Helaine Graziele Santos Vieira

Garvan Institute of Medical Research

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Eva Maria Novoa

Centre for Genomic Regulation

Ivan Milenkovic

Centre for Genomic Regulation

Morghan C. Lucas

Pompeu Fabra University

Glória Regina Franco

Universidade Federal de Minas Gerais

Sonia Cruciani

Pompeu Fabra University

Huanle Liu

Shanxi University

John S. Mattick

UNSW Sydney

Schraga Schwartz

Weizmann Institute of Science

Aldema Sas‐Chen

Tel Aviv University

Noelia Camacho

Institute for Research in Biomedicine

Cooperative Institutions

Universidade Federal de Minas Gerais

Centre for Genomic Regulation

UNSW Sydney

Garvan Institute of Medical Research

Pompeu Fabra University

Institute for Research in Biomedicine

St Vincent's Clinic

Barcelona Institute for Science and Technology

Fundação Oswaldo Cruz

Universidade de São Paulo

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Caracterização do perfil protéico e da atividade mitocondrial de Trypanosoma cruzi sob o estresse de radiação gama

Helaine Graziele Santos Vieira

O Trypanosoma cruzi e considerado um dos eucariotos mais resistentes a Radiacao ionizante, suportando doses sub-letais de ate 1,0 kGy. A radiacao ionizante do tipo gama promove a quebra das fitas simples e duplas do DNA, alem de gerar especies reativas de oxigenio (ROS). Apos a irradiacao, os parasitos cessam o seu crescimento por ate 96 horas, retomando-o em seguida e atingindo a fase estacionaria em ate 240 horas apos irradiacao. O padrao de bandas cromossomais, que sao extensamente fragmentadas pela radiacao, e restabelecido cerca de 48 horas apos o estresse. Para tolerar tais danos, e necessario um eficiente aparato de reconhecimento e reparo de lesoes. Neste trabalho, investigamos a radioresistencia em T.cruzi, mediante a analise do seu perfil de expressao proteica e da sua atividade mitocondrial em diferentes tempos apos uma dose de 500 Gy de radiacao gama. Utilizando a metodologia 2D-DIGE, foram encontrados 543 spots proteicos diferencialmente expressos apos a exposicao do T. cruzi a radiacao. A partir destes spots, 53 proteinas foram identificadas por espectrometria de massas (MS/MS) e classificadas de acordo com sua funcao biologica. Analisando a anotacao funcional das 53 proteinas, observamos uma tendencia para a superexpressao de proteinas com peso molecular abaixo do predito. Esta tendencia indica a possivel existencia de modificacoes pos-traducionais e/ou de mecanismos de processamento das proteinas identificadas, que geraria polipeptidios mais curtos do que o predito em resposta a radiacao gama. Outras tendencias observadas em nossas analises de proteomica incluem alteracoes nos seguintes processos biologicos: regulacao positiva do processo de sintese de proteinas, repressao de proteinas envolvidas no processo de enovelamento (exceto pela inducao de duas chaperonas do reticulo endoplasmatico), repressao das vias de geracao de ATP por fosforilacao oxidativa, da glicolise, e do metabolismo de aminoacidos. Nossos resultados tambem indicam que a sintese proteica de#novo e essencial para a recuperacao do T. cruzi apos o estresse por exposicao a radiacao gama, pois o tratamento com inibidores de traducao interfere drasticamente no retorno ao crescimento dos parasitos irradiados. Ao avaliarmos a atividade mitocondrial de T. cruzi, xvi! verificamos que parasitos irradiados produzem mais ATP do que parasitos nao irradiados, alem de consumirem mais O2. Adicionalmente, a analise da expressao genica de T. cruzi irradiado por qRT-PCR mostrou que os genes do maxicirculo mitocondrial foram induzidos apos a irradiacao. Ambos os resultados indicam uma maior atividade da mitocondria apos o estresse. Entretanto, observamos uma menor producao de H2O2 nos parasitos irradiados, indicando assim a existencia de um mecanismo eficiente de eliminacao de radicais livres. O presente estudo revela a resposta peculiar de T. cruzi a radiacao ionizante e suscita questionamentos pertinentes sobre como este organismo e capaz de modificar seu perfil de expressao proteica e sua atividade mitocondrial para possibilitar a sobrevivencia em condicoes tao adversas.

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Subcellular relocalization and nuclear redistribution of the RNA methyltransferases TRMT1 and TRMT1L upon neuronal activation

RNA Biology (2021)

Nicky Jonkhout Sonia Cruciani Helaine Graziele Santos Vieira Julia Tran Huanle Liu

RNA modifications are dynamic chemical entities that expand the RNA lexicon and regulate RNA fate. The most abundant modification present in mRNAs, N6-methyladenosine (m6A), has been implicated in neurogenesis and memory formation. However, whether additional RNA modifications may be playing a role in neuronal functions and in response to environmental queues is largely unknown. Here we characterize the biochemical function and cellular dynamics of two human RNA methyltransferases previously associated with neurological dysfunction, TRMT1 and its homolog, TRMT1-like (TRMT1L). Using a combination of next-generation sequencing, LC-MS/MS, patient-derived cell lines and knockout mouse models, we confirm the previously reported dimethylguanosine (m2,2G) activity of TRMT1 in tRNAs, as well as reveal that TRMT1L, whose activity was unknown, is responsible for methylating a subset of cytosolic tRNAAla(AGC) isodecoders at position 26. Using a cellular in vitro model that mimics neuronal activation and long term potentiation, we find that both TRMT1 and TRMT1L change their subcellular localization upon neuronal activation. Specifically, we observe a major subcellular relocalization from mitochondria and other cytoplasmic domains (TRMT1) and nucleoli (TRMT1L) to different small punctate compartments in the nucleus, which are as yet uncharacterized. This phenomenon does not occur upon heat shock, suggesting that the relocalization of TRMT1 and TRMT1L is not a general reaction to stress, but rather a specific response to neuronal activation. Our results suggest that subcellular relocalization of RNA modification enzymes may play a role in neuronal plasticity and transmission of information, presumably by addressing new targets.

10.1080/15476286.2021.1881291

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Análise da expressão das enzimas desubiquitinadoras 5, 14 e 16 durante o ciclo de vida do Schistosoma mansoni.

Helaine Graziele Santos Vieira

Schistosoma mansoni presents a peculiar life cycle, in which several morphologic and biochemical alterations occur in different phases of development. These changes are evolutive adaptations found by the parasite to survive in distinct environments. These adaptations are promoted by a set of genes expressed coordinately in the six life periods of the parasite, being a target to elaboration of vaccines and new drugs. In this context, the post-transductional modifications of proteins, as ubiquitination, must be operating in some crucial cellular processes in the parasite. Ubiquitin (Ub) binds to target protein covalently, through an isopeptide linkage between the C-terminal glicina residue (Gly 76) of Ub and the amino group of Lysine (Lys). The reversible process of ubiquitination is the deubiquitination. DUBs (deubiquitin enzymes) are cystein proteases that specifically cleave off Ub from Ub-protein conjugates, Ub precursors and Ub adducts. In the present work, our objective was to validate the in silico analyses of DUB's 5, 14 and 16 in S. mansoni and evaluate the expression patterns of these enzymes in cercariae, in vitro schistosomulum obtained by times of 3 hours, 18,5 hours; 24 hours, 3 days, in adult worms and eggs. The comparative expression patterns of DUB' s 5, 14 and 16 shown that they are more expressed in cercariae, adult worm and egg phases, and in basal level in different periods of in vitro cultivate schistosomulum. Our results corroborate the low total DUB activity in phases of schistosomulum when compared with the other phases. This expression pattern also corroborates with the results obtained by our group, where the presence of ubiquitin conjugates was observed in all the phases of S. mansoni, with a relative abundance during the different culture times of schistosomulum (3h, 4h, 8h, 18h, 24h, 3 days, 5 days, 8 days and 10 days). These data suggest that the occurrence of ubiquitinating/deubiquitinating cycles can be involved in critical cellular processes during the full biological S. mansoni life cycle

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Quantitative profiling of pseudouridylation dynamics in native RNAs with nanopore sequencing

Nature Biotechnology (2021)

Oguzhan Begik Morghan C. Lucas Leszek P. Pryszcz José Miguel Ramírez Rebeca Medina

Nanopore

Pseudouridine

Small nucleolar RNA

10.1038/s41587-021-00915-6

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Citations (231)

Trypanosoma cruzi RNA-binding protein ALBA30 aggregates into cytoplasmic foci under nutritional stress

Parasitology Research (2020)

Daniela Ferreira Chame Daniela de Laet Souza Helaine Graziele Santos Vieira Erich Birelli Tahara Andréa Mara Macedo

Stress granule

10.1007/s00436-019-06554-w

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Trypanosoma cruzi RNA-binding protein DRBD3: perinuclear foci formation during benznidazole exposure

Anais da Academia Brasileira de Ciências (2024)

Daniela Ferreira Chame DANIELA DE LAET-SOUZA Helaine Graziele Santos Vieira E. B. Tahara Andréa Mara Macedo

Benznidazole (BZ) is the trypanocidal compound of choice for Chagas disease, a neglected tropical disease in the Americas. However, this drug often fails to cure the infection. The regulation of gene expression in Trypanosoma cruzi, the causative agent of Chagas disease, is based on post-transcriptional mechanisms. When environmental changes cause translational arrest, RNA-binding proteins, and their target mRNAs assemble into cytoplasmic bodies, known as RNA granules, which act as RNA sorting centers. We have characterized the T. cruzi RNA-binding protein DRBD3, which has two RRMs domains, and a C-terminal low-complexity sequence rich in proline and glutamines. Using a tagged form of TcDRBD3 (rTcDRBD3), we showed that this protein resides in the cytoplasm, but localizes into perinuclear cytoplasmic foci after BZ exposure. RNA staining after BZ also showed that this molecule accumulates into perinuclear cytoplasmic foci. Moreover, BZ and puromycin treatment enhanced the colocalization of rTcDRBD3 and RNA, suggesting that TcDRBD3 granules repertoire harbors RNAs released from polysomes. Under starvation, rTcDRBD3 granules localized throughout the cytoplasm and also increased in number in the presence of puromycin. Our results suggest that TcDRBD3 accumulates into perinuclear granules that harbor RNA and also that its localization varies according to the type of stress.

Benznidazole

10.1590/0001-3765202420240321

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Familial STAG2 germline mutation defines a new human cohesinopathy

npj Genomic Medicine (2017)

Fernanda Caroline Soardi Alice Machado-Silva Natália D. Linhares Zheng Ge Qianhui Qu

Abstract We characterize a novel human cohesinopathy originated from a familial germline mutation of the gene encoding the cohesin subunit STAG2, which we propose to call STAG2 -related X-linked Intellectual Deficiency. Five individuals carry a STAG2 p.Ser327Asn (c.980 G > A) variant that perfectly cosegregates with a phenotype of syndromic mental retardation in a characteristic X-linked recessive pattern. Although patient-derived cells did not show overt sister-chromatid cohesion defects, they exhibited altered cell cycle profiles and gene expression patterns that were consistent with cohesin deficiency. The protein level of STAG2 in patient cells was normal. Interestingly, STAG2 S327 is located at a conserved site crucial for binding to SCC1 and cohesin regulators. When expressed in human cells, the STAG2 p.Ser327Asn mutant is defective in binding to SCC1 and other cohesin subunits and regulators. Thus, decreased amount of intact cohesin likely underlies the phenotypes of STAG2 -SXLID. Intriguingly, recombinant STAG2 p.Ser327Asn binds normally to SCC1, WAPL, and SGO1 in vitro, suggesting the existence of unknown in vivo mechanisms that regulate the interaction between STAG2 and SCC1.

10.1038/s41525-017-0009-4

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Proteomic Analysis of Trypanosoma cruzi Response to Ionizing Radiation Stress

PLoS ONE (2014)

Helaine Graziele Santos Vieira Priscila Grynberg Mainá Bitar Simone da Fonseca Pires Heron Oliveira Hilário

Trypanosoma cruzi, the causative agent of Chagas disease, is extremely resistant to ionizing radiation, enduring up to 1.5 kGy of gamma rays. Ionizing radiation can damage the DNA molecule both directly, resulting in double-strand breaks, and indirectly, as a consequence of reactive oxygen species production. After a dose of 500 Gy of gamma rays, the parasite genome is fragmented, but the chromosomal bands are restored within 48 hours. Under such conditions, cell growth arrests for up to 120 hours and the parasites resume normal growth after this period. To better understand the parasite response to ionizing radiation, we analyzed the proteome of irradiated (4, 24, and 96 hours after irradiation) and non-irradiated T. cruzi using two-dimensional differential gel electrophoresis followed by mass spectrometry for protein identification. A total of 543 spots were found to be differentially expressed, from which 215 were identified. These identified protein spots represent different isoforms of only 53 proteins. We observed a tendency for overexpression of proteins with molecular weights below predicted, indicating that these may be processed, yielding shorter polypeptides. The presence of shorter protein isoforms after irradiation suggests the occurrence of post-translational modifications and/or processing in response to gamma radiation stress. Our results also indicate that active translation is essential for the recovery of parasites from ionizing radiation damage. This study therefore reveals the peculiar response of T. cruzi to ionizing radiation, raising questions about how this organism can change its protein expression to survive such a harmful stress.

Proteome

Chagas Disease

10.1371/journal.pone.0097526

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Decoding ribosomal RNA modification dynamics at single molecule resolution

bioRxiv (Cold Spring Harbor Laboratory) (2020)

Oguzhan Begik Morghan C. Lucas José Miguel Ramírez Ivan Milenkovic Sonia Cruciani

SUMMARY A broad diversity of modifications decorate RNA molecules. Originally conceived as static components, evidence is accumulating that some RNA modifications may be dynamic, contributing to cellular responses to external signals and environmental circumstances. A major difficulty in studying these modifications, however, is the need of tailored protocols to map each modification individually. Here, we present a new approach that uses direct RNA nanopore sequencing to identify diverse RNA modification types present in native RNA molecules, using rRNA as the exemplar, and show that each RNA modification type results in distinct and characteristic base-calling ‘error’ signatures. We demonstrate the value of these signatures for de novo prediction of pseudouridine (Y) modifications transcriptome-wide, confirming known Y modifications in rRNAs, snRNAs and mRNAs, and uncovering a novel Pus4-dependent Y modification in yeast mitochondrial rRNA. Using a machine learning classifier, we show that the stoichiometry of modified sites can be quantified by identifying current intensity alterations in individual RNA reads. Finally, we explore the dynamics of pseudouridylation across a battery of environmental stresses, revealing novel heat-sensitive Y-modified sites in both snRNAs and snoRNAs. Altogether, our work demonstrates that Y RNA modifications can be predicted de novo and in a quantitative manner using native RNA nanopore sequencing.

Pseudouridine

Nucleic acid structure

Nanopore

Small nucleolar RNA

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Human tRNAs with inosine 34 are essential to efficiently translate eukarya-specific low-complexity proteins

Nucleic Acids Research (2021)

Adrian Gabriel Torres Marta Rodríguez-Escribà Marina Marcet‐Houben Helaine Graziele Santos Vieira Noelia Camacho

The modification of adenosine to inosine at the wobble position (I34) of tRNA anticodons is an abundant and essential feature of eukaryotic tRNAs. The expansion of inosine-containing tRNAs in eukaryotes followed the transformation of the homodimeric bacterial enzyme TadA, which generates I34 in tRNAArg and tRNALeu, into the heterodimeric eukaryotic enzyme ADAT, which modifies up to eight different tRNAs. The emergence of ADAT and its larger set of substrates, strongly influenced the tRNA composition and codon usage of eukaryotic genomes. However, the selective advantages that drove the expansion of I34-tRNAs remain unknown. Here we investigate the functional relevance of I34-tRNAs in human cells and show that a full complement of these tRNAs is necessary for the translation of low-complexity protein domains enriched in amino acids cognate for I34-tRNAs. The coding sequences for these domains require codons translated by I34-tRNAs, in detriment of synonymous codons that use other tRNAs. I34-tRNA-dependent low-complexity proteins are enriched in functional categories related to cell adhesion, and depletion in I34-tRNAs leads to cellular phenotypes consistent with these roles. We show that the distribution of these low-complexity proteins mirrors the distribution of I34-tRNAs in the phylogenetic tree.

Inosine

10.1093/nar/gkab461

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Citations (24)