Selective Loading and Variations in the miRNA Profile of Extracellular Vesicles from Endothelial-like Cells Cultivated under Normoxia and Hypoxia.

Endothelial-like cells may be obtained from CD133+ mononuclear cells isolated from human umbilical cord blood (hUCB) and expanded using endothelial-inducing medium (E-CD133 cells). Their use in regenerative medicine has been explored by the potential not only to form vessels but also by the secretion of bioactive elements. Extracellular vesicles (EVs) are prominent messengers of this paracrine activity, transporting bioactive molecules that may guide cellular response under different conditions. Using RNA-Seq, we characterized the miRNA content of EVs derived from E-CD133 cells cultivated under normoxia (N-EVs) and hypoxia (H-EVs) and observed that changing the O2 status led to variations in the selective loading of miRNAs in the EVs. In silico analysis showed that among the targets of differentially loaded miRNAs, there are transcripts involved in pathways related to cell growth and survival, such as FoxO and HIF-1 pathways. The data obtained reinforce the pro-regenerative potential of EVs obtained from E-CD133 cells and shows that fine tuning of their properties may be regulated by culture conditions.

The Nuclear RNA-binding Protein RBSR1 Interactome in Trypanosoma cruzi.

Trypanosoma cruzi, the etiological agent of Chagas disease, has been widely studied, reflecting both its medical importance and the particular features that make this pathogen an attractive model for basic biological studies. The repression of transcripts by messenger ribonucleoprotein (mRNP) complexes is an important pathway of post-transcriptional regulation in eukaryotes, including T. cruzi. RBSR1 is a serine-arginine (SR)-rich RNA-binding protein (RBP) in T. cruzi that contains one RNA-recognition motif (RRM); this protein has a primarily nuclear localization and is developmentally regulated, not being detected in metacyclic trypomastigotes. RBSR1 interacts with other RBPs, such as UBP1 and UBP2, and the nuclear SR-protein TRRM1. Phylogenetic analysis indicated that RBSR1 is orthologous to the human splicing factor SRSF7, what might indicate its possible involvement in pre-RNA processing. Accordingly, ribonomics data showed the enrichment of snoRNAs and snRNAs in the RBSR1 immunoprecipiatation complex, hence reinforcing the supposition that this protein might be involved in RNA processing in the nucleus.

Extracellular Vesicles in Viral Infections: Two Sides of the Same Coin?

Extracellular vesicles are small membrane structures containing proteins and nucleic acids that are gaining a lot of attention lately. They are produced by most cells and can be detected in several body fluids, having a huge potential in therapeutic and diagnostic approaches. EVs produced by infected cells usually have a molecular signature that is very distinct from healthy cells. For intracellular pathogens like viruses, EVs can have an even more complex function, since the viral biogenesis pathway can overlap with EV pathways in several ways, generating a continuum of particles, like naked virions, EVs containing infective viral genomes and quasi-enveloped viruses, besides the classical complete viral particles that are secreted to the extracellular space. Those particles can act in recipient cells in different ways. Besides being directly infective, they also can prime neighbor cells rendering them more susceptible to infection, block antiviral responses and deliver isolated viral molecules. On the other hand, they can trigger antiviral responses and cytokine secretion even in uninfected cells near the infection site, helping to fight the infection and protect other cells from the virus. This protective response can also backfire, when a massive inflammation facilitated by those EVs can be responsible for bad clinical outcomes. EVs can help or harm the antiviral response, and sometimes both mechanisms are observed in infections by the same virus. Since those pathways are intrinsically interlinked, understand the role of EVs during viral infections is crucial to comprehend viral mechanisms and respond better to emerging viral diseases.

Golgi Reassembly and Stacking Protein (GRASP) Participates in Vesicle-Mediated RNA Export in Cryptococcus Neoformans.

Golgi reassembly and stacking protein (GRASP) is required for polysaccharide secretion and virulence in Cryptococcus neoformans. In fungal species, extracellular vesicles (EVs) participate in the export of polysaccharides, proteins and RNA. In the present work, we investigated if EV-mediated RNA export is functionally connected with GRASP in C. neoformans using a graspΔ mutant. Since GRASP-mediated unconventional secretion involves autophagosome formation in yeast, we included the atg7Δ mutant with defective autophagic mechanisms in our analysis. All fungal strains exported EVs but deletion of GRASP or ATG7 profoundly affected vesicular dimensions. The mRNA content of the graspΔ EVs differed substantially from that of the other two strains. The transcripts associated to the endoplasmic reticulum were highly abundant transcripts in graspΔ EVs. Among non-coding RNAs (ncRNAs), tRNA fragments were the most abundant in both mutant EVs but graspΔ EVs alone concentrated 22 exclusive sequences. In general, our results showed that the EV RNA content from atg7Δ and WT were more related than the RNA content of graspΔ, suggesting that GRASP, but not the autophagy regulator Atg7, is involved in the EV export of RNA. This is a previously unknown function for a key regulator of unconventional secretion in eukaryotic cells.

Caracterização da proteína TcRRM2 de Trypanosoma cruzi: envolvimento na regulação da expressão gênica e resposta a estresse

Trypanosoma cruzi, o agente etiológico da doença de Chagas, é um organismoamplamente estudado devido a sua importância médica e também por possuircaracterísticas peculiares que o tornam um bom modelo de estudo para questõesbiológicas básicas. A repressão de RNAs mensageiros em grânulos citoplasmáticoscompostos de complexos mRNA-proteína (mRNPs) é uma importante via de regulaçãopós transcricional em eucariotos e, recentemente, foi demonstrado que grânulos deRNA estão presentes em T. cruzi. Alguns ortólogos de proteínas humanas envolvidasem mecanismos de regulação foram encontrados nestas estruturas e caracterizados,mas a função e composição de grânulos de mRNA neste modelo experimentalpermanecem desconhecidas. Em humanos e outros eucariotos, condições de estressecomo calor, radiação UV, presença de agentes citotóxicos ou deficiência de glucosepodem induzir a formação de grânulos de estresse, um tipo de estrutura citoplasmáticaenvolvida em repressão, separação e armazenamento de mRNA durante condiçõesadversas. Foram encontradas, em banco de dados de T. cruzi, três sequências deproteínas que possuem certa similaridade estrutural com as proteínas humanas TIA1 eTIAR. Os genes correspondentes foram clonados utilizando-se a tecnologia Gateway®,para a obtenção de proteínas recombinantes. As proteínas purificadas foram utilizadaspara produzir anticorpos policlonais em camundongos, os quais puderam elucidar alocalização celular e padrões de expressão destas proteínas durante o ciclo de vida doparasita. A caracterização destas proteínas pode ajudar a elucidar melhor osmecanismos de regulação pós transcricional em T. cruzi.