Inter-Strain Epigenomic Profiling Reveals a Candidate IAP Master Copy in C3H Mice.

Insertions of endogenous retroviruses cause a significant fraction of mutations in inbred mice but not all strains are equally susceptible. Notably, most new Intracisternal A particle (IAP) ERV mutagenic insertions have occurred in C3H mice. We show here that strain-specific insertional polymorphic IAPs accumulate faster in C3H/HeJ mice, relative to other sequenced strains, and that IAP transcript levels are higher in C3H/HeJ embryonic stem (ES) cells compared to other ES cells. To investigate the mechanism for high IAP activity in C3H mice, we identified 61 IAP copies in C3H/HeJ ES cells enriched with H3K4me3 (a mark of active promoters) and, among those tested, all are unmethylated in C3H/HeJ ES cells. Notably, 13 of the 61 are specific to C3H/HeJ and are members of the non-autonomous 1Δ1 IAP subfamily that is responsible for nearly all new insertions in C3H. One copy is full length with intact open reading frames and hence potentially capable of providing proteins i

BrumiR: A toolkit for de novo discovery of microRNAs from sRNA-seq data

International audienceAbstract MicroRNAs (miRNAs) are small noncoding RNAs that are key players in the regulation of gene expression. In the past decade, with the increasing accessibility of high-throughput sequencing technologies, different methods have been developed to identify miRNAs, most of which rely on preexisting reference genomes. However, when a reference genome is absent or is not of high quality, such identification becomes more difficult. In this context, we developed BrumiR, an algorithm that is able to discover miRNAs directly and exclusively from small RNA (sRNA) sequencing (sRNA-seq) data. We benchmarked BrumiR with datasets encompassing animal and plant species using real and simulated sRNA-seq experiments. The results demonstrate that BrumiR reaches the highest recall for miRNA discovery, while at the same time being much faster and more efficient than the state-of-the-art tools evaluated. The latter allows BrumiR to analyze a large number of sRNA-seq experiments, from plants or animal species. Moreover, BrumiR detects additional information regarding other expressed sequences (sRNAs, isomiRs, etc.), thus maximizing the biological insight gained from sRNA-seq experiments. Additionally, when a reference genome is available, BrumiR provides a new mapping tool (BrumiR2reference) that performs an a posteriori exhaustive search to identify the precursor sequences. Finally, we also provide a machine learning classifier based on a random forest model that evaluates the sequence-derived features to further refine the prediction obtained from the BrumiR-core. The code of BrumiR and all the algorithms that compose the BrumiR toolkit are freely available at https://github.com/camoragaq/BrumiR

Transcriptomic-based selection of reference genes for quantitative real-time PCR in an insect endosymbiotic model

Reference genes are a fundamental tool for analyses of gene expression by real-time quantitative PCR (qRT-PCR), in that they ensure the correct comparison between conditions, stages, or treatments. Because of this, selection of appropriate genes to use as references is crucial for proper application of the technique. Nevertheless, efforts to find appropriate, stably expressed transcripts are still lacking, in particular in the field of insect science. Here, we took advantage of a massive transcriptomic high-throughput analysis of various developmental stages of the gut and associated-bacteriomes of the cereal weevil Sitophilus oryzae and identified a subset of stably expressed genes with the potential to be used as housekeeping genes from the larva to the adult stage. We employed several normalization techniques to select the most suitable genes among our subset. Our final selection includes two genes–TAO, and YTH3–which can also be used to compare transcript abundance at various developmental stages in symbiotic insects, and in insects devoid of endosymbionts (aposymbiotic). Since they are well conserved, these genes have the potential to be useful for many other insect species. This work confirms the interest in using large-scale, unbiased methods for reference gene selection

Insights on the virulence of swine respiratory tract mycoplasmas through genome-scale metabolic modeling

Background: The respiratory tract of swine is colonized by several bacteria among which are three Mycoplasma species: Mycoplasma flocculare, Mycoplasma hyopneumoniae and Mycoplasma hyorhinis. While colonization by M. flocculare is virtually asymptomatic, M. hyopneumoniae is the causative agent of enzootic pneumonia and M. hyorhinis is present in cases of pneumonia, polyserositis and arthritis. The genomic resemblance among these three Mycoplasma species combined with their different levels of pathogenicity is an indication that they have unknown mechanisms of virulence and differential expression, as for most mycoplasmas. Methods: In this work, we performed whole-genome metabolic network reconstructions for these three mycoplasmas. Cultivation tests and metabolomic experiments through nuclear magnetic resonance spectroscopy (NMR) were also performed to acquire experimental data and further refine the models reconstructed in silico. Results: Even though the refined models have similar metabolic capabilities, interesting differences include a wider range of carbohydrate uptake in M. hyorhinis, which in turn may also explain why this species is a widely contaminant in cell cultures. In addition, the myo-inositol catabolism is exclusive to M. hyopneumoniae and may be an important trait for virulence. However, the most important difference seems to be related to glycerol conversion to dihydroxyacetone-phosphate, which produces toxic hydrogen peroxide. This activity, missing only in M. flocculare, may be directly involved in cytotoxicity, as already described for two lung pathogenic mycoplasmas, namely Mycoplasma pneumoniae in human and Mycoplasma mycoides subsp. mycoides in ruminants. Metabolomic data suggest that even though these mycoplasmas are extremely similar in terms of genome and metabolism, distinct products and reaction rates may be the result of differential expression throughout the species. Conclusions: We were able to infer from the reconstructed networks that the lack of pathogenicity of M. flocculare if compared to the highly pathogenic M. hyopneumoniae may be related to its incapacity to produce cytotoxic hydrogen peroxide. Moreover, the ability of M. hyorhinis to grow in diverse sites and even in different hosts may be a reflection of its enhanced and wider carbohydrate uptake. Altogether, the metabolic differences highlighted in silico and in vitro provide important insights to the different levels of pathogenicity observed in each of the studied species

Totoro: Identifying Active Reactions During the Transient State for Metabolic Perturbations

International audienceMotivation: The increasing availability of metabolomic data and their analysis are improving the understanding of cellular mechanisms and how biological systems respond to different perturbations. Currently, there is a need for novel computational methods that facilitate the analysis and integration of increasing volume of available data. Results: In this paper, we present Totoro a new constraint-based approach that integrates quantitative non-targeted metabolomic data of two different metabolic states into genome-wide metabolic models and predicts reactions that were most likely active during the transient state. We applied Totoro to real data of three different growth experiments (pulses of glucose, pyruvate, succinate) from Escherichia coli and we were able to predict known active pathways and gather new insights on the different metabolisms related to each substrate. We used both the E. coli core and the iJO1366 models to demonstrate that our approach is applicable to both smaller and larger networks. Availability: Totoro is an open source method (available at https://gitlab.inria.fr/erable/totoro ) suitable for any organism with an available metabolic model. It is implemented in C++ and depends on IBM CPLEX which is freely available for academic purposes

Investigação da função de grânulos de TcDHH1 na regulação da estabilidade e tradução de mRNAS alvos em Trypanosoma Cruzi

Orientadora : Drª Andréa Rodrigues ÁvilaCoorientadora: Profa.Dra. Fabíola Barbieri HoletzDissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Biologia Celular e Molecular. Defesa: Curitiba, 23/02/2012Bibliografia: fla. 150-154Resumo: A regulação da expressão gênica em tripanossomatídeos ocorre principalmente em nível póstranscricional. Assim, a abundância do mRNA depende de fatores que alterem a estabilidade, localização e acesso destes mRNAs aos polissomos. Diversos estudos têm demonstrado que grânulos de mRNA, presentes em eucariotos têm papel fundamental na regulação da expressão gênica em nível pós-transcricional. Tais grânulos participam ativamente nos processos de estocagem, tradução e degradação do mRNA. Grânulos de estresse e P-bodies são tipos diferentes de grânulos de RNA, e algumas proteínas que constituem o cerne dessas estruturas, em leveduras e mamíferos, já foram identificadas em T. cruzi. Dentre elas, podemos citar a proteína DHH1, uma DEAD-box RNA helicase que estimula a degradação de mRNAs. Em T. cruzi esta proteína tem expressão constitutiva e faz parte de grânulos de RNA que estão presentes no parasita tanto em fase logarítmica de crescimento quanto nas formas submetidas a estresse nutricional, uma característica de formação semelhante aos P-bodies. Contudo, o conteúdo protéico dos grânulos que contém a proteína TcDHH1 em T. cruzi, é formado por componentes específicos de grânulos de estresse. Por isso, o foco desse estudo foi desvendar as funções de grânulos que contém DHH1 em T. cruzi e avaliar se teriam papel majoritário na estocagem, como grânulos de estresse ou no processamento e degradação de mRNAs, como P-bodies. Para isso, várias ferramentas se fizeram necessárias, desde a produção de anticorpos até a padronização de técnicas mais elaboradas. Estudos prévios em epimastigotas utilizando microarranjo de DNA identificaram mRNAs alvos da proteína TcDHH1, os quais em sua maioria representam genes estágio-específicos expressos em outros estágios do ciclo de vida do parasita. Dessa forma, para tentar desvendar se os grânulos de TcDHH1 participam da estocagem ou degradação de mRNAs, os parasitas foram tratados com drogas que inibem os processos de transcrição (actinomicina D) e de trans-splicing (sinefungina) e a estabilidade dos mRNAs alvos da TcDHH1 foi avaliada pela técnica de PCR quantitativa. Os dados obtidos em epimastigotas demonstram que a proteína TcDHH1 interage tanto com alvos estáveis (geralmente mais expressos em epimastigotas) quanto com alvos instáveis (em sua maior parte, mais expressos em outros estágios do ciclo de vida). Paralelamente, demonstramos a colocalização dos grânulos que contém a proteína TcDHH1 e os seus mRNAs alvo pela técnica de FISH, em epimastigotas. Além disso, a fim de elucidar melhor a composição dos grânulos de T. cruzi, escolhemos duas proteínas específicas de degradação de mRNAs para estudo: a exonuclease 5'-3'TcXRNA, que é regulada durante o ciclo de vida e a desadenilase TcPOP2, componente do complexo CCR4-NOT. Ambas as proteínas colocalizam parcialmente com TcDHH1 em grânulos citoplasmáticos nas formas epimastigotas, sugerindo que o destino dos mRNAs alvos desta proteína seja dependente da combinação protéica dos grânulos, corroborando a complexidade do processo de regulação da expressão gênica neste parasita.Abstract: In trypanosomatids, gene expression is regulated mainly post-transcriptionally by mechanisms involving changes in mRNA stability or access to polysomes. Recent studies have demonstrated that cytoplasmic RNA granules, present in several eukaryotes, have key role in regulation of gene expression. These granules are involved in mRNA sorting, storage and degradation and can be divided in different classes, including P-bodies and stress granules and some of the proteins that are included in the core of these structures, in yeast and mammals, have been identified in T. cruzi. The highly conserved DEAD-box helicase Dhh1p stimulates mRNA degradation. In T. cruzi, expression of DHH1 is not regulated throughout the parasite's life cycle or under stress conditions. This protein is present in polysome-independent complexes and is located to discrete cytoplasmic foci, resembling P-bodies. However, the protein content of these granules shows grater similarity to stress granules. Thus, the main focus of this study is to unravel if TcDHH1 granules have a major role on storage (similar to stress granule) or on degradation of transcripts (similar to P-bodies). In order to perform this study, several tools were necessary, from the production of antibodies to standardization of sophisticated techniques. Previous studies in epimastigotes revealed that several mRNA targets of TcDHH1, identified by ribonomic microarray and confirmed by RT-PCR, codify mainly proteins that are regulated in a stage-specific manner and expressed in other stages of life cycle. In order to determine if these TcDHH1 granules participate in storage/stabilization or degradation of mRNAs, the parasites were treated with drugs that inhibit transcription (actinomycin D) and trans-splicing (sinefungin) and the stability of target mRNAs were evaluated by quantitative PCR. The data obtained demonstrate that TcDHH1 interacts with both stable (usually upregulated in epimastigotes) and unstable (upregulated in other stages of life cycle) transcripts. In parallel, we demonstrated the colocalization between TcDHH1 granules and its target mRNAs in epimastigotes, by FISH. Furthermore, in order to better elucidate de granules' content in T. cruzi, we chose two degradation specific proteins: the 5' -3' exonuclease TcXRNA, which is regulated throughout the life cycle, and the deadenylase TcPOP2, a component of the CCR4-NOT complex. Both proteins partially colocalize with TcDHH1 in cytoplasmic granules in epimastigotes, suggesting that the destiny of these proteins' targets is dependent on the combination with other proteins in these granules and corroborating the complexity of the regulation of gene expression in these parasites

Metabolic investigation of the mycoplasmas from the respiratory tract of swines

Resumo não disponíve

Metabolic investigation of the mycoplasmas from the respiratory tract of swines

Resumo não disponíve