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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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Stress-induced nucleolar localization of RBPs is inhibited by blocking phosphorylation but not dephosphorylation.Epimastigotes were incubated either with both okadaic acid (Oka) and ActD for 24 h or first preincubated with staurosporine (Stau) for 16 h and then with ActD for 24 h. Panel (A) shows TcSR62 (green), L1C6 (red) and DAPI. The fourth column on the right is an overlap of the TcSR62 and L1C6 and DNA stain. Green and red pixels overlapped in the digital images yielded yellow signals. (B) TcPTB2 (green), (C) TcPABP1 (green), each counterstained with DAPI. The third column on the right is an overlap of each protein and DNA stain. Each inhibitor treatment alone is shown as control. Nuclei were counterstained with DAPI (blue). Size bars represent 2 µm. Representative nuclei are shown. The graphic in panel (D) is a quantitative analysis of the experiments shown in panels (A) and (B). The graphic in panel (E) is a quantitative analysis of TcPABP1 behaviour. Results are expressed as mean +/− SD from at least three independent experiments.
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pone-0019920-g005: Stress-induced nucleolar localization of RBPs is inhibited by blocking phosphorylation but not dephosphorylation.Epimastigotes were incubated either with both okadaic acid (Oka) and ActD for 24 h or first preincubated with staurosporine (Stau) for 16 h and then with ActD for 24 h. Panel (A) shows TcSR62 (green), L1C6 (red) and DAPI. The fourth column on the right is an overlap of the TcSR62 and L1C6 and DNA stain. Green and red pixels overlapped in the digital images yielded yellow signals. (B) TcPTB2 (green), (C) TcPABP1 (green), each counterstained with DAPI. The third column on the right is an overlap of each protein and DNA stain. Each inhibitor treatment alone is shown as control. Nuclei were counterstained with DAPI (blue). Size bars represent 2 µm. Representative nuclei are shown. The graphic in panel (D) is a quantitative analysis of the experiments shown in panels (A) and (B). The graphic in panel (E) is a quantitative analysis of TcPABP1 behaviour. Results are expressed as mean +/− SD from at least three independent experiments.

Mentions: It is well known that phosphorylation/dephosphorylation processes play an important role in protein localization as well as in the modulation of the interaction of proteins such as SF2/ASF (a well-studied SR protein), PTB and PABP [46]–[50]. To test the possibility that nucleolar relocalization of RBPs in T. cruzi might be also regulated by such mechanism, we investigated their behaviour in parasites subjected to ActD treatment in the presence of either okadaic acid (a specific inhibitor of protein serine/threonine phosphatases 1, 2A and 2B) [51] or staurosporine (a broad-spectrum protein kinase inhibitor) [52]. Although we are aware that these drugs affect the overall cellular phosphorylation/dephosphorylation cycle, they have been --and still are-- used extensively to investigate whether changes in the phosphorylation state of proteins are responsible for altering their activity and/or their dynamics within cells [53]–[56]. The results shown in Figure 5 demonstrate that neither okadaic acid (Figure 5, panel 3) nor staurosporine alone (Figure 5, panel 5) affected the speckled pattern of TcSR62 or TcPTB2 (Figure 5A and 5B, respectively) or the cytoplasmic localization of TcPABP1 (Figure 5C). However, ActD-induced nucleolar accumulation of these proteins was inhibited upon staurosporine but not okadaic acid treatment (see panels 6 and 4, respectively). Figure 5D shows a quantification of the experiments illustrated in Figure 5A and 5B. Interestingly, upon treatment of parasites with ActD and staurosporine, TcPABP1 was able to mobilize from the cytoplasm to the nucleus, accumulating throughout the nucleoplasm, but it was unable to accumulate into the nucleolus in most cells (see Figure 5E for a quantitative analysis). It should be pointed out that nucleolar accumulation inhibition of RBPs by staurosporine was effective only when the cells were pretreated with this drug 16 h before ActD was added and further incubated for 24 h. Simultaneous treatment with both drugs could not prevent nucleolar relocalization of the RBPs analyzed.


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)

Stress-induced nucleolar localization of RBPs is inhibited by blocking phosphorylation but not dephosphorylation.Epimastigotes were incubated either with both okadaic acid (Oka) and ActD for 24 h or first preincubated with staurosporine (Stau) for 16 h and then with ActD for 24 h. Panel (A) shows TcSR62 (green), L1C6 (red) and DAPI. The fourth column on the right is an overlap of the TcSR62 and L1C6 and DNA stain. Green and red pixels overlapped in the digital images yielded yellow signals. (B) TcPTB2 (green), (C) TcPABP1 (green), each counterstained with DAPI. The third column on the right is an overlap of each protein and DNA stain. Each inhibitor treatment alone is shown as control. Nuclei were counterstained with DAPI (blue). Size bars represent 2 µm. Representative nuclei are shown. The graphic in panel (D) is a quantitative analysis of the experiments shown in panels (A) and (B). The graphic in panel (E) is a quantitative analysis of TcPABP1 behaviour. Results are expressed as mean +/− SD from at least three independent experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3101214&req=5

pone-0019920-g005: Stress-induced nucleolar localization of RBPs is inhibited by blocking phosphorylation but not dephosphorylation.Epimastigotes were incubated either with both okadaic acid (Oka) and ActD for 24 h or first preincubated with staurosporine (Stau) for 16 h and then with ActD for 24 h. Panel (A) shows TcSR62 (green), L1C6 (red) and DAPI. The fourth column on the right is an overlap of the TcSR62 and L1C6 and DNA stain. Green and red pixels overlapped in the digital images yielded yellow signals. (B) TcPTB2 (green), (C) TcPABP1 (green), each counterstained with DAPI. The third column on the right is an overlap of each protein and DNA stain. Each inhibitor treatment alone is shown as control. Nuclei were counterstained with DAPI (blue). Size bars represent 2 µm. Representative nuclei are shown. The graphic in panel (D) is a quantitative analysis of the experiments shown in panels (A) and (B). The graphic in panel (E) is a quantitative analysis of TcPABP1 behaviour. Results are expressed as mean +/− SD from at least three independent experiments.
Mentions: It is well known that phosphorylation/dephosphorylation processes play an important role in protein localization as well as in the modulation of the interaction of proteins such as SF2/ASF (a well-studied SR protein), PTB and PABP [46]–[50]. To test the possibility that nucleolar relocalization of RBPs in T. cruzi might be also regulated by such mechanism, we investigated their behaviour in parasites subjected to ActD treatment in the presence of either okadaic acid (a specific inhibitor of protein serine/threonine phosphatases 1, 2A and 2B) [51] or staurosporine (a broad-spectrum protein kinase inhibitor) [52]. Although we are aware that these drugs affect the overall cellular phosphorylation/dephosphorylation cycle, they have been --and still are-- used extensively to investigate whether changes in the phosphorylation state of proteins are responsible for altering their activity and/or their dynamics within cells [53]–[56]. The results shown in Figure 5 demonstrate that neither okadaic acid (Figure 5, panel 3) nor staurosporine alone (Figure 5, panel 5) affected the speckled pattern of TcSR62 or TcPTB2 (Figure 5A and 5B, respectively) or the cytoplasmic localization of TcPABP1 (Figure 5C). However, ActD-induced nucleolar accumulation of these proteins was inhibited upon staurosporine but not okadaic acid treatment (see panels 6 and 4, respectively). Figure 5D shows a quantification of the experiments illustrated in Figure 5A and 5B. Interestingly, upon treatment of parasites with ActD and staurosporine, TcPABP1 was able to mobilize from the cytoplasm to the nucleus, accumulating throughout the nucleoplasm, but it was unable to accumulate into the nucleolus in most cells (see Figure 5E for a quantitative analysis). It should be pointed out that nucleolar accumulation inhibition of RBPs by staurosporine was effective only when the cells were pretreated with this drug 16 h before ActD was added and further incubated for 24 h. Simultaneous treatment with both drugs could not prevent nucleolar relocalization of the RBPs analyzed.

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