The protozoan parasite Trypanosoma cruzi has a complex life cycle characterized by intracellular and extracellular forms alternating between invertebrate and mammals. To cope with these changing environments, T. cruzi undergoes rapid changes in gene expression, which are achieved essentially at the posttranscriptional level. At present, expanding families of small RNAs are recognized as key players in novel forms of posttranscriptional gene regulation in most eukaryotes. However, T. cruzi lacks canonical small RNA pathways. In a recent work, we reported the presence of alternate small RNA pathways in T. cruzi mainly represented by a homogeneous population of tRNA-derived small RNAs (tsRNAs). In T. cruzi epimastigotes submitted to nutrient starvation, tsRNAs colocalized with an argonaute protein distinctive of trypanosomatids (TcPIWI-tryp) and were recruited to particular cytoplasmic granules. Using epifluorescence and electronic microscopy, we observed that tsRNAs and the TcPIWI-tryp protein were recruited mainly to reservosomes and other intracellular vesicles including endosome-like vesicles and vesicular structures resembling the Golgi complex. These data suggested that, in T. cruzi, tsRNA biogenesis is probably part of endocytic/exocytic routes. We also demonstrated that epimastigotes submitted to nutrient starvation shed high levels of vesicles to the extracellular medium, which carry small tRNAs and TcPIWI-tryp proteins as cargo. At least a fraction of extracellular vesicle cargo was transferred between parasites and to mammalian susceptible cells. Our data afford experimental evidence, indicating that extracellular vesicles shed by T. cruzi promote not only life cycle transition of epimastigotes to trypomastigote forms but also infection susceptibility of mammalian cells.

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With the advent of new and improved high-throughput sequencing technologies in the last few years, a growing number of novel classes of small RNA, other than miRNAs or siRNA, has emerged, which appear as new actors in gene expression regulation. tRNA-derived small RNAs represent one of these novel members that are, surprisingly, among the most conserved class of small RNAs throughout evolution. They could represent the most primitive small RNA pathways from which the well-known canonical RNA silencing pathways reported in higher eukaryotes evolved. This review aims to make a compilation of the most relevant research literature in this field with the purpose of shedding light on the relation of these primitive tRNA-derived molecules with the gene silencing machinery.

At present, noncoding small RNAs are recognized as key players in novel forms of posttranscriptional gene regulation in most eukaryotes. However, canonical small RNA pathways seem to be lost or excessively simplified in some unicellular organisms including Trypanosoma cruzi which lack functional RNAi pathways. Recently, we reported the presence of alternate small RNA pathways in T. cruzi mainly represented by homogeneous populations of tRNA- and rRNA-derived small RNAs, which are secreted to the extracellular medium included in extracellular vesicles. Extracellular vesicle cargo could be delivered to other parasites and to mammalian susceptible cells promoting metacyclogenesis and conferring susceptibility to infection, respectively. Here we analyzed the changes in gene expression of host HeLa cells induced by extracellular vesicles from T. cruzi. As assessed by microarray assays a large set of genes in HeLa cells were differentially expressed upon incorporation of T. cruzi-derived extracellular vesicles. The elicited response modified mainly host cell cytoskeleton, extracellular matrix, and immune responses pathways. Some genes were also modified by the most abundant tRNA-derived small RNAs included in extracellular vesicles. These data suggest that microvesicles secreted by T. cruzi could be relevant players in early events of the T. cruzi host cell interplay.

A common feature seen in acute infections is a severe atrophy of the thymus. This occurs in the murine model of acute Chagas disease. Moreover, in thymuses from Trypanosoma cruzi acutely infected mice, thymocytes exhibit an increase in the density of fibronectin and laminin integrin-type receptors, with an increase in migratory response ex vivo. Thymic epithelial cells (TEC) play a major role in the intrathymic T cell differentiation. To date, the consequences of molecular changes promoted by parasite infection upon thymus have not been elucidated. Considering the importance of microRNA for gene expression regulation, 85 microRNAs (mRNAs) were analyzed in TEC from T. cruzi acutely infected mice. The infection significantly modulated 29 miRNAs and modulation of 9 was also dependent whether TEC sorted out from the thymus exhibited cortical or medullary phenotype. In silico analysis revealed that these miRNAs may control target mRNAs known to be responsible for chemotaxis, cell adhesion, and cell death. Considering that we sorted TEC in the initial phase of thymocyte loss, it is conceivable that changes in TEC miRNA expression profile are functionally related to thymic atrophy, providing new clues to better understanding the mechanisms of the thymic involution seen in experimental Chagas disease.

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There is increasing interest among cancer researchers in the study of Piwi-interacting RNAs (piRNAs), a group of small RNAs important for maintaining genome stability in the germline. Aberrant expression of piRNAs in cancer could imply an involvement of these regulatory RNAs in neoplastic transformation. On top of that, it could enable early cancer diagnosis based on RNA analysis in liquid biopsies, as piRNAs are not expected to widely circulate in the bloodstream of healthy individuals. Indeed, it has recently been shown that serum piR-54265 allows for excellent discrimination between colorectal cancer patients and healthy controls. However, we have also shown that most somatic piRNAs reported to date in mammals are actually fragments of other noncoding RNAs. Herein, we show that reports positioning piR-54265 as a noninvasive biomarker for colorectal cancer were actually measuring variations in the levels of a full-length (72 nt) small nucleolar RNA in serum. This should place a cautionary note for future research in somatic and cancer-specific piRNAs. We deeply encourage this line of research but discuss proper ways to identify somatic piRNAs without the interference of erroneous entries contained in piRNA databases. We also introduce the concept of miscellaneous-piRNAs (m-piRNAs) to distinguish between canonical piRNAs and other small RNAs circumstantially associated with PIWI proteins in somatic cells.