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Nucleolar localization of RNA binding proteins induced by actinomycin D and heat shock in Trypanosoma cruzi.

Názer E, Verdún RE, Sánchez DO - PLoS ONE (2011)

Bottom Line: ATP depletion as well as kinase inhibition markedly reduced the nucleolar localization response, suggesting that an energy-dependent transport modulated by the phosphorylation status of the parasite might be required.Interestingly, we showed that in addition to RBPs, poly(A)+ RNA is also accumulated into the nucleolus in response to ActD treatment.Together, these results suggest that the nucleolus of an early divergent eukaryote is either able to sequester key factors related to mRNA metabolism in response to transcriptional stress or behaves as a RBP processing center, arguing in favour to the hypothesis that the non-traditional features of the nucleolus could be acquired early during evolution.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Investigaciones Biotecnólogicas-Instituto Tecnológico Chascomús, UNSAM-CONICET, San Martín, Provincia de Buenos Aires, Argentina.

ABSTRACT
In this work we show that under Actinomycin D (ActD) treatment, several RNA Binding Proteins (RBPs) involved in mRNA metabolism are relocalized into the nucleolus in Trypanosoma cruzi as a specific stress response. ATP depletion as well as kinase inhibition markedly reduced the nucleolar localization response, suggesting that an energy-dependent transport modulated by the phosphorylation status of the parasite might be required. Deletion analyses in one of such proteins, TcSR62, showed that a domain bearing basic amino acids located in the COOH terminal region was sufficient to promote its nucleolar relocalization. Interestingly, we showed that in addition to RBPs, poly(A)+ RNA is also accumulated into the nucleolus in response to ActD treatment. Finally, we found out that nucleolar relocalization of RBPs is also triggered by severe heat shock in a reversible way. Together, these results suggest that the nucleolus of an early divergent eukaryote is either able to sequester key factors related to mRNA metabolism in response to transcriptional stress or behaves as a RBP processing center, arguing in favour to the hypothesis that the non-traditional features of the nucleolus could be acquired early during evolution.

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Nucleolar accumulation of RBPs upon ActD treatment depends on an active transport mechanism.(A) Epimastigotes were incubated with both sodium Azide (Az) and 2-Deoxy-Glucose (2De) and ActD at 28°C for 24 h. (B) Epimastigotes were incubated with ActD at 4°C for 24 h. Then, immunofluorescence studies were performed using antibodies against TcSR62 (green) and TcPTB2 (red). Recovery of both treatments was allowed either by washing up the cultures and then incubating with fresh medium (−Az−2De+ActD) or reincubating at 28°C (↑28°+ActD) for 24 h. Each inhibitor treatment alone or cell culture at 4°C, are shown as controls. Nuclei were counterstained with DAPI (blue). The fourth column on the right is an overlap of the TcSR62 and TcPTB2 and DNA stain. Green and red pixels overlapped in the digital images yielded yellow signals. Size bars represent 2 µm. Representative nuclei are shown. The graphic in panel (C) is a quantification of the experiments shown in panels (A) and (B). Results are expressed as mean +/− SD from at least three independent experiments.
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pone-0019920-g004: Nucleolar accumulation of RBPs upon ActD treatment depends on an active transport mechanism.(A) Epimastigotes were incubated with both sodium Azide (Az) and 2-Deoxy-Glucose (2De) and ActD at 28°C for 24 h. (B) Epimastigotes were incubated with ActD at 4°C for 24 h. Then, immunofluorescence studies were performed using antibodies against TcSR62 (green) and TcPTB2 (red). Recovery of both treatments was allowed either by washing up the cultures and then incubating with fresh medium (−Az−2De+ActD) or reincubating at 28°C (↑28°+ActD) for 24 h. Each inhibitor treatment alone or cell culture at 4°C, are shown as controls. Nuclei were counterstained with DAPI (blue). The fourth column on the right is an overlap of the TcSR62 and TcPTB2 and DNA stain. Green and red pixels overlapped in the digital images yielded yellow signals. Size bars represent 2 µm. Representative nuclei are shown. The graphic in panel (C) is a quantification of the experiments shown in panels (A) and (B). Results are expressed as mean +/− SD from at least three independent experiments.

Mentions: The observation of nucleolar migration following ActD treatment led us to examine whether this response depends on an active nucleolar transport, or passive diffusion towards the nucleolus and retention in this organelle by binding to a nucleolar component. To distinguish between these two possibilities, we took advantage of two metabolic inhibitors, namely 2-deoxy-D-glucose (2De) and sodium Azide (Az) for ATP depletion. The former inhibits glycolysis, whereas the latter inhibits the production of ATP by oxidative phosphorylation [43]. Previous works have demonstrated that these two metabolic inhibitors promote nuclear transport inhibition in HeLa cells [43], yeast [44], and also in T. brucei [45]. We evaluated the behaviour of TcSR62 and TcPTB2 upon ATP depletion in untreated and ActD-treated epimastigote cells (Figure 4A). Neither protein changed its subnuclear localization pattern after treating parasites simultaneously with both metabolic inhibitors for 24 h (Figure 4A, compare panels 1 and 2). However, nucleolar relocalization of both proteins, promoted by ActD, was almost completely abolished in the presence of these metabolic inhibitors (Figure 4A, compare panels 3 and 4). Finally, to know whether the nucleolar transport inhibition was reversible, we first treated parasites with ActD in the presence of both inhibitors for 24 h, washed them twice with PBS, refed them with fresh medium and then incubated them for another 24 h. As it can be seen in Figure 4A (panels 4 and 5), both proteins were relocalized to the nucleolus in the majority of the cells, suggesting that an active metabolism is essential for transporting these proteins from nuclear speckles to the nucleolus.


Nucleolar localization of RNA binding proteins induced by actinomycin D and heat shock in Trypanosoma cruzi.

Názer E, Verdún RE, Sánchez DO - PLoS ONE (2011)

Nucleolar accumulation of RBPs upon ActD treatment depends on an active transport mechanism.(A) Epimastigotes were incubated with both sodium Azide (Az) and 2-Deoxy-Glucose (2De) and ActD at 28°C for 24 h. (B) Epimastigotes were incubated with ActD at 4°C for 24 h. Then, immunofluorescence studies were performed using antibodies against TcSR62 (green) and TcPTB2 (red). Recovery of both treatments was allowed either by washing up the cultures and then incubating with fresh medium (−Az−2De+ActD) or reincubating at 28°C (↑28°+ActD) for 24 h. Each inhibitor treatment alone or cell culture at 4°C, are shown as controls. Nuclei were counterstained with DAPI (blue). The fourth column on the right is an overlap of the TcSR62 and TcPTB2 and DNA stain. Green and red pixels overlapped in the digital images yielded yellow signals. Size bars represent 2 µm. Representative nuclei are shown. The graphic in panel (C) is a quantification of the experiments shown in panels (A) and (B). Results are expressed as mean +/− SD from at least three independent experiments.
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Related In: Results  -  Collection

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pone-0019920-g004: Nucleolar accumulation of RBPs upon ActD treatment depends on an active transport mechanism.(A) Epimastigotes were incubated with both sodium Azide (Az) and 2-Deoxy-Glucose (2De) and ActD at 28°C for 24 h. (B) Epimastigotes were incubated with ActD at 4°C for 24 h. Then, immunofluorescence studies were performed using antibodies against TcSR62 (green) and TcPTB2 (red). Recovery of both treatments was allowed either by washing up the cultures and then incubating with fresh medium (−Az−2De+ActD) or reincubating at 28°C (↑28°+ActD) for 24 h. Each inhibitor treatment alone or cell culture at 4°C, are shown as controls. Nuclei were counterstained with DAPI (blue). The fourth column on the right is an overlap of the TcSR62 and TcPTB2 and DNA stain. Green and red pixels overlapped in the digital images yielded yellow signals. Size bars represent 2 µm. Representative nuclei are shown. The graphic in panel (C) is a quantification of the experiments shown in panels (A) and (B). Results are expressed as mean +/− SD from at least three independent experiments.
Mentions: The observation of nucleolar migration following ActD treatment led us to examine whether this response depends on an active nucleolar transport, or passive diffusion towards the nucleolus and retention in this organelle by binding to a nucleolar component. To distinguish between these two possibilities, we took advantage of two metabolic inhibitors, namely 2-deoxy-D-glucose (2De) and sodium Azide (Az) for ATP depletion. The former inhibits glycolysis, whereas the latter inhibits the production of ATP by oxidative phosphorylation [43]. Previous works have demonstrated that these two metabolic inhibitors promote nuclear transport inhibition in HeLa cells [43], yeast [44], and also in T. brucei [45]. We evaluated the behaviour of TcSR62 and TcPTB2 upon ATP depletion in untreated and ActD-treated epimastigote cells (Figure 4A). Neither protein changed its subnuclear localization pattern after treating parasites simultaneously with both metabolic inhibitors for 24 h (Figure 4A, compare panels 1 and 2). However, nucleolar relocalization of both proteins, promoted by ActD, was almost completely abolished in the presence of these metabolic inhibitors (Figure 4A, compare panels 3 and 4). Finally, to know whether the nucleolar transport inhibition was reversible, we first treated parasites with ActD in the presence of both inhibitors for 24 h, washed them twice with PBS, refed them with fresh medium and then incubated them for another 24 h. As it can be seen in Figure 4A (panels 4 and 5), both proteins were relocalized to the nucleolus in the majority of the cells, suggesting that an active metabolism is essential for transporting these proteins from nuclear speckles to the nucleolus.

Bottom Line: ATP depletion as well as kinase inhibition markedly reduced the nucleolar localization response, suggesting that an energy-dependent transport modulated by the phosphorylation status of the parasite might be required.Interestingly, we showed that in addition to RBPs, poly(A)+ RNA is also accumulated into the nucleolus in response to ActD treatment.Together, these results suggest that the nucleolus of an early divergent eukaryote is either able to sequester key factors related to mRNA metabolism in response to transcriptional stress or behaves as a RBP processing center, arguing in favour to the hypothesis that the non-traditional features of the nucleolus could be acquired early during evolution.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Investigaciones Biotecnólogicas-Instituto Tecnológico Chascomús, UNSAM-CONICET, San Martín, Provincia de Buenos Aires, Argentina.

ABSTRACT
In this work we show that under Actinomycin D (ActD) treatment, several RNA Binding Proteins (RBPs) involved in mRNA metabolism are relocalized into the nucleolus in Trypanosoma cruzi as a specific stress response. ATP depletion as well as kinase inhibition markedly reduced the nucleolar localization response, suggesting that an energy-dependent transport modulated by the phosphorylation status of the parasite might be required. Deletion analyses in one of such proteins, TcSR62, showed that a domain bearing basic amino acids located in the COOH terminal region was sufficient to promote its nucleolar relocalization. Interestingly, we showed that in addition to RBPs, poly(A)+ RNA is also accumulated into the nucleolus in response to ActD treatment. Finally, we found out that nucleolar relocalization of RBPs is also triggered by severe heat shock in a reversible way. Together, these results suggest that the nucleolus of an early divergent eukaryote is either able to sequester key factors related to mRNA metabolism in response to transcriptional stress or behaves as a RBP processing center, arguing in favour to the hypothesis that the non-traditional features of the nucleolus could be acquired early during evolution.

Show MeSH
Related in: MedlinePlus