Nucleolar Accumulation of RNA Binding Proteins Induced by ActinomycinD Is Functional in Trypanosoma cruzi and Leishmania mexicana but Not in T. brucei

We have recently shown in T. cruzi that a group of RNA Binding Proteins (RBPs), involved in mRNA metabolism, are accumulated into the nucleolus in response to Actinomycin D (ActD) treatment. In this work, we have extended our analysis to other members of the trypanosomatid lineage. In agreement with our previous study, the mechanism seems to be conserved in L. mexicana, since both endogenous RBPs and a transgenic RBP were relocalized to the nucleolus in parasites exposed to ActD. In contrast, in T. brucei, neither endogenous RBPs (TbRRM1 and TbPABP2) nor a transgenic RBP from T. cruzi were accumulated into the nucleolus under such treatment. Interestingly, when a transgenic TbRRM1was expressed in T. cruzi and the parasites exposed to ActD, TbRRM1 relocated to the nucleolus, suggesting that it contains the necessary sequence elements to be targeted to the nucleolus. Together, both experiments demonstrate that the mechanism behind nucleolar localization of RBPs, which is present in T. cruzi and L. mexicana, is not functional in T. brucei, suggesting that it has been lost or retained differentially during the evolution of the trypanosomatid lineage

Poly(A)+ RNA in response to heat shock is located in cytoplasmic granules but not into the nucleolus in <i>T. brucei</i>.

<p>Poly(A)+ RNA subcellular localization in <i>T. brucei</i> procyclic forms (A) untreated, (B) exposed to heat shock at 40°C for 2 h, (C) pre-treated with RNase A after heat shock, and (D) incubated with ActD for 4 h. The white arrows indicate the nucleolus. Nuclei were counterstained with DAPI (blue). N: nucleus, K: kinetoplast, Nu: nucleolus. Size bars represent 2 µm. Representative parasites are shown.</p

The <i>T. cruzi</i> mRNA population is partially accumulated into the nucleolus in response to severe heat shock.

<p>Localization of the mRNA population using either an (A) oligo(dT) or (B) mini-exon probe in untreated, heat-shocked and recovered <i>T. cruzi</i> epimastigotes. In addition, cells were pre-treated with RNase A before performing FISH (bottom panels). Nuclei were counterstained with DAPI (blue). The white arrows indicate the nucleolar localization for both probes. (C) RNA FISH using a Cy3-labelled oligo(dA)30 probe in untreated parasites and parasites exposed to 40°C for 2 h. N: nucleus, K: kinetoplast, Nu: nucleolus. Size bars represent 2 µm. Representative nuclei are shown. (D) A quantitative analysis of experiments is shown in panels (A) and (B). The results are expressed as mean +/− SD from at least three independent experiments (a minimum of 100 parasites per experiment were counted).</p

Integrative model showing the behaviour of RBPs and mRNAs in response to severe heat shock in <i>T. cruzi</i>.

<p>Under normal conditions, the mRNAs are exported to the cytoplasm to be target either to translation, storage or degradation. When epimastigotes are exposed to severe heat shock, there is a decrease in transcription which likely triggers the nucleolar accumulation of certain RBPs (for instance, TcSR62 and TcPTB2). In addition, mRNAs are relocalized into the nucleolus where they might be stored/protected until favourable environmental conditions are resumed. On the other hand, the Hsp70 mRNA could bypass such nucleolar retention, being transported to the cytoplasm where its translation can continue even under severe heat shock. Unidentified, but probable, factors/events are indicated with a question mark. See the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043715#s3" target="_blank">discussion</a> section for details. SL, spliced leader.</p

Hsp70 mRNA is able to bypass the nucleolar accumulation.

<p>FISH analysis for (A) α-Tub, (B) Smug and (C) Hsp70 mRNAs in parasites subjected to heat shock at 40°C for 2 h. In addition, cells were pre-treated with RNase A before performing FISH (bottom panels). The white arrows indicate the nucleolus. Nuclei were counterstained with DAPI (blue). Size bars represent 2 µm. Representative nuclei are shown. (D) A quantitative analysis of the experiments shown in panels (A), (B) and (C). The results are expressed as mean +/− SD from at least three independent experiments (a minimum of 100 parasites per experiment were counted).</p

Nucleolar Localization of RNA Binding Proteins Induced by Actinomycin D and Heat Shock in Trypanosoma cruzi

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 acquire