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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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Expression of ectopic TopBP1 induces shut-down of rRNA transcription and nucleolar segregation. (A) Expression of eGFP-TopBP1 was left non-induced (eGFP-TopBP1 Off), induced for 24 h (eGFP-TopBP1 On), or cells were treated with ActD. Nascent transcripts were labelled with a short pulse of FUrd. Confocal images of FUrd, endogenous RNA Pol I, UBF, NPM (Nucleophosmin) and NCL (Nucleolin) and eGFP-TopBP1 are shown. Merge shows an overlay of panels (DNA excluded). Scale bar is 10 μm. (B) Pre-rRNA synthesis levels were determined with qRT-PCR from eGFP-TopBP1 cells left non-induced (Off), induced for the indicated times or treated with ActD. Percentages of pre-rRNA levels normalized to ACTB transcription are shown relative to non-induced cells.
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Figure 1: Expression of ectopic TopBP1 induces shut-down of rRNA transcription and nucleolar segregation. (A) Expression of eGFP-TopBP1 was left non-induced (eGFP-TopBP1 Off), induced for 24 h (eGFP-TopBP1 On), or cells were treated with ActD. Nascent transcripts were labelled with a short pulse of FUrd. Confocal images of FUrd, endogenous RNA Pol I, UBF, NPM (Nucleophosmin) and NCL (Nucleolin) and eGFP-TopBP1 are shown. Merge shows an overlay of panels (DNA excluded). Scale bar is 10 μm. (B) Pre-rRNA synthesis levels were determined with qRT-PCR from eGFP-TopBP1 cells left non-induced (Off), induced for the indicated times or treated with ActD. Percentages of pre-rRNA levels normalized to ACTB transcription are shown relative to non-induced cells.

Mentions: When cells expressing eGFP-TopBP1 were examined under fluorescent microscope, strong localization of eGFP-TopBP1 into nuclear foci was observed. More close inspection suggested that the foci were associated with the nucleoli, which were identified by weak staining of DNA with Hoechst 33258. We then looked at the localization of nucleolar marker proteins Upstream binding factor (UBF), RNA Pol I, Nucleolin (NCL) and Nucleophosmin (NPM). To our surprise, we found changes in localization of all these marker proteins upon induction of eGFP-TopBP1 (Figure 1A). UBF and RNA Pol I both co-localized extensively with the eGFP-TopBP1 foci (Figure 1A, middle panels), while in non-induced cells they localized in necklace-like structures (Figure 1A, top panels). NPM and NCL signals in the nucleoplasm were increased and both proteins formed ring-like structures around the nucleolar body in cells expressing eGFP-TopBP1, while in non-induced cells they were concentrated in the nucleoli. The relocalization of nucleolar proteins in cells expressing eGFP-TopBP1 bring to mind nucleolar segregation that is induced by inhibition of rRNA transcription after ultraviolet light irradiation or ActD treatment. Indeed, the similarity in localization of nucleolar marker proteins between eGFP-TopBP1 expressing cells and ActD-treated cells was apparent (Figure 1A, middle and bottom panels). ActD binds to the DNA duplex and inhibits specifically RNA Pol I, when used in nanomolar concentrations. Higher concentrations of ActD inhibit also RNA polymerase II. To confirm this specific inhibition of RNA pol I, we gave cells a short pulse of ribonucleotide analogue 5′-fluorouridine (FUrd) that incorporates into nascent RNA. As expected, cells treated with 30 nM ActD lost FUrd signal in nucleoli, while transcription elsewhere was visible as dispersed foci throughout the nucleoplasm (Figure 1A). Most cells displaying eGFP-TopBP1-induced segregation were devoid of nucleolar transcription (Figure 1A and Supplementary Figure S2), while transcription elsewhere in nucleoplasm looked normal, resembling also in this respect ActD-treated cells. We then further studied transcription of ribosomal RNA genes by measuring pre-ribosomal rRNA (pre-rRNA) with quantitative reverse-transcriptase PCR (qRT-PCR) assay. Upon induction of eGFP-TopBP1 expression, the pre-rRNA synthesis level dropped gradually from 100% at 1 h down to 31% at 24 h (Figure 1B). This pattern follows the microscopically observed accumulation of eGFP-TopBP1 protein levels, which appeared earliest at around 6 h and was saturated at 24 h after the induction (not shown). In cells treated with 30 nM ActD for 30 min the level of pre-rRNA was 58% compared to that of non-treated cells.


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)

Expression of ectopic TopBP1 induces shut-down of rRNA transcription and nucleolar segregation. (A) Expression of eGFP-TopBP1 was left non-induced (eGFP-TopBP1 Off), induced for 24 h (eGFP-TopBP1 On), or cells were treated with ActD. Nascent transcripts were labelled with a short pulse of FUrd. Confocal images of FUrd, endogenous RNA Pol I, UBF, NPM (Nucleophosmin) and NCL (Nucleolin) and eGFP-TopBP1 are shown. Merge shows an overlay of panels (DNA excluded). Scale bar is 10 μm. (B) Pre-rRNA synthesis levels were determined with qRT-PCR from eGFP-TopBP1 cells left non-induced (Off), induced for the indicated times or treated with ActD. Percentages of pre-rRNA levels normalized to ACTB transcription are shown relative to non-induced cells.
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Figure 1: Expression of ectopic TopBP1 induces shut-down of rRNA transcription and nucleolar segregation. (A) Expression of eGFP-TopBP1 was left non-induced (eGFP-TopBP1 Off), induced for 24 h (eGFP-TopBP1 On), or cells were treated with ActD. Nascent transcripts were labelled with a short pulse of FUrd. Confocal images of FUrd, endogenous RNA Pol I, UBF, NPM (Nucleophosmin) and NCL (Nucleolin) and eGFP-TopBP1 are shown. Merge shows an overlay of panels (DNA excluded). Scale bar is 10 μm. (B) Pre-rRNA synthesis levels were determined with qRT-PCR from eGFP-TopBP1 cells left non-induced (Off), induced for the indicated times or treated with ActD. Percentages of pre-rRNA levels normalized to ACTB transcription are shown relative to non-induced cells.
Mentions: When cells expressing eGFP-TopBP1 were examined under fluorescent microscope, strong localization of eGFP-TopBP1 into nuclear foci was observed. More close inspection suggested that the foci were associated with the nucleoli, which were identified by weak staining of DNA with Hoechst 33258. We then looked at the localization of nucleolar marker proteins Upstream binding factor (UBF), RNA Pol I, Nucleolin (NCL) and Nucleophosmin (NPM). To our surprise, we found changes in localization of all these marker proteins upon induction of eGFP-TopBP1 (Figure 1A). UBF and RNA Pol I both co-localized extensively with the eGFP-TopBP1 foci (Figure 1A, middle panels), while in non-induced cells they localized in necklace-like structures (Figure 1A, top panels). NPM and NCL signals in the nucleoplasm were increased and both proteins formed ring-like structures around the nucleolar body in cells expressing eGFP-TopBP1, while in non-induced cells they were concentrated in the nucleoli. The relocalization of nucleolar proteins in cells expressing eGFP-TopBP1 bring to mind nucleolar segregation that is induced by inhibition of rRNA transcription after ultraviolet light irradiation or ActD treatment. Indeed, the similarity in localization of nucleolar marker proteins between eGFP-TopBP1 expressing cells and ActD-treated cells was apparent (Figure 1A, middle and bottom panels). ActD binds to the DNA duplex and inhibits specifically RNA Pol I, when used in nanomolar concentrations. Higher concentrations of ActD inhibit also RNA polymerase II. To confirm this specific inhibition of RNA pol I, we gave cells a short pulse of ribonucleotide analogue 5′-fluorouridine (FUrd) that incorporates into nascent RNA. As expected, cells treated with 30 nM ActD lost FUrd signal in nucleoli, while transcription elsewhere was visible as dispersed foci throughout the nucleoplasm (Figure 1A). Most cells displaying eGFP-TopBP1-induced segregation were devoid of nucleolar transcription (Figure 1A and Supplementary Figure S2), while transcription elsewhere in nucleoplasm looked normal, resembling also in this respect ActD-treated cells. We then further studied transcription of ribosomal RNA genes by measuring pre-ribosomal rRNA (pre-rRNA) with quantitative reverse-transcriptase PCR (qRT-PCR) assay. Upon induction of eGFP-TopBP1 expression, the pre-rRNA synthesis level dropped gradually from 100% at 1 h down to 31% at 24 h (Figure 1B). This pattern follows the microscopically observed accumulation of eGFP-TopBP1 protein levels, which appeared earliest at around 6 h and was saturated at 24 h after the induction (not shown). In cells treated with 30 nM ActD for 30 min the level of pre-rRNA was 58% compared to that of non-treated cells.

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