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High levels of TopBP1 induce ATR-dependent shut-down of rRNA transcription and nucleolar segregation.

Sokka M, Rilla K, Miinalainen I, Pospiech H, Syväoja JE - Nucleic Acids Res. (2015)

Bottom Line: We found that a basal level of TopBP1 protein associates with ribosomal DNA repeat.Our findings demonstrate that TopBP1 and ATR are able to inhibit the synthesis of rRNA and to activate nucleolar stress pathway; yet the p53-mediated cell cycle arrest is thwarted in cells expressing high levels of TopBP1.We suggest that inhibition of rRNA transcription by different stress regulators is a general mechanism for cells to initiate nucleolar stress pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Eastern Finland, FI-80101 Joensuu, Finland Institute of Biomedicine, University of Eastern Finland, FI-70211 Kuopio, Finland miiko.sokka@uef.fi.

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Both endogenous and ectopic TopBP1 display nucleolar localization. eGFP-TopBP1 was left non-induced (Off) or induced (On) for 24 h before fixing the cells for immunoelectron microscopy. TopBP1 was immunostained with 10 nm, and RNA pol I, UBF and NCL with 5 nm gold particles. (A) Examples of nucleoli in a non-induced and an induced cell at lower magnification. (B) Higher magnification of nucleoli in non-induced and induced cells. Endogenous TopBP1 partly co-localizes with RNA Pol I, UBF and NCL in the electron-dense regions of the nucleolus, but in induced cells it strongly concentrates in distinct regions immediately adjacent to main body of nucleolus (arrowheads). Scale bars are 100, 500 or 1000 nm, as indicated.
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Figure 2: Both endogenous and ectopic TopBP1 display nucleolar localization. eGFP-TopBP1 was left non-induced (Off) or induced (On) for 24 h before fixing the cells for immunoelectron microscopy. TopBP1 was immunostained with 10 nm, and RNA pol I, UBF and NCL with 5 nm gold particles. (A) Examples of nucleoli in a non-induced and an induced cell at lower magnification. (B) Higher magnification of nucleoli in non-induced and induced cells. Endogenous TopBP1 partly co-localizes with RNA Pol I, UBF and NCL in the electron-dense regions of the nucleolus, but in induced cells it strongly concentrates in distinct regions immediately adjacent to main body of nucleolus (arrowheads). Scale bars are 100, 500 or 1000 nm, as indicated.

Mentions: Next we looked the nucleoli at the ultrastructural level using immuno-EM. In non-induced cells nucleoli were visible as strongly contrasted, round regions in the nuclei (Figure 2A). Interestingly, we found endogenous TopBP1 co-localizing with RNA Pol I, UBF and NCL inside the nucleoli in non-induced cells (Figure 2B, upper panels).The levels of endogenous TopBP1 staining by immuno-EM in the nucleolus appeared even to exceed the general staining in the nucleus. When induced to express eGFP-TopBP1, we found TopBP1 concentrating in regions that were closely associated with the nucleolus, but which appeared distinct from the more electron-dense body of nucleolus (Figure 2A and B, lower panels). These regions obviously correspond to the nucleolar TopBP1 foci visible in fluorescence microscopy.


High levels of TopBP1 induce ATR-dependent shut-down of rRNA transcription and nucleolar segregation.

Sokka M, Rilla K, Miinalainen I, Pospiech H, Syväoja JE - Nucleic Acids Res. (2015)

Both endogenous and ectopic TopBP1 display nucleolar localization. eGFP-TopBP1 was left non-induced (Off) or induced (On) for 24 h before fixing the cells for immunoelectron microscopy. TopBP1 was immunostained with 10 nm, and RNA pol I, UBF and NCL with 5 nm gold particles. (A) Examples of nucleoli in a non-induced and an induced cell at lower magnification. (B) Higher magnification of nucleoli in non-induced and induced cells. Endogenous TopBP1 partly co-localizes with RNA Pol I, UBF and NCL in the electron-dense regions of the nucleolus, but in induced cells it strongly concentrates in distinct regions immediately adjacent to main body of nucleolus (arrowheads). Scale bars are 100, 500 or 1000 nm, as indicated.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4446431&req=5

Figure 2: Both endogenous and ectopic TopBP1 display nucleolar localization. eGFP-TopBP1 was left non-induced (Off) or induced (On) for 24 h before fixing the cells for immunoelectron microscopy. TopBP1 was immunostained with 10 nm, and RNA pol I, UBF and NCL with 5 nm gold particles. (A) Examples of nucleoli in a non-induced and an induced cell at lower magnification. (B) Higher magnification of nucleoli in non-induced and induced cells. Endogenous TopBP1 partly co-localizes with RNA Pol I, UBF and NCL in the electron-dense regions of the nucleolus, but in induced cells it strongly concentrates in distinct regions immediately adjacent to main body of nucleolus (arrowheads). Scale bars are 100, 500 or 1000 nm, as indicated.
Mentions: Next we looked the nucleoli at the ultrastructural level using immuno-EM. In non-induced cells nucleoli were visible as strongly contrasted, round regions in the nuclei (Figure 2A). Interestingly, we found endogenous TopBP1 co-localizing with RNA Pol I, UBF and NCL inside the nucleoli in non-induced cells (Figure 2B, upper panels).The levels of endogenous TopBP1 staining by immuno-EM in the nucleolus appeared even to exceed the general staining in the nucleus. When induced to express eGFP-TopBP1, we found TopBP1 concentrating in regions that were closely associated with the nucleolus, but which appeared distinct from the more electron-dense body of nucleolus (Figure 2A and B, lower panels). These regions obviously correspond to the nucleolar TopBP1 foci visible in fluorescence microscopy.

Bottom Line: We found that a basal level of TopBP1 protein associates with ribosomal DNA repeat.Our findings demonstrate that TopBP1 and ATR are able to inhibit the synthesis of rRNA and to activate nucleolar stress pathway; yet the p53-mediated cell cycle arrest is thwarted in cells expressing high levels of TopBP1.We suggest that inhibition of rRNA transcription by different stress regulators is a general mechanism for cells to initiate nucleolar stress pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Eastern Finland, FI-80101 Joensuu, Finland Institute of Biomedicine, University of Eastern Finland, FI-70211 Kuopio, Finland miiko.sokka@uef.fi.

Show MeSH
Related in: MedlinePlus