TAR DNA-Binding protein 43 (TDP-43) regulates stress granule dynamics via differential regulation of G3BP and TIA-1

TDP-43 est une protéine multifonctionnelle possédant des rôles dans la transcription, l'épissage des pré-ARNm, la stabilité et le transport des ARNm. TDP-43 interagit avec d'autres hnRNP, incluant hnRNP A2, via son extrémité C-terminale. Plusieurs membres de la famille des hnRNP étant impliqués dans la réponse au stress cellulaire, alors nous avons émis l’hypothèse que TDP-43 pouvait y participer aussi. Nos résultats démontrent que TDP-43 et hnRNP A2 sont localisés au niveau des granules de stress, à la suite d’un stress oxydatif, d’un choc thermique, et lors de l’exposition à la thapsigargine. TDP-43 contribue à la fois à l'assemblage et au maintien des granules de stress en réponse au stress oxydatif. TDP-43 régule aussi de façon différentielle les composants clés des granules de stress, notamment TIA-1 et G3BP. L'agrégation contrôlée de TIA-1 est perturbée en l'absence de TDP-43. En outre, TDP-43 régule le niveau d`ARNm de G3BP, un facteur de granule de stress de nucléation. La mutation associée à la sclérose latérale amyotrophique, TDP-43R361S, compromet la formation de granules de stress. Ainsi, la fonction cellulaire de TDP-43 s'étend au-delà de l’épissage; TDP-43 est aussi un composant de la réponse cellulaire au stress central et un acteur actif dans le stockage des ARNs.

Stress-induced targeting of molecular chaperones in the yeast Saccharomyces cerevisiae

The eukaryotic stress response is an essential mechanism that helps protect cells from a variety of environmental stresses. Cell death can result if cells are not able to properly adapt and protect themselves against adverse stress conditions. Failure to properly deal with stress has implications in human diseases including neurodegenerative disorders and distinct cancers, emphasizing the importance of understanding the eukaryotic stress response in detail. As part of this response, expression of a battery of heat shock proteins (HSP) is induced, which act as molecular chaperones to assist in the repair or triage of unfolded proteins. The 90-kDa HSP (Hsp90) operates in the context of a multi-chaperone complex to promote the maturation of nuclear and cytoplasmic clients. I have discovered that Hsp90 and the co-chaperone Sba1 accumulate in the nucleus of quiescent Saccharomyces cerevisiae cells in a karyopherin-dependent manner. I isolated nuclear accumulation- defective HSP82 mutant alleles to probe the nature of this targeting event and identified a mutant with a single amino acid substitution (I578F) sufficient to prevent nuclear accumulation of Hsp90 in quiescent cells. Diploid hsp82-I578F cells exhibited pronounced defects in spore wall construction and maturation, resulting in catastrophic sporulation. The mislocalization and sporulation phenotypes were shared by another previously identified HSP82 mutant allele, further linking localization to Hsp90 functional status. Pharmacological inhibition of Hsp90 with macbecin in sporulating diploid cells also blocked spore formation, underscoring the importance of this chaperone in this developmental program. The yeast molecular chaperone Hsp104 is a member of the Hsp100 superfamily of AAA+ ATPases. Unlike the Hsp90 family of chaperones, Hsp104 is not restricted to a specific set of client proteins, but rather assists in reactivating stress-denatured proteins by solubilizing protein aggregates. I have discovered that Hsp104, along with the Hsp70 chaperone, Ssa1, and the sHSP Hsp26 accumulate into RNA processing bodies (P- bodies) and stress granules, sites of mRNA metabolism. I found that Hsp104 recruits both Ssa1 and Hsp26 to P-bodies and that these three chaperones are required for stress granule formation. These findings suggest a possible role for chaperones in mRNA metabolism by aiding in the assembly, disassembly or conversion of these enigmatic mRNP complexes. Taken together, the work presented in this dissertation serves to better understand the eukaryotic stress response by illustrating the importance of subcellular-chaperone localization in key biological processes.

C9ORF72 Regulates Stress Granule Formation and Its Deficiency Impairs Stress Granule Assembly, Hypersensitizing Cells to Stress

Hexanucleotide repeat expansions in the C9ORF72 gene are causally associated with frontotemporal lobar dementia (FTLD) and/or amyotrophic lateral sclerosis (ALS). The physiological function of the normal C9ORF72 protein remains unclear. In this study, we characterized the subcellular localization of C9ORF72 to processing bodies (P-bodies) and its recruitment to stress granules (SGs) upon stress-related stimuli. Gain of function and loss of function experiments revealed that the long isoform of C9ORF72 protein regulates SG assembly. CRISPR/Cas9-mediated knockdown of C9ORF72 completely abolished SG formation, negatively impacted the expression of SG-associated proteins such as TIA-1 and HuR, and accelerated cell death. Loss of C9ORF72 expression further compromised cellular recovery responses after the removal of stress. Additionally, mimicking the pathogenic condition via the expression of hexanucleotide expansion upstream of C9ORF72 impaired the expression of the C9ORF72 protein, caused an abnormal accumulation of RNA foci, and led to the spontaneous formation of SGs. Our study identifies a novel function for normal C9ORF72 in SG assembly and sheds light into how the mutant expansions might impair SG formation and cellular-stress-related adaptive responses.

Role of cyclooxygenase-2 in Trypanosoma cruzisurvival in the early stages of parasite host-cell interaction

Chagas disease, caused by the intracellular protozoan Trypanosoma cruzi, is a serious health problem in Latin America. During this parasitic infection, the heart is one of the major organs affected. The pathogenesis of tissue remodelling, particularly regarding cardiomyocyte behaviour after parasite infection and the molecular mechanisms that occur immediately following parasite entry into host cells are not yet completely understood. When cells are infected with T. cruzi, they develop an inflammatory response, in which cyclooxygenase-2 (COX-2) catalyses rate-limiting steps in the arachidonic acid pathway. However, how the parasite interaction modulates COX-2 activity is poorly understood. In this study, the H9c2 cell line was used as our model and we investigated cellular and biochemical aspects during the initial 48 h of parasitic infection. Oscillatory activity of COX-2 was observed, which correlated with the control of the pro-inflammatory environment in infected cells. Interestingly, subcellular trafficking was also verified, correlated with the control of Cox-2 mRNA or the activated COX-2 protein in cells, which is directly connected with the assemble of stress granules structures. Our collective findings suggest that in the very early stage of the T. cruzi-host cell interaction, the parasite is able to modulate the cellular metabolism in order to survives.

Role of Sam68 in post-transcriptional gene regulation.

The STAR family of proteins links signaling pathways to various aspects of post-transcriptional regulation and processing of RNAs. Sam68 belongs to this class of heteronuclear ribonucleoprotein particle K (hnRNP K) homology (KH) single domain-containing family of RNA-binding proteins that also contains some domains predicted to bind critical components in signal transduction pathways. In response to phosphorylation and other post-transcriptional modifications, Sam68 has been shown to have the ability to link signal transduction pathways to downstream effects regulating RNA metabolism, including transcription, alternative splicing or RNA transport. In addition to its function as a docking protein in some signaling pathways, this prototypic STAR protein has been identified to have a nuclear localization and to take part in the formation of both nuclear and cytosolic multi-molecular complexes such as Sam68 nuclear bodies and stress granules. Coupling with other proteins and RNA targets, Sam68 may play a role in the regulation of differential expression and mRNA processing and translation according to internal and external signals, thus mediating important physiological functions, such as cell death, proliferation or cell differentiation.

Exogenous mRNA delivery and bioavailability in gene transfer mediated by piggyBac transposition.

BACKGROUND: Up to now, the different uptake pathways and the subsequent intracellular trafficking of plasmid DNA have been largely explored. By contrast, the mode of internalization and the intracellular routing of an exogenous mRNA in transfected cells are poorly investigated and remain to be elucidated. The bioavailability of internalized mRNA depends on its intracellular routing and its potential accumulation in dynamic sorting sites for storage: stress granules and processing bodies. This question is of particular significance when a secure transposon-based system able to integrate a therapeutic transgene into the genome is used. Transposon vectors usually require two components: a plasmid DNA, carrying the gene of interest, and a source of transposase allowing the integration of the transgene. The principal drawback is the lasting presence of the transposase, which could remobilize the transgene once it has been inserted. Our study focused on the pharmacokinetics of the transposition process mediated by the piggyBac transposase mRNA transfection. Exogenous mRNA internalization and trafficking were investigated towards a better apprehension and fine control of the piggyBac transposase bioavailability. RESULTS: The mRNA prototype designed in this study provides a very narrow expression window of transposase, which allows high efficiency transposition with no cytotoxicity. Our data reveal that exogenous transposase mRNA enters cells by clathrin and caveolae-mediated endocytosis, before finishing in late endosomes 3 h after transfection. At this point, the mRNA is dissociated from its carrier and localized in stress granules, but not in cytoplasmic processing bodies. Some weaker signals have been observed in stress granules at 18 h and 48 h without causing prolonged production of the transposase. So, we designed an mRNA that is efficiently translated with a peak of transposase production 18 h post-transfection without additional release of the molecule. This confines the integration of the transgene in a very small time window. CONCLUSION: Our results shed light on processes of exogenous mRNA trafficking, which are crucial to estimate the mRNA bioavailability, and increase the biosafety of transgene integration mediated by transposition. This approach provides a new way for limiting the transgene copy in the genome and their remobilization by mRNA engineering and trafficking.

Regulation of protein synthesis and the role of eIF3 in cancer

Maintenance of cell homeostasis and regulation of cell proliferation depend importantly on regulating the process of protein synthesis. Many disease states arise when disregulation of protein synthesis occurs. This review focuses on mechanisms of translational control and how disregulation results in cell malignancy. Most translational controls occur during the initiation phase of protein synthesis, with the initiation factors being the major target of regulation through their phosphorylation. In particular, the recruitment of mRNAs through the m7G-cap structure and the binding of the initiator methionyl-tRNAi are frequent targets. However, translation, especially of specific mRNAs, may also be regulated by sequestration into processing bodies or stress granules, by trans-acting proteins or by microRNAs. When the process of protein synthesis is hyper-activated, weak mRNAs are translated relatively more efficiently, leading to an imbalance of cellular proteins that promotes cell proliferation and malignant transformation. This occurs, for example, when the cap-binding protein, eIF4E, is overexpressed, or when the methionyl-tRNAi-binding factor, eIF2, is too active. In addition, enhanced activity of eIF3 contributes to oncogenesis. The importance of the translation initiation factors as regulators of protein synthesis and cell proliferation makes them potential therapeutic targets for the treatment of cancer.

Efecto de la hipoxia-reoxigenación y las radiaciones ionizantes en la captación de glucosa en líneas tumorales de seno y colon cocultivadas con células endoteliales.

La captación de glucosa y su conversión en lactato juega un papel fundamental en el metabolismo tumoral, independientemente de la concentración de oxígeno presente en el tejido (efecto Warburg). Sin embrago, dicha captación varía de un tipo tumoral a otro, y dentro del mismo tumor, situación que podría depender de las características microambientales tumorales (fluctuaciones de oxígeno, presencia de otros tipos celulares) y de factores estresores asociados a los tratamientos. Se estudió el efecto de la hipoxia-reoxigenación (HR) y las radiaciones ionizantes (RI) sobre la captación de glucosa, en cultivos de líneas tumorales MCF-7 y HT-29, cultivadas de forma aislada o en cocultivo con la línea celular EAhy296. Se encontró que la captación de glucosa en HR es diferente para lo descrito en condiciones de hipoxia permanente y que es modificada en el cocultivo. Se identificaron poblaciones celulares dentro de la misma línea celular, de alta y baja captación de glucosa, lo que implicaría una simbiosis metabólica de la célula como respuesta adaptativa a las condiciones tumorales. Se evaluó la expresión de NRF2 y la translocación nuclear de NRF2 y HIF1a, como vías de respuesta a estrés celular e hipoxia. La translocación nuclear de las proteínas evaluadas explicaría el comportamiento metabólico de las células tumorales de seno, pero no de colon, por lo cual deben existir otras vías metabólicas implicadas. Las diferencias en el comportamiento de las células tumorales en HR en relación con hipoxia permitirá realizar planeaciones dosimétricas más dinámicas, que reevalúen las condiciones de oxigenación tumoral constantemente.

A novel function for CUGBP2 in controlling the pro-inflammatory stimulus in H9c2 cells: Subcellular trafficking of messenger molecules

Accumulating evidence demonstrates that chronic inflammation plays an important role in heart hypertrophy and cardiac diseases. However, the fine-tuning of cellular and molecular mechanisms that connect inflammatory process and cardiac diseases is still under investigation. Many reports have demonstrated that the overexpression of the cyclooxygenase-2 (COX-2), a key enzyme in the conversion of arachidonic acid to prostaglandins and other prostanoids, is correlated with inflammatory processes. Increased level of prostaglandin E2 was also found in animal model of left ventricle of hypertrophy. Based on previous observations that demonstrated a regulatory loop between COX-2 and the RNA-binding protein CUGBP2, we studied cellular and molecular mechanisms of a pro-inflammatory stimulus in a cardiac cell to verify if the above two molecules could be correlated with the inflammatory process in the heart. A cellular model of investigation was established and H9c2 was used.We also demonstrated a regulatory connection between COX-2 and CUGBP2 in the cardiac cells. Based on a set of different assays including gene silencing and fluorescence microscopy, we describe a novel function for the RNA-binding protein CUGBP2 in controlling the pro-inflammatory stimulus: subcellular trafficking of messenger molecules to specific cytoplasmic stress granules to maintain homeostasis. © 2013 International Federation for Cell Biology.

Análise de interação funcional de elF5A com a tradução, repressão da tradução e degradação de mRNA em Saccharomyces cerevisiae

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Role of cyclooxygenase-2 in Trypanosoma cruzi survival in the early stages of parasite host-cell interaction

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Functional characterization of the trypanosome translational repressor SCD6

The storage of translationally inactive mRNAs in cytosolic granules enables cells to react flexibly to environmental changes. In eukaryotes, Scd6 (suppressor of clathrin deficiency 6)/Rap55 (RNA-associated protein 55), a member of the LSm14 (like-Sm14) family, is an important factor in the formation and activity of P-bodies, where mRNA decay factors accumulate, in stress granules that store mRNAs under adverse conditions and in granules that store developmentally regulated mRNAs. SCD6 from Trypanosoma brucei (TbSCD6) shares the same domain architecture as orthologous proteins in other organisms and is also present in cytosolic granules (equivalent to P-bodies). We show that TbSCD6 is a general repressor of translation and that its depletion by RNAi results in a global increase in protein synthesis. With few exceptions, the steady-state levels of proteins are unchanged. TbSCD6 is not required for the formation of starvation-induced granules in trypanosomes, and unlike Scd6 from yeast, Plasmodium and all multicellular organisms analysed to date, it does not form a complex with the helicase Dhh1 (DExD/H-box helicase 1). In common with Xenopus laevis RAP55, TbSCD6 co-purifies with two arginine methyltransferases; moreover, TbSCD6 itself is methylated on three arginine residues. Finally, a detailed analysis identified roles for the Lsm and N-rich domains in both protein localization and tr

The Structure of theCyprinid herpesvirus 3ORF112-Zα·Z-DNA Complex Reveals a Mechanism of Nucleic Acids Recognition Conserved with E3L, a Poxvirus Inhibitor of Interferon Response

In vertebrate species, the innate immune system down-regulates protein translation in response to viral infection through the action of the double-stranded RNA (dsRNA)-activated protein kinase (PKR). In some teleost species another protein kinase, Z-DNA-dependent protein kinase (PKZ), plays a similar role but instead of dsRNA binding domains, PKZ has Zα domains. These domains recognize the left-handed conformer of dsDNA and dsRNA known as Z-DNA/Z-RNA. Cyprinid herpesvirus 3 infects common and koi carp, which have PKZ, and encodes the ORF112 protein that itself bears a Zα domain, a putative competitive inhibitor of PKZ. Here we present the crystal structure of ORF112-Zα in complex with an 18-bp CpG DNA repeat, at 1.5 Å. We demonstrate that the bound DNA is in the left-handed conformation and identify key interactions for the specificity of ORF112. Localization of ORF112 protein in stress granules induced in Cyprinid herpesvirus 3-infected fish cells suggests a functional behavior similar to that of Zα domains of the interferon-regulated, nucleic acid surveillance proteins ADAR1 and DAI.

TDP-43 régule la dynamique et la fonction des Granules de Stress via G3BP1

Les Granule de Stress (GS) sont des inclusions cytoplasmiques contenant des protéines et des ARNm qui s’assemblent en réponse à l’exposition à un stress. Leur formation fait partie intégrante de la réponse cellulaire au stress et est considérée comme une étape déterminante pour la résistance au stress et la survie cellulaire. Actuellement, les GS sont reliés à divers pathologies allant des infections virales aux maladies neurovégétatives. L’une d’entre elle, la Sclérose Latérale Amyotrophique (SLA) est particulièrement agressive, caractérisée par une perte des neurones moteurs aboutissant à la paralysie et à la mort du patient en cinq ans en moyenne. Les mécanismes de déclenchement de la pathologie restent encore à déterminer. TDP-43 (TAR DNA binding protein 43) et FUS (Fused in liposarcoma) sont deux protéines reliées à la pathologie qui présentent des similarités de structure et de fonction, suggérant un mécanisme commun de toxicité. TDP-43 et FUS sont toutes les deux recrutées au niveau des GS en condition de stress. Nous avons démontré pour la première fois que la fonction des GS est de protéger les ARNm de la dégradation induite par l’exposition au stress. Cette fonction n’était que suspectée jusqu’alors. De plus nous avons mis en évidence que G3BP1 (Ras GTPase-activating protein-binding protein 1) est l’effectrice de cette fonction via son implication dans la dynamique de formation des GS. TDP-43 étant un régulateur de G3BP1, nous prouvons ainsi que la perte de fonction de TDP-43/G3BP1 aboutit à un défaut de réponse au stress aboutissant à une vulnérabilisation cellulaire. Le mécanisme de toxicité emprunter par FUS diffère de celui de TDP-43 et ne semble pas passer par une perte de fonction dans le cadre de la réponse au stress.