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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., piRNA biogenesis requires RBP‐9 mediated transcriptional termination at complex piRNA genes.

Methylation of carbon‐5 of cytosines (m5C) is a post‐transcriptional nucleotide modification of RNA found in all kingdoms of life. While individual m5C‐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 m5C in RNA, demonstrating that this modification is non‐essential. Using this genetic tool, we determine the localisation and enzymatic specificity of m5C 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 m5C impacts the decoding of some triplets of these two amino acids, leading to reduced translation efficiency. Upon heat stress, m5C loss leads to ribosome stalling at UUG triplets, the only codon translated by an m5C34‐modified tRNA. This leads to reduced translation efficiency of UUG‐rich transcripts and impaired fertility, suggesting a role of m5C tRNA wobble methylation in the adaptation to higher temperatures., Analysis of worm mutants devoid of m5C RNA modifications uncovers their contribution to efficient Leu/Pro codon translation, and requirement for fertility‐supporting Leu‐UUG decoding at higher temperatures.

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 nematodeCaenorhabditis elegans. rpb-9mutants fail to initiate heritable piRNA-mediated gene silencing. Furthermore, we show that RPB-9 is required to repress two DNA transposon families and a subset of somatic genes in theC. elegansgermline. We provide genetic and biochemical evidence that RPB-9 is required for piRNA biogenesis. We demonstrate that RPB-9 acts to promote transcriptional elongation/termination at endogenous piRNA loci. We conclude that as a part of its rapid evolution the piRNA pathway has co-opted another ancient machinery, this time for high-fidelity transcription.

Methylation of carbon-5 of cytosines (m5C) is a post-transcriptional nucleotide modification of RNA found in all kingdoms of life. While individual m5C-methyltransferases have been studied, the impact of the global cytosine-5 methylome on development, homeostasis and stress remains unknown. Here, usingCaenorhabditis elegans, we generated the first organism devoid of m5C in RNA, demonstrating that this modification is non-essential. We determined the localisation and enzymatic specificity of m5C sites in RNAin vivoand showed that animals devoid of m5C are sensitive to temperature stress. At the molecular level, we showed that loss of m5C specifically impacts decoding of leucine and proline thus reducing the translation efficiency of transcripts enriched in these amino acids. Finally, we found translation of leucine UUG codons to be the most strongly affected upon heat shock, suggesting a role of m5C tRNA wobble methylation in the adaptation to heat stress.

BackgroundFunctional characterisation of the compact genome of the model organism Caenorhabditis elegans remains incomplete despite its sequencing twenty 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.ResultsBy analysing hundreds of published transcriptome datasets, we annotated 3,397 potential lncRNAs including 146 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.ConclusionsOur results highlight that a large section of the non-coding regions of the C. elegans genome remain 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 biological function at either the genomic or the transcript level.

Long noncoding RNAs (lncRNAs) modulate gene expression at the epigenetic, transcriptional, and posttranscriptional levels. Dysregulation of the lncRNA known as myocardial infarction-associated transcript (MIAT) has been associated with myocardial infarction. Chagas disease causes a severe inflammatory dilated chronic cardiomyopathy (CCC). We investigated the role of MIAT in CCC. A whole-transcriptome analysis of heart biopsy specimens and formalin-fixed, paraffin-embedded samples revealed that MIAT was overexpressed in patients with CCC, compared with subjects with noninflammatory dilated cardiomyopathy and controls. These results were confirmed in a mouse model. Results suggest that MIAT is a specific biomarker of CCC.

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 prognósticas Chagas 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 tools

Background 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.

Background/methods Chagas disease is caused by an intracellular parasite, Trypanosoma cruzi , and it is a leading cause of heart failure in Latin America. The main clinical consequence of the infection is the development of a Chronic Chagas disease Cardiomyopathy (CCC), which is characterized by myocarditis, hypertrophy and fibrosis and affects about 30% of infected patients. CCC has a worse prognosis than other cardiomyopathies, like idiopathic dilated cardiomyopathy (DCM). It is well established that myocardial gene expression patterns are altered in CCC, but the molecular mechanisms underlying these differences are not clear. MicroRNAs are recently discovered regulators of gene expression, and are recognized as important factors in heart development and cardiovascular disorders (CD). We analyzed the expression of nine different miRNAs in myocardial tissue samples of CCC patients in comparison to DCM patients and samples from heart transplant donors. Using the results of a cDNA microarray database on CCC and DCM myocardium, signaling networks were built and nodal molecules were identified. Results We observed that five miRNAs were significantly altered in CCC and three in DCM; importantly, three miRNAs were significantly reduced in CCC as compared to DCM. We observed that multiple gene targets of the differentially expressed miRNAs showed a concordant inverse expression in CCC. Significantly, most gene targets and involved networks belong to crucial disease-related signaling pathways. Conclusion These results suggest that miRNAs may play a major role in the regulation of gene expression in CCC pathogenesis, with potential implication as diagnostic and prognostic tools.

Chagas disease, caused by the protozoan parasiteTrypanosoma cruzi(T. cruzi), is characterized by immunopathology driven by IFN-γsecreting Th1-like T cells.T. cruzihas 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 thatT. cruzior 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-γprimed murine macrophages. However, little is known about whetherT. cruzior GPI-mucins exert a similar action in human cells. We therefore decided to further investigate thein vitrocytokine production profile from human mononuclear cells from uninfected donors exposed toT. cruzias well as tGPI-mucins. We observed that both livingT. cruzitrypomastigotes 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 inT. cruziinfected patients might be a long-term effect of IL-12 production induced by lifelong exposure toT. cruzitGPI-mucins.