Identification of functional long non-coding RNAs in C. elegans.

BACKGROUND: Functional characterisation of the compact genome of the model organism Caenorhabditis elegans remains incomplete despite its sequencing 20 years ago. The last decade of research has seen a tremendous increase in the number of non-coding RNAs identified in various organisms. While we have mechanistic understandings of small non-coding RNA pathways, long non-coding RNAs represent a diverse class of active transcripts whose function remains less well characterised. RESULTS: By analysing hundreds of published transcriptome datasets, we annotated 3392 potential lncRNAs including 143 multi-exonic loci that showed increased nucleotide conservation and GC content relative to other non-coding regions. Using CRISPR/Cas9 genome editing, we generated deletion mutants for ten long non-coding RNA loci. Using automated microscopy for in-depth phenotyping, we show that six of the long non-coding RNA loci are required for normal development and fertility. Using RNA interference-mediated gene knock-down, we provide evidence that for two of the long non-coding RNA loci, the observed phenotypes are dependent on the corresponding RNA transcripts. CONCLUSIONS: Our results highlight that a large section of the non-coding regions of the C. elegans genome remains unexplored. Based on our in vivo analysis of a selection of high-confidence lncRNA loci, we expect that a significant proportion of these high-confidence regions is likely to have a biological function at either the genomic or the transcript level

Translational adaptation to heat stress is mediated by RNA 5-methylcytosine in Caenorhabditis elegans.

Methylation of carbon-5 of cytosines (m5 C) is a post-transcriptional nucleotide modification of RNA found in all kingdoms of life. While individual m5 C-methyltransferases have been studied, the impact of the global cytosine-5 methylome on development, homeostasis and stress remains unknown. Here, using Caenorhabditis elegans, we generated the first organism devoid of m5 C in RNA, demonstrating that this modification is non-essential. Using this genetic tool, we determine the localisation and enzymatic specificity of m5 C sites in the RNome in vivo. We find that NSUN-4 acts as a dual rRNA and tRNA methyltransferase in C. elegans mitochondria. In agreement with leucine and proline being the most frequently methylated tRNA isoacceptors, loss of m5 C impacts the decoding of some triplets of these two amino acids, leading to reduced translation efficiency. Upon heat stress, m5 C loss leads to ribosome stalling at UUG triplets, the only codon translated by an m5 C34-modified tRNA. This leads to reduced translation efficiency of UUG-rich transcripts and impaired fertility, suggesting a role of m5 C tRNA wobble methylation in the adaptation to higher temperatures

The RNA polymerase II subunit RPB-9 recruits the integrator complex to terminate Caenorhabditis elegans piRNA transcription.

PIWI-interacting RNAs (piRNAs) are genome-encoded small RNAs that regulate germ cell development and maintain germline integrity in many animals. Mature piRNAs engage Piwi Argonaute proteins to silence complementary transcripts, including transposable elements and endogenous genes. piRNA biogenesis mechanisms are diverse and remain poorly understood. Here, we identify the RNA polymerase II (RNA Pol II) core subunit RPB-9 as required for piRNA-mediated silencing in the nematode Caenorhabditis elegans. We show that rpb-9 initiates heritable piRNA-mediated gene silencing at two DNA transposon families and at a subset of somatic genes in the germline. We provide genetic and biochemical evidence that RPB-9 is required for piRNA biogenesis by recruiting the Integrator complex at piRNA genes, hence promoting transcriptional termination. We conclude that, as a part of its rapid evolution, the piRNA pathway has co-opted an ancient machinery for high-fidelity transcription

Expression profile of microRNAs in myocardium during acute infection with Trypanosoma cruzi in mice

A doença de Chagas é uma doença crônica causada pela infecção pelo protozoário Trypanosoma cruzi (T.cruzi). A sua principal consequência clínica é o desenvolvimento da cardiomiopatia chagásica crônica (CCC), que acomete 30% dos pacientes. Não foi determinado um indicador de evolução para a CCC ou permanência na forma indeterminada assintomática da doença de Chagas. Diversos trabalhos têm mostrado alterações no perfil de expressão gênica e proteômica ocorridas na fase aguda e crônica da doença de Chagas experimental e humana. Tais alterações advêm da regulação estabelecida em diversos estágios da expressão gênica e podem ser fatores relevantes no prognóstico da doença. Neste contexto, os microRNAs (miRs), podem exercer uma importante função reguladora. Sua ação se dá pela associação a um RNA mensageiro (RNAm) alvo, inibindo sua tradução ou degradando este transcrito. Assim, a hipótese deste trabalho é a de que a infecção aguda por T. cruzi modula a expressão de miRs no miocárdio de camundongos. Foi avaliado por qRT-PCR o perfil de expressão de miRs 15, 30 e 45 dias após a infecção. O perfil de expressão de miRs resultante foi suficiente para segregar os grupos de acordo com o tempo da infecção. O número de miRs diferencialmente expressos aumentou com a progressão da infecção. Além disso, seis miRs tiveram sua expressão correlacionada à piora na parasitemia e intervalo QTc dos animais: miR-142-3p miR-142-5p, miR-145, miR-146b, miR-149 e miR-21. Análises de correlação realizadas com todos os miRs avaliados ressaltaram este mesmo grupo de miRs entre os mais significativamente correlacionados, além de outros 73 correlacionados com a parasitemia, 67 com o intervalo QTc e 16 com ambos os parâmetros simultaneamente. Nas análises in silico, TNF-alfa e ciclina-D1 foram moléculas nodais recorrentes nas redes criadas com alvos dos miRs diferencialmente expressos em todos os tempos avaliados. Na única rede criada com os miRs correlacionados às alterações na parasitemia e intervalo QTc, TNF-alfa, TGF-beta, Rac1 e Src foram as moléculas nodais. Este trabalho apresenta de maneira inédita o envolvimento dos miRs durante a infecção aguda por T. cruzi, proporcionando novas perspectivas em relação a potenciais ferramentas terapêuticas e prognósticasChagas disease is a chronic illness caused by infection with the protozoan Trypanosoma cruzi (T. cruzi). Its main clinical outcome is the development of chronic Chagas cardiomyopathy (CCC), which affects 30% of the patients. The factors that define the progression to CCC or maintenance in the asymptomatic indeterminate form of the disease are still poorly understood. Several studies have presented changes occurred in the gene and proteomic expression profiles in both acute and chronic phases of experimental and human Chagas disease. Such changes result from regulation established at different stages of gene expression and may be relevant for the disease prognosis. In this context, microRNAs (miRs) may play an important regulatory function. miRs act by association to a target messenger RNA (mRNA), inhibiting translation or degrading the transcript. Thus, our hypothesis is that acute infection by T. cruzi modulates the expression of microRNAs in the myocardium of mice. The miR expression profile was evaluated by qRT-PCR 15, 30 or 45 days after the infection. This profile was sufficient to segregate the samples according to the time of infection. The number of differentially expressed miRs was higher as the infection progressed. Moreover, six miRs had their expression correlated with worsening of parasitaemia and QTc interval: miR-142-3p miR-142- 5p, miR-145, miR-146b, miR-149 and miR-21. Secondary unbiased correlation analyses showed this cluster of miRs among the most significant and other 73 miRs correlated with parasitaemia, 67 with QTc and 16 with both parameters simultaneously. In silico target prediction analyses showed TNF-alfa and cyclin-D1 as recurrent nodal molecules of the networks created with miRs targets from all time points. The network generated with miRs correlated to changes in parasitaemia and QTc interval showed TNF-alfa, TGF-beta, Rac1 and Src as nodal molecules. This work points out for the first time the involvement of miRs in the acute infection by T. cruzi, providing new insights about potential diagnostic and prognostic tool

Interferon-gamma and other inflammatory mediators in cardiomyocyte signaling during Chagas disease cardiomyopathy.

International audienceno abstrac

Induction of IL-12 Production in Human Peripheral Monocytes by Trypanosoma cruzi Is Mediated by Glycosylphosphatidylinositol-Anchored Mucin-Like Glycoproteins and Potentiated by IFN- and CD40-CD40L Interactions.

Submitted by Nuzia Santos ([email protected]) on 2015-03-02T18:01:21Z No. of bitstreams: 1 2014_132.pdf: 1484334 bytes, checksum: d096f23e5a1f9d4cd062bfcebc0ee3c2 (MD5)Approved for entry into archive by Nuzia Santos ([email protected]) on 2015-03-02T18:01:57Z (GMT) No. of bitstreams: 1 2014_132.pdf: 1484334 bytes, checksum: d096f23e5a1f9d4cd062bfcebc0ee3c2 (MD5)Approved for entry into archive by Nuzia Santos ([email protected]) on 2015-03-02T18:05:44Z (GMT) No. of bitstreams: 1 2014_132.pdf: 1484334 bytes, checksum: d096f23e5a1f9d4cd062bfcebc0ee3c2 (MD5)Made available in DSpace on 2015-03-02T18:05:44Z (GMT). No. of bitstreams: 1 2014_132.pdf: 1484334 bytes, checksum: d096f23e5a1f9d4cd062bfcebc0ee3c2 (MD5) Previous issue date: 2014Universidade de São Paulo. Escola de Medicina. Instituto do Coração. Laboratory of Immunology. São Paulo, SP, BrazilUniversidade de São Paulo. Escola de Medicina. Instituto do Coração. Laboratory of Immunology. São Paulo, SP, Brazil/Universidade de São Paulo. Escola de Medicina. Clinca de Imunologia e Alergia. Sao Paulo, SP, Brazil/Institutos Nacionais de Ciência e Tecnologia. Instituto de Investigação em Imunologia. Sao Paulo, SP, BrazilUniversidade de São Paulo. Escola de Medicina. Instituto do Coração. Laboratory of Immunology. São Paulo, SP, Brazil/Universidade de São Paulo. Escola de Medicina. Clinca de Imunologia e Alergia. Sao Paulo, SP, Brazil/Institutos Nacionais de Ciência e Tecnologia. Instituto de Investigação em Imunologia. Sao Paulo, SP, BrazilUniversidade de São Paulo. Escola de Medicina. Instituto do Coração. Laboratory of Immunology. São Paulo, SP, Brazil/Universidade de São Paulo. Escola de Medicina. Clinca de Imunologia e Alergia. Sao Paulo, SP, Brazil/Institutos Nacionais de Ciência e Tecnologia. Instituto de Investigação em Imunologia. Sao Paulo, SP, BrazilUniversidade de São Paulo. Escola de Medicina. Instituto do Coração. Laboratory of Immunology. São Paulo, SP, Brazil/Universidade de São Paulo. Escola de Medicina. Clinca de Imunologia e Alergia. Sao Paulo, SP, Brazil/Institutos Nacionais de Ciência e Tecnologia. Instituto de Investigação em Imunologia. Sao Paulo, SP, BrazilFundação Oswaldo Cruz. Centro de Pesquisa Rene Rachou. Belo Horizonte, MG, Brazil/Universidade Federal de Minas Gerais. Instituto de Ciencias Biologicas.Departamento de Bioquimica e Imunologia. Laboratorio de Imunopatologia. Belo Horizonte, MG, Brazil/University of Massachusetts Medical School. Department of Medicine. Division of Infectious Diseases and Immunology. Worcester, MA, USAHospital Israelita Albert Einstein. Sao Paulo, SP, BrazilUniversidade de São Paulo. Escola de Medicina. Instituto do Coração. Laboratory of Immunology. São Paulo, SP, Brazil/Universidade de São Paulo. Escola de Medicina. Clinca de Imunologia e Alergia. Sao Paulo, SP, Brazil/Institutos Nacionais de Ciência e Tecnologia. Instituto de Investigação em Imunologia. Sao Paulo, SP, BrazilChagas disease, caused by the protozoan parasite Trypanosoma cruzi (T. cruzi), is characterized by immunopathology driven by IFN-γ secreting Th1-like T cells. T. cruzi has a thick coat of mucin-like glycoproteins covering its surface, which plays an important role in parasite invasion and host immunomodulation. It has been extensively described that T. cruzi or its products-like GPI anchors isolated from GPI-anchored mucins from the trypomastigote life cycle stage (tGPI-mucins)-are potent inducers of proinflammatory responses (i.e., cytokines and NO production) by IFN-γ primed murine macrophages. However, little is known about whether T. cruzi or GPI-mucins exert a similar action in human cells. We therefore decided to further investigate the in vitro cytokine production profile from human mononuclear cells from uninfected donors exposed to T. cruzi as well as tGPI-mucins. We observed that both living T. cruzi trypomastigotes and tGPI-mucins are potent inducers of IL-12 by human peripheral blood monocytes and this effect depends on CD40-CD40L interaction and IFN-γ. Our findings suggest that the polarized T1-type cytokine profile seen in T. cruzi infected patients might be a long-term effect of IL-12 production induced by lifelong exposure to T. cruzi tGPI-mucins

Enhancement of experimental cutaneous leishmaniasis by Leishmania extract: identification of a disease-associated antibody specificity

Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2015-07-08T18:21:29Z No. of bitstreams: 1 Silva VMG Enhancement....pdf: 742748 bytes, checksum: 810c79b14459ec853dad04db055bbc58 (MD5)Approved for entry into archive by Ana Maria Fiscina Sampaio ([email protected]) on 2015-07-08T18:32:08Z (GMT) No. of bitstreams: 1 Silva VMG Enhancement....pdf: 742748 bytes, checksum: 810c79b14459ec853dad04db055bbc58 (MD5)Made available in DSpace on 2015-07-08T18:32:08Z (GMT). No. of bitstreams: 1 Silva VMG Enhancement....pdf: 742748 bytes, checksum: 810c79b14459ec853dad04db055bbc58 (MD5) Previous issue date: 2015Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / Universidade Estadual do Sudoeste da Bahia. Departamento de Ciências Biológicas. Jequié, BA, Brasil, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / Escola Bahiana de Medicina e Saúde Pública. Salvador, BA, BrasilBACKGROUND: Both Leishmania braziliensis and Leishmania amazonensis induce cutaneous disease when injected in the skin of BALB/c mice. However, L. amazonensis may also visceralize in that strain of mice, infecting mainly the liver and spleen. In addition, whereas BALB/c mice die with a progressive cutaneous disease when infected by L. amazonensis, the infection by L. braziliensis is spontaneously cured. In a previous work, we have found that intravenous injections of L. amazonensis amastigote extract (LaE) potentiated a L. braziliensis infection in BALB/c mice, and that this infection-promoting activity could be inhibited by the addition of protease inhibitors to the extract. METHODS: In order to detect markers of disease evolution, in the present work we analyzed the specificity of the anti-L. amazonensis antibody response of L. braziliensis-infected BALB/c mice injected intravenously with saline or LaE, supplemented or not with protease inhibitors, by the Western blot technique. RESULTS: IgG1 antibodies recognizing an antigen with apparent molecular weight of 116 kDa were specifically detected in BALB/c mice that had been turned susceptible to L. braziliensis infection by injections of LaE. CONCLUSION: A Th2 immune response (IgG1 antibody-producing) against this 116 kDa antigen, therefore, could be associated with susceptibility to severe Leishmania infection

MicroRNA Transcriptome Profiling in Heart of Trypanosoma cruzi-Infected Mice: Parasitological and Cardiological Outcomes

International audienceChagas disease is caused by the parasite Trypanosoma cruzi, and it begins with a short acute phase characterized by high parasitemia followed by a life-long chronic phase with scarce parasitism. Cardiac involvement is the most prominent manifestation, as 30% of infected subjects will develop abnormal ventricular repolarization with myocarditis, fibrosis and cardiomyocyte hypertrophy by undefined mechanisms. Nevertheless, follow-up studies in chagasic patients, as well as studies with murine models, suggest that the intensity of clinical symptoms and pathophysiological events that occur during the acute phase of disease are associated with the severity of cardiac disease observed during the chronic phase. In the present study we investigated the role of microRNAs (miRNAs) in the disease progression in response to T. cruzi infection, as alterations in miRNA levels are known to be associated with many cardiovascular disorders. We screened 641 rodent miRNAs in heart samples of mice during an acute infection with the Colombiana T.cruzi strain and identified multiple miRNAs significantly altered upon infection. Seventeen miRNAs were found significantly deregulated in all three analyzed time points post infection. Among these, six miRNAs had their expression correlated with clinical parameters relevant to the disease, such as parasitemia and maximal heart rate-corrected QT (QTc) interval. Computational analyses identified that the gene targets for these six miRNAs were involved in networks and signaling pathways related to increased ventricular depolarization and repolarization times, important factors for QTc interval prolongation. The data presented here will guide further studies about the contribution of microRNAs to Chagas heart disease pathogenesis

Integration of miRNA and gene expression profiles suggest a role for miRNAs in the pathobiological processes of acute Trypanosoma cruzi infection

International audienceChagas disease, caused by the parasite Trypanosoma cruzi, is endemic in Latin America. Its acute phase is associated with high parasitism, myocarditis and profound myocardial gene expression changes. A chronic phase ensues where 30% develop severe heart lesions. Mouse models of T. cruzi infection have been used to study heart damage in Chagas disease. The aim of this study was to provide an interactome between miRNAs and their targetome in Chagas heart disease by integrating gene and microRNA expression profiling data from hearts of T. cruzi infected mice. Gene expression profiling revealed enrichment in biological processes and pathways associated with immune response and metabolism. Pathways, functional and upstream regulator analysis of the intersections between predicted targets of differentially expressed microRNAs and differentially expressed mRNAs revealed enrichment in biological processes and pathways such as IFNγ, TNFα, NF-kB signaling signatures, CTL-mediated apoptosis, mitochondrial dysfunction, and Nrf2-modulated antioxidative responses. We also observed enrichment in other key heart disease-related processes like myocarditis, fibrosis, hypertrophy and arrhythmia. Our correlation study suggests that miRNAs may be implicated in the pathophysiological processes taking place the hearts of acutely T. cruzi-infected mice