Severe heat shock induces nucleolar accumulation of mRNAs in Trypanosoma cruzi.
ABSTRACT: Several lines of evidence have shown that, besides its traditional function in ribosome biogenesis, the nucleolus is also involved in regulating other cellular processes such as mRNA metabolism, and that it also plays an important role as a sensor and coordinator of the stress response. We have recently shown that a subset of RNA Binding Proteins and the poly(A)+ RNA are accumulated into the Trypanosoma cruzi nucleolus after inducing transcription inhibition with Actinomycin D. In this study, we investigated the behaviour of the T. cruzi mRNA population in parasites subjected to severe heat shock, an environmental stress that also decreases the rate of RNA synthesis. We found that the bulk of poly(A)+ RNA is reversibly accumulated into the nucleolus when exposing T. cruzi epimastigote forms to severe heat shock. However, the Hsp70 mRNA was able to bypass such nucleolar accumulation. Together, these data reinforce the idea about the involvement of the T. cruzi nucleolus in mRNA metabolism during an environmental stress response. Interestingly, T. brucei procyclic forms did not induce nucleolar accumulation of poly(A)+ RNA under such stress condition, suggesting that different trypanosomatids have adopted different responses to deal with the same stress conditions.
Project description: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.
Project description: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 TbRRM1 was 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.
Project description:Although bystin has been identified as a protein potentially involved in embryo implantation (a process unique to mammals) in humans, the bystin gene is evolutionarily conserved from yeast to humans. DNA microarray data indicates that bystin is overexpressed in human cancers, suggesting that it promotes cell growth. We undertook RT (reverse transcription)-PCR and immunoblotting, and confirmed that bystin mRNA and protein respectively are expressed in human cancer cell lines, including HeLa. Subcellular fractionation identified bystin protein as nuclear and cytoplasmic, and immunofluorescence showed that nuclear bystin localizes mainly in the nucleolus. Sucrose gradient ultracentrifugation of total cytoplasmic ribosomes revealed preferential association of bystin with the 40S subunit fractions. To analyse its function, bystin expression in cells was suppressed by RNAi (RNA interference). Pulse-chase analysis of ribosomal RNA processing suggested that bystin knockdown delays processing of 18S ribosomal RNA, a component of the 40S subunit. Furthermore, this knockdown significantly inhibited cell proliferation. Our findings suggest that bystin may promote cell proliferation by facilitating ribosome biogenesis, specifically in the production of the 40S subunit. Localization of bystin to the nucleolus, the site of ribosome biogenesis, was blocked by low concentrations of actinomycin D, a reagent that causes nucleolar stress. When bystin was transiently overexpressed in HeLa cells subjected to nucleolar stress, nuclear bystin was included in particles different from the nuclear stress granules induced by heat shock. In contrast, cytoplasmic bystin was barely affected by nucleolar stress. These results suggest that, while bystin may play multiple roles in mammalian cells, a conserved function is to facilitate ribosome biogenesis required for cell growth.
Project description:Maintenance of epigenetic modifiers is of utmost importance to preserve the epigenome and consequently appropriate cellular functioning. Here, we analyzed Polycomb group protein (PcG) complex integrity in response to heat shock (HS). Upon HS, various Polycomb Repressive Complex (PRC)1 and PRC2 subunits, including CBX proteins, but also other chromatin regulators, are found to accumulate in the nucleolus. In parallel, binding of PRC1/2 to target genes is strongly reduced, coinciding with a dramatic loss of H2AK119ub and H3K27me3 marks. Nucleolar-accumulated CBX proteins are immobile, but remarkably both CBX protein accumulation and loss of PRC1/2 epigenetic marks are reversible. This post-heat shock recovery of pan-nuclear CBX protein localization and reinstallation of epigenetic marks is HSP70 dependent. Our findings demonstrate that the nucleolus is an essential protein quality control center, which is indispensable for recovery of epigenetic regulators and maintenance of the epigenome after heat shock.
Project description:The nucleolus, whose primary function is ribosome biogenesis, plays an essential role in p53 activation. Ribosome biogenesis is inhibited in response to cellular stress and several nucleolar proteins translocate from the nucleolus to the nucleoplasm, where they activate p53. In this study, we analysed precisely how impaired ribosome biogenesis regulates the activation of p53 by depleting nucleolar factors involved in rRNA transcription or rRNA processing. Nucleolar RNA content decreased when rRNA transcription was inhibited. In parallel with the reduced levels of nucleolar RNA content, the nucleolar protein Myb-binding protein 1?A (MYBBP1A) translocated to the nucleoplasm and increased p53 acetylation. The acetylated p53 enhanced p21 and BAX expression and induced apoptosis. In contrast, when rRNA processing was inhibited, MYBBP1A remained in the nucleolus and nonacetylated p53 accumulated, causing cell cycle arrest at the G1 phase by inducing p21 but not BAX. We propose that the nucleolus functions as a stress sensor to modulate p53 protein levels and its acetylation status, determining cell fate between cell cycle arrest and apoptosis by regulating MYBBP1A translocation.
Project description:Frontotemporal dementia and amyotrophic lateral sclerosis patients with C9orf72 mutation show cytoplasmic poly-GR and poly-PR aggregates. Short poly-(Gly-Arg) and poly-(Pro-Arg) (poly-GR/PR) repeats localizing to the nucleolus are toxic in various model systems, but no interactors have been validated in patients. Here, the neuronal interactomes of cytoplasmic GFP-(GR)149 and nucleolar (PR)175-GFP revealed overlapping RNA-binding proteins, including components of stress granules, nucleoli, and ribosomes. Overexpressing the poly-GR/PR interactors STAU1/2 and YBX1 caused cytoplasmic aggregation of poly-GR/PR in large stress granule-like structures, whereas NPM1 recruited poly-GR into the nucleolus. Poly-PR expression reduced ribosome levels and translation consistent with reduction of synaptic proteins detected by proteomics. Surprisingly, truncated GFP-(GR)53, but not GFP-(GR)149, localized to the nucleolus and reduced ribosome levels and translation similar to poly-PR, suggesting that impaired ribosome biogenesis may be driving the acute toxicity observed in vitro. In patients, only ribosomes and STAU2 co-aggregated with poly-GR/PR. Partial sequestration of ribosomes may chronically impair protein synthesis even in the absence of nucleolar localization and contribute to pathogenesis.
Project description:Maintenance of the epigenome is of utmost importance to warrant appropriate cellular functioning Here we analyzed PRC1/2 function, in response to cellular stress. We observe that heat shock (HS) leads to nucleolar accumulation of CBX proteins, and are strongly immobilized in a 1,6-hexanediol insensitive manner, suggestive of a HS-induced liquid-to-solid phase transition of the nucleolus. CBX protein recovery from the nucleolus is dependent on heat shock protein (HSP) activity. LC-MS/MS analyses of nucleoli shows HS-induced nucleolar accumulation of PRC1/2 subunits, various chromatin regulators, HSPs, and the 26S proteasome. Finally, we find that HS leads to a strong reduction of PRC1/2 chromatin binding at target genes and loss of H2AK119ub and H3K27me3. Our findings demonstrate that thermal stress results in a rapid accumulation of epigenetic regulators in the nucleolus, and a concomitant loss of PRC1/2 complex chromatin binding and associated epimarks, underlining that environmental stress directly alters the epigenome. Overall design: ChIP-seq analysis of CBX proteins using control and Heatshock induced cells was performed using illumina high-throughput sequencing
Project description:The RDM1 gene encodes a RNA recognition motif (RRM)-containing protein involved in the cellular response to the anti-cancer drug cisplatin in vertebrates. We previously reported a cDNA encoding the full-length human RDM1 protein. Here, we describe the identification of 11 human cDNAs encoding RDM1 protein isoforms. This repertoire is generated by alternative pre-mRNA splicing and differential usage of two translational start sites, resulting in proteins with long or short N-terminus and a great diversity in the exonic composition of their C-terminus. By using tagged proteins and fluorescent microscopy, we examined the subcellular distribution of full-length RDM1 (renamed RDM1alpha), and other RDM1 isoforms. We show that RDM1alpha undergoes subcellular redistribution and nucleolar accumulation in response to proteotoxic stress and mild heat shock. In unstressed cells, the long N-terminal isoforms displayed distinct subcellular distribution patterns, ranging from a predominantly cytoplasmic to almost exclusive nuclear localization, suggesting functional differences among the RDM1 proteins. However, all isoforms underwent stress-induced nucleolar accumulation. We identified nuclear and nucleolar localization determinants as well as domains conferring cytoplasmic retention to the RDM1 proteins. Finally, RDM1 null chicken DT40 cells displayed an increased sensitivity to heat shock, compared to wild-type (wt) cells, suggesting a function for RDM1 in the heat-shock response.
Project description:A number of external and internal insults disrupt nucleolar structure, and the resulting nucleolar stress stabilizes and activates p53. We show here that nucleolar disruption induces acetylation and accumulation of p53 without phosphorylation. We identified three nucleolar proteins, MYBBP1A, RPL5, and RPL11, involved in p53 acetylation and accumulation. MYBBP1A was tethered to the nucleolus through nucleolar RNA. When rRNA transcription was suppressed by nucleolar stress, MYBBP1A translocated to the nucleoplasm and facilitated p53-p300 interaction to enhance p53 acetylation. We also found that RPL5 and RPL11 were required for rRNA export from the nucleolus. Depletion of RPL5 or RPL11 blocked rRNA export and counteracted reduction of nucleolar RNA levels caused by inhibition of rRNA transcription. As a result, RPL5 or RPL11 depletion inhibited MYBBP1A translocation and p53 activation. Our observations indicated that a dynamic equilibrium between RNA generation and export regulated nucleolar RNA content. Perturbation of this balance by nucleolar stress altered the nucleolar RNA content and modulated p53 activity.
Project description:The nucleolus is a plurifunctional organelle in which structure and function are intimately linked. Its structural plasticity has long been appreciated, particularly in response to transcriptional inhibition and other cellular stresses, although the mechanism and physiological relevance of these phenomena are unclear. Using MCF-7 and other mammalian cell lines, we describe a structural and functional adaptation of the nucleolus, triggered by heat shock or physiological acidosis, that depends on the expression of ribosomal intergenic spacer long noncoding RNA (IGS lncRNA). At the heart of this process is the de novo formation of a large subnucleolar structure, termed the detention center (DC). The DC is a spatially and dynamically distinct region, characterized by an 8-anilino-1-naphthalenesulfonate-positive hydrophobic signature. Its formation is accompanied by redistribution of nucleolar factors and arrest in ribosomal biogenesis. Silencing of regulatory IGS lncRNA prevents the creation of this structure and allows the nucleolus to retain its tripartite organization and transcriptional activity. Signal termination causes a decrease in IGS transcript levels and a return to the active nucleolar conformation. We propose that the induction of IGS lncRNA by environmental signals operates as a molecular switch that regulates the structure and function of the nucleolus.