeSH
• p80-coilin MeSH Prohlížeč
• ribonukleoproteiny malé jaderné MeSH
• RNA malá jaderná MeSH