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Trypanosoma cruzi is the etiological agent for Chagas disease, a neglected parasitic disease in Latin America. Gene transcription control governs the eukaryotic cell replication but is absent in trypanosomatids; thus, it must be replaced by posttranscriptional regulatory events. We investigated the entrance into the T. cruzi replicative cycle using ribosome profiling and proteomics on G1/S epimastigote cultures synchronized with hydroxyurea. We identified 1,784 translationally regulated genes (change > 2, false-discovery rate [FDR] 1.5, FDR

BackgroundDefining the cellular mechanisms that drive Alzheimer’s disease (AD) pathogenesis and progression will be aided by studies defining how gene expression patterns change during pre-symptomatic AD and the ensuing periods of steadily declining cognition. Previous studies have emphasized changes in transcriptional regulation, but not translational regulation, leaving the ultimate results of gene expression alterations relatively unexplored in the context of AD.ObjectiveTo identify genes whose expression might be regulated at the transcriptional, and especially at the translational levels in AD, we analyzed gene expression in cerebral cortex of two AD model mouse strains, CVN (APPSwDI;NOS2-/-) and Tg2576 (APPSw), and their companion wild type (WT) strains at 6 months of age by tandem RNA-Seq and Ribo-Seq (ribosome profiling).MethodsIdentical starting pools of bulk RNA were used for RNA-Seq and Ribo-Seq. Differential gene expression analysis was performed at the transcriptional and translational levels separately, and also at the translational efficiency level. Regulated genes were functionally evaluated by gene ontology tools.ResultsCompared to WT mice, AD model mice had similar levels of transcriptional regulation, but displayed differences in translational regulation. A specific microglial signature associated with early stages of Aβ accumulation was up-regulated at both transcriptome and translatome levels in CVN mice. Although the two mice strains did not share many regulated genes, they showed common regulated pathways related to APP metabolism associated with neurotoxicity and neuroprotection.ConclusionThis work represents the first genome-wide study of brain translational regulation in animal models of AD, and provides evidence of a tight and early translational regulation of gene expression controlling the balance between neuroprotective and neurodegenerative processes in brain.

Motivation Nanopore sequencing technologies are rapidly gaining popularity, in part, due to the massive amounts of genomic data they produce in short periods of time (up to 8.5 TB of data in Results We introduce RENANO, a reference-based lossless data compressor specifically tailored to FASTQ files generated with nanopore sequencing technologies. RENANO improves on its predecessor ENANO, currently the state of the art, by providing a more efficient base call sequence compression component. Two compression algorithms are introduced, corresponding to the following scenarios: (1) a reference genome is available without cost to both the compressor and the decompressor and (2) the reference genome is available only on the compressor side, and a compacted version of the reference is included in the compressed file. We compare the compression performance of RENANO against ENANO on several publicly available nanopore datasets. RENANO improves the base call sequences compression of ENANO by 39.8% in scenario (1), and by 33.5% in scenario (2), on average, over all the datasets. As for total file compression, the average improvements are 12.7% and 10.6%, respectively. We also show that RENANO consistently outperforms the recent general-purpose genomic compressor Genozip. Availability and implementation RENANO is freely available for download at: https://github.com/guilledufort/RENANO. Supplementary information Supplementary data are available at Bioinformatics online.

Axonal protein synthesis has been shown to play a role in developmental and regenerative growth, as well as in the maintenance of the axoplasm in a steady state. Recent studies have begun to identify the mRNAs localized in axons, which could be translated locally under different conditions. Despite that by now hundreds or thousands of mRNAs have been shown to be localized into the axonal compartment of cultured neurons in vitro, knowledge of which mRNAs are localized in mature myelinated axons is quite limited. With the purpose of characterizing the transcriptome of mature myelinated motor axons of peripheral nervous systems, we modified the axon microdissection method devised by Koenig, enabling the isolation of the axoplasm RNA to perform RNA-seq analysis. The transcriptome analysis indicates that the number of RNAs detected in mature axons is lower in comparison with in vitro data, depleted of glial markers, and enriched in neuronal markers. The mature myelinated axons are enriched for mRNAs related to cytoskeleton, translation, and oxidative phosphorylation. Moreover, it was possible to define core genes present in axons when comparing our data with transcriptomic data of axons grown in different conditions. This work provides evidence that axon microdissection is a valuable method to obtain genome-wide data from mature and myelinated axons of the peripheral nervous system, and could be especially useful for the study of axonal involvement in neurodegenerative pathologies of motor neurons such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophies (SMA).

Cell and developmental processes are complex, and profoundly dependent on spatial relationships that change over time. Innovative educational or teaching strategies are always needed to foster deep comprehension of these processes and their dynamic features. However, laboratory exercises in cell and developmental biology at the undergraduate level do not often take into account the time dimension. In this article, we provide a laboratory exercise focused in cell migration, aiming to stimulate thinking in time and space dimensions through a simplification of more complex processes occurring in cell or developmental biology. The use of open-source tools for the analysis, as well as the whole package of raw results (available at http://github.com/danielprieto/keratocyte) make it suitable for its implementation in courses with very diverse budgets. Aiming to facilitate the student's transition from science-students to science-practitioners we propose an exercise of scientific thinking, and an evaluation method. This in turn is communicated here to facilitate the finding of common caveats and weaknesses in the process of producing simple scientific communications describing the results achieved. © 2017 by The International Union of Biochemistry and Molecular Biology, 45(6):475-482, 2017.

vtRNA2-1 is a vault RNA initially classified as microRNA precursor hsa-mir-886 and recently proposed as &ldquo, nc886&rdquo, a new type of non-coding RNA involved in cancer progression acting as an oncogene and tumor suppressor gene in different tissues. We have shown that vtRNA2-1/nc886 is epigenetically repressed in neoplastic cells, increasing cell proliferation and invasion in prostate tissue. Here we investigate the ability of vtRNA2-1/nc886 to produce small-RNAs and their biological effect in prostate cells. The interrogation of public small-RNA transcriptomes of prostate and other tissues uncovered two small RNAs, snc886-3p and snc886-5p, derived from vtRNA2-1/nc886 (previously hsa-miR-886-3p and hsa-miR-886-5p). Re-analysis of PAR-CLIP and knockout of microRNA biogenesis enzymes data showed that these small RNAs are products of DICER, independent of DROSHA, and associate with Argonaute proteins, satisfying microRNA attributes. In addition, the overexpression of snc886-3p provokes the downregulation of mRNAs bearing sequences complementary to its &ldquo, seed&rdquo, in their 3&prime, UTRs. Microarray and in vitro functional assays in DU145, LNCaP and PC3 cell lines revealed that snc886-3p reduced cell cycle progression and increases apoptosis, like its precursor vtRNA2-1/nc886. Finally, we found a list of direct candidate targets genes of snc886-3p upregulated and associated with disease condition and progression in PRAD-TCGA data. Overall, our findings suggest that vtRNA2-1/nc886 and its processed product snc886-3p are synthesized in prostate cells, exerting a tumor suppressor action.

Programmed cell death 4 (PDCD4) protein is a tumor suppressor that inhibits translation through the mTOR-dependent initiation factor EIF4A, but its functional role and mRNA targets in neurons remain largely unknown. Our work identified that PDCD4 is highly expressed in axons and dendrites of CNS and PNS neurons. Using loss- and gain-of-function experiments in cortical and dorsal root ganglia primary neurons, we demonstrated the capacity of PDCD4 to negatively control axonal growth. To explore PDCD4 transcriptome and translatome targets, we used Ribo-seq and uncovered a list of potential targets with known functions as axon/neurite outgrowth regulators. In addition, we observed that PDCD4 can be locally synthesized in adult axons in vivo, and its levels decrease at the site of peripheral nerve injury and before nerve regeneration. Overall, our findings demonstrate that PDCD4 can act as a new regulator of axonal growth via the selective control of translation, providing a target mechanism for axon regeneration and neuronal plasticity processes in neurons.

The discovery of a large number of long noncoding RNAs (lncRNAs), and the finding that they may play key roles in different biological processes, have started to provide a new perspective in the understanding of gene regulation. It has been shown that the testes express the highest amount of lncRNAs among different vertebrate tissues. However, although some studies have addressed the characterization of lncRNAs along spermatogenesis, an exhaustive analysis of the differential expression of lncRNAs at its different stages is still lacking. Here, we present the results for lncRNA transcriptome profiling along mouse spermatogenesis, employing highly pure flow sorted spermatogenic stage-specific cell populations, strand-specific RNAseq, and a combination of up-to-date bioinformatic pipelines for analysis. We found that the vast majority of testicular lncRNA genes are expressed at post-meiotic stages (i.e. spermiogenesis), which are characterized by extensive post-transcriptional regulation. LncRNAs at different spermatogenic stages shared common traits in terms of transcript length, exon number, and biotypes. Most lncRNAs were lincRNAs, followed by a high representation of antisense (AS) lncRNAs. Co-expression analyses showed a high correlation along the different spermatogenic stage transitions between the expression patterns of AS lncRNAs and their overlapping protein-coding genes, raising possible clues about lncRNA-related regulatory mechanisms. Interestingly, we observed the co-localization of an AS lncRNA and its host sense mRNA in the chromatoid body, a round spermatids-specific organelle that has been proposed as a reservoir of RNA-related regulatory machinery. An additional, intriguing observation is the almost complete lack of detectable expression for Y-linked testicular lncRNAs, despite that a high number of lncRNA genes are annotated for this chromosome.

The protein Deleted in Breast Cancer-1 is a regulator of several transcription factors and epigenetic regulators, including HDAC3, Rev-erb-alpha, PARP1 and SIRT1. It is well known that DBC1 regulates its targets, including SIRT1, by protein-protein interaction. However, little is known about how DBC1 biological activity is regulated. In this work, we show that in quiescent cells DBC1 is proteolytically cleaved, producing a protein (DN-DBC1) that misses the S1-like domain and no longer binds to SIRT1. DN-DBC1 is also found in vivo in mouse and human tissues. Interestingly, DN-DBC1 is cleared once quiescent cells re-enter to the cell cycle. Using a model of liver regeneration after partial hepatectomy, we found that DN-DBC1 is down-regulated in vivo during regeneration. In fact, WT mice show a decrease in SIRT1 activity during liver regeneration, coincidentally with DN-DBC1 downregulation and the appearance of full length DBC1. This effect on SIRT1 activity was not observed in DBC1 KO mice. Finally, we found that DBC1 KO mice have altered cell cycle progression and liver regeneration after partial hepatectomy, suggesting that DBC1/DN-DBC1 transitions play a role in normal cell cycle progression in vivo after cells leave quiescence. We propose that quiescent cells express DN-DBC1, which either replaces or coexist with the full-length protein, and that restoring of DBC1 is required for normal cell cycle progression in vitro and in vivo. Our results describe for the first time in vivo a naturally occurring form of DBC1, which does not bind SIRT1 and is dynamically regulated, thus contributing to redefine the knowledge about its function.

Differences in social status are often mediated by agonistic encounters between competitors. Robust literature has examined social status-dependent brain gene expression profiles across vertebrates, yet social status and reproductive state are often confounded. It has therefore been challenging to identify the neuromolecular mechanisms underlying social status independent of reproductive state. Weakly electric fish, Gymnotus omarorum, display territorial aggression and social dominance independent of reproductive state. We use wild-derived G. omarorum males to conduct a transcriptomic analysis of non-breeding social dominance relationships. After allowing paired rivals to establish a dominance hierarchy, we profiled the transcriptomes of brain sections containing the preoptic area (region involved in regulating aggressive behaviour) in dominant and subordinate individuals. We identified 16 differentially expressed genes (FDR G. omarorum as a powerful model system for understanding the neuromolecular bases of social status independent of reproductive state.

Early stages in tumor development involve growth in confined spaces, where oxygen diffusion is limited and metabolic waste products accumulate. This hostile microenvironment imposes strong selective pressures on tumor cells, leading eventually to the survival and expansion of aggressive subclones that condition further tumor evolution. To model features of this microenvironment in vitro, a diffusional barrier can be introduced in the form of a coverslip placed on top of cells, a method termed coverslip hypoxia. Using a variant of this method, with larger volume between coverslip and cells and with oxygen diffusion occurring only through a small hole in the center of the coverslip, we have visualized alterations in LNCaP tumor cells as a function of their distance to the oxygen source at the center. We observed remarkable morphological changes in LNCaP cells as the distance from the center increases, with cells becoming highly spread, displaying dynamic membrane protrusions and occasionally adopting a migratory phenotype. Concomitantly, cells farther from the center displayed marked increases in the hypoxia marker hypoxyprobe, whereas extracellular pH decreased in the same direction. Cells with altered morphology displayed prominent increases in fibrillar actin, as well as swollen mitochondria with distorted cristae and accumulation of neutral lipid‐containing intracellular vesicles. These results show that an in vitro microenvironment that models diffusional barriers encountered by tumors in situ can have profound effects on tumor cells. The coverslip hypoxia variant we describe can be used to characterize in vitro the response of tumor cells to environmental conditions that play crucial roles in early tumor development.

Prostate cancer is a major health problem worldwide due to its high incidence morbidity and mortality. There is currently a need of improved biomarkers, capable to distinguish mild versus aggressive forms of the disease, and thus guide therapeutic decisions. Although miRNAs deregulated in cancer represent exciting candidates as biomarkers, its scientific literature is frequently fragmented in dispersed studies. This problem is aggravated for miRNAs belonging to miRNA gene clusters with shared target genes. The miRNA cluster composed by hsa-mir-130b and hsa-mir-301b precursors was recently involved in prostate cancer pathogenesis, yet different studies assigned it opposite effects on the disease. We sought to elucidate the role of the human miR-130b/301b miRNA cluster in prostate cancer through a comprehensive data analysis of most published clinical cohorts. We interrogated methylomes, transcriptomes and patient clinical data, unifying previous reports and adding original analysis using the largest available cohort (TCGA-PRAD). We found that hsa-miR-130b-3p and hsa-miR-301b-3p are upregulated in neoplastic vs normal prostate tissue, as well as in metastatic vs primary sites. However, this increase in expression is not due to a decrease of the global DNA methylation of the genes in prostate tissues, as the promoter of the gene remains lowly methylated in normal and neoplastic tissue. A comparison of the levels of human miR-130b/301b and all the clinical variables reported for the major available cohorts, yielded positive correlations with malignance, specifically significant for T-stage, residual tumor status and primary therapy outcome. The assessment of the correlations between the hsa-miR-130b-3p and hsa-miR-301b-3p and candidate target genes in clinical samples, supports their repression of tumor suppressor genes in prostate cancer. Altogether, these results favor an oncogenic role of miR-130b/301b cluster in prostate cancer. Electronic supplementary material The online version of this article (10.1186/s40164-018-0102-0) contains supplementary material, which is available to authorized users.

Protein translation is a key step in gene expression. The development of Ribosome Profiling has allowed the global analysis of this process at sub-codon resolution. In the last years the method has been applied to several models ranging from bacteria to mammalian cells yielding a surprising amount of insight on the mechanism and the regulation of translation. In this review we describe the key aspects of the experimental protocol and comment on the main conclusions raised in different models. Keywords: Ribo-seq, Translation, Translatome, Transcriptome, Ribosome profiling

Background Nc886 is a 102 bp non-coding RNA transcript initially classified as a microRNA precursor (Pre-miR-886), later as a divergent homologue of the vault RNAs (vtRNA 2–1) and more recently as a novel type of RNA (nc886). Although nc886/vtRNA2–1/Pre-miR-886 identity is still controversial, it was shown to be epigenetically controlled, presenting both tumor suppressor and oncogenic function in different cancers. Here, we study for the first time the role of nc886 in prostate cancer. Methods Nc886 promoter methylation status and its correlation with patient clinical parameters or DNMTs levels were evaluated in TCGA and specific GEO prostate tissue datasets. Nc886 level was measured by RT-qPCR to compare normal/neoplastic prostate cells from radical prostatectomies and cell lines, and to assess nc886 response to demethylating agents. The effect of nc886 recovery in cell proliferation (in vitro and in vivo) and invasion (in vitro) was evaluated using lentiviral transduced DU145 and LNCaP cell lines. The association between the expression of nc886 and selected genes was analyzed in the TCGA-PRAD cohort. Results Nc886 promoter methylation increases in tumor vs. normal prostate tissue, as well as in metastatic vs. normal prostate tissue. Additionally, nc886 promoter methylation correlates with prostate cancer clinical staging, including biochemical recurrence, Clinical T-value and Gleason score. Nc886 transcript is downregulated in tumor vs. normal tissue -in agreement with its promoter methylation status- and increases upon demethylating treatment. In functional studies, the overexpression of nc886 in the LNCaP and DU145 cell line leads to a decreased in vitro cell proliferation and invasion, as well as a reduced in vivo cell growth in NUDE-mice tumor xenografts. Finally, nc886 expression associates with the prostate cancer cell cycle progression gene signature in TCGA-PRAD. Conclusions Our data suggest a tumor suppressor role for nc886 in the prostate, whose expression is epigenetically silenced in cancer leading to an increase in cell proliferation and invasion. Nc886 might hold clinical value in prostate cancer due to its association with clinical parameters and with a clinically validated gene signature. Electronic supplementary material The online version of this article (10.1186/s12885-018-4049-7) contains supplementary material, which is available to authorized users.

BackgroundPoly-ADP-ribose (PAR) is a polymer synthesized by poly-ADP-ribose polymerases (PARPs) as a postranslational protein modification and catabolized mainly by poly-ADP-ribose glycohydrolase (PARG). In spite of the existence of cytoplasmic PARPs and PARG, research has been focused on nuclear PARPs and PAR, demonstrating roles in the maintenance of chromatin architecture and the participation in DNA damage responses and transcriptional regulation. We have recently detected non-nuclear PAR structurally and functionally associated to the E-cadherin richzonula adherensand the actin cytoskeleton of VERO epithelial cells. Myelinating Schwann cells (SC) are stabilized by E-cadherin rich autotypicadherens junctions (AJ). We wondered whether PAR would map to these regions. Besides, we have demonstrated an altered microfilament pattern in peripheral nerves of Trembler-J (Tr-J) model of CMT1-E. We hypothesized that cytoplasmic PAR would accompany such modified F-actin pattern.MethodsWild-type (WT) and Tr-J mice sciatic nerves cryosections were subjected to immunohistofluorescence with anti-PAR antibodies (including antibody validation), F-actin detection with a phalloidin probe and DAPI/DNA counterstaining. Confocal image stacks were subjected to a colocalization highlighter and to semi-quantitative image analysis.ResultsWe have shown for the first time the presence of PAR in sciatic nerves. Cytoplasmic PAR colocalized with F-actin at non-compact myelin regions in WT nerves. Moreover, in Tr-J, cytoplasmic PAR was augmented in close correlation with actin. In addition, nuclear PAR was detected in WT SC and was moderately increased in Tr-J SC.DiscussionThe presence of PAR associated to non-compact myelin regions (which constitute E-cadherin rich autotypicAJ/actin anchorage regions) and the co-alterations experienced by PAR and the actin cytoskeleton in epithelium and nerves, suggest that PAR may be a constitutive component ofAJ/actin anchorage regions. Is PAR stabilizing theAJ-actin complexes? This question has strong implications in structural cell biology and cell signaling networks. Moreover, if PAR played a stabilizing role, such stabilization could participate in the physiological control of axonal branching. PARP and PAR alterations exist in several neurodegenerative pathologies including Alzheimer’s, Parkinson’s and Hungtington’s diseases. Conversely, PARP inhibition decreases PAR and promotes neurite outgrowth in cortical neuronsin vitro. Coherently, the PARP inhibitor XAV939 improves myelinationin vitro,ex vivoandin vivo. Until now such results have been interpreted in terms of nuclear PARP activity. Our results indicate for the first time the presence of PARylation in peripheral nerve fibers, in a healthy environment. Besides, we have evidenced a PARylation increase in Tr-J, suggesting that the involvement of cytoplasmic PARPs and PARylation in normal and neurodegenerative conditions should be re-evaluated.

A substantial number of studies over a period of four decades have indicated that axons contain mRNAs and ribosomes, and are metabolically active in synthesizing proteins locally. For the most part, little attention has been paid to these findings until recently when the concept of targeting of specific mRNAs and translation in subcellular domains in polarized cells emerged to contribute to the likelihood and acceptance of mRNA targeting to axons as well. Trans-acting factor proteins bind to cis-acting sequences in the untranslated region of mRNAs integrated in ribonucleoprotein (RNPs) complexes determine its targeting in neurons. In vitro studies in immature axons have shown that molecular motors proteins (kinesins and myosins) associate to RNPs suggesting they would participate in its transport to growth cones. Tau and actin mRNAs are transported as RNPs, and targeted to axons as well as ribosomes. Periaxoplasmic ribosomal plaques (PARPs), which are systematically distributed discrete peripheral ribosome-containing, actin-rich formations in myelinated axons, also are enriched with actin and myosin Va mRNAs and additional regulatory proteins. The localization of mRNAs in PARPs probably means that PARPs are local centers of translational activity, and that these domains are the final destination in the axon compartment for targeted macromolecular traffic originating in the cell body. The role of glial cells as a potentially complementary source of axonal mRNAs and ribosomes is discussed in light of early reports and recent ultrastructural observations related to the possibility of glial-axon trans-endocytosis.

In the protozoan parasite Trypanosoma cruzi, as in other trypanosomatids, transcription of protein coding genes occurs in a constitutive fashion, producing large polycistronic transcription units. These units are composed of non-functionally related genes which are pervasively processed to yield each mRNA. Therefore, post-transcriptional processes are crucial to regulate gene expression. Considering that nuclear compartmentalization could contribute to gene expression regulation, we comparatively studied the nuclear, cytoplasmic and whole cell transcriptomes of the non-infective epimastigote stage of T. cruzi, using RNA-Seq. We found that the cytoplasmic transcriptome tightly correlates with the whole cell transcriptome and both equally correlate with the proteome. Nonetheless, 1,200 transcripts showed differential abundance between the nuclear and cytoplasmic fractions. For the genes with transcript content augmented in the nucleus, significant structural and compositional differences were found. The analysis of the reported epimastigote translatome and proteome, revealed scarce ribosome footprints and encoded proteins for them. Ontology analyses unveiled that many of these genes are distinctive of other parasite life-cycle stages. Finally, the relocalization of transcript abundance in the metacyclic trypomastigote infective stage was confirmed for specific genes. While gene expression is strongly dependent on transcript steady-state level, we here highlight the importance of the distribution of transcripts abundance between compartments in T. cruzi. Particularly, we show that nuclear compartmentation is playing an active role in the developmental stage determination preventing off-stage expression.

Commitment of adult stem cells involves the activation of specific gene networks regulated from transcription to protein synthesis. Here, we used ribosome profiling to identify mRNAs regulated at the translational level, through both differential association to polysomes and modulation of their translational rates. We observed that translational regulation during the differentiation of human adipose-derived stromal cells (hASCs, also known as adipose-derived mesenchymal stem cells), a subset of which are stem cells, to adipocytes was a major regulatory event. hASCs showed a significant reduction of whole protein synthesis after adipogenic induction and a downregulation of the expression and translational efficiency of ribosomal proteins. Additionally, focal adhesion and cytoskeletal proteins were downregulated at the translational level. This negative regulation of the essential biological functions of hASCs resulted in a reduction in cell size and the potential of hASCs to migrate. We analyzed whether the inactivation of key translation initiation factors was involved in this observed major repression of translation. We showed that there was an increase in the hypo phosphorylated forms of 4E-BP1, a negative regulator of translation, during early adipogenesis. Our results showed that extensive translational regulation occurred during the early stage of the adipogenic differentiation of hASCs. Fil: Marcon, Bruna H.. Fundación Oswaldo Cruz; Brasil Fil: Holetz, Fabíola B.. Fundación Oswaldo Cruz; Brasil Fil: Eastman, Guillermo. Instituto de Investigaciones Biológicas "Clemente Estable"; Uruguay Fil: Origa Alves, Ana Carolina. Fundación Oswaldo Cruz; Brasil Fil: Amorós, Mariana Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: de Aguiar, Alessandra Melo. Fundación Oswaldo Cruz; Brasil Fil: Rebelatto, Carmen K.. Pontificia Universidade Catolica Do Parana; Brasil Fil: Brofman, Paulo R.S.. Pontificia Universidade Catolica Do Parana; Brasil Fil: Sotelo-Silveira, Jose. Instituto de Investigaciones Biológicas "Clemente Estable"; Uruguay. Universidad de la República; Uruguay Fil: Dallagiovanna, Bruno. Fundación Oswaldo Cruz; Brasil

Stem cells can either differentiate into more specialized cells or undergo self-renewal. Several lines of evidence from different organisms suggest that these processes depend on the post-transcriptional regulation of gene expression. The presence of the PUF [Pumilio/FBF (fem-3 binding factor)] domain defines a conserved family of RNA binding proteins involved in repressing gene expression. It has been suggested that a conserved function of PUF proteins is to repress differentiation and sustain the mitotic proliferation of stem cells. In humans, Pumilio-2 (PUM2) is expressed in embryonic stem cells and adult germ cells. Here we show that PUM2 is expressed in a subpopulation of adipose-derived stem cell (ASC) cultures, with a granular pattern of staining in the cytoplasm. Protein levels of PUM2 showed no changes during the differentiation of ASCs into adipocytes. Moreover, RNAi knockdown of pum2 did not alter the rate of adipogenic differentiation compared with wild-type control cells. A ribonomic approach was used to identify PUM2-associated mRNAs. Microarray analysis showed that PUM2-bound mRNAs are part of gene networks involved in cell proliferation and gene expression control. We studied pum2 expression in cell cultures with low or very high levels of proliferation and found that changes in pum2 production were dependent on the proliferation status of the cell. Transient knockdown of pum2 expression by RNAi impaired proliferation of ASCs in vitro. Our results suggest that PUM2 does not repress differentiation of ASCs but rather is involved in the positive control of ASCs division and proliferation.

We detected Zika virus in breast milk of a woman in Brazil infected with the virus during the 36th week of pregnancy. Virus was detected 33 days after onset of signs and symptoms and 9 days after delivery. No abnormalities were found during fetal assessment or after birth of the infant.

Recent genomewide association studies have found multiple genetic variants on chromosome 8q24 that are significantly associated with an increased susceptibility to prostate, colorectal, and breast cancer. These risk loci are located in a ”gene desert,” a few hundred kilobases telomeric to the Myc gene. To date, the biological mechanism(s) underlying these associations remain unclear. It has been speculated that these 8q24 genetic variant(s) might affect Myc expression by altering its regulation or amplification status. Here, we show that multiple enhancer elements are present within this region and that they can regulate transcription of Myc. We also demonstrate that one such enhancer element physically interacts with the Myc promoter via transcription factor Tcf-4 binding and acts in an allele specific manner to regulate Myc expression.

We studied the subcellular distribution of mitochondria and superoxide dismutase-1 (SOD1) in whole mounts of microdissected motor axons of rats expressing the ALS-linked SOD1-G93A mutation. The rationale was to determine whether physical interactions between the enzyme and mitochondria were linked to the axonopathy of motor fibers occurring in amyotrophic lateral sclerosis (ALS). Mitochondria and SOD1 displayed a homogeneous distribution along motor axons both in nontransgenic rats and in those overexpressing wild-type SOD1. In contrast, axons from SOD1-G93A rats (older than 35 days) showed accumulation of mitochondria in discrete clusters located at regular intervals. Most of SOD1 immunoreactivity was enriched in these clusters and colocalized with mitochondria, suggesting a recruitment of SOD1-G93A to the organelle. The SOD1/mitochondrial clusters were abundant in motor axons but scarcely seen in sensory axons. Clusters also were stained for neuronal nitric oxide synthase, nitrotyrosine, and cytochrome c. The later also was detected surrounding clusters. Ubiquitin colocalized with clusters only at late stages of the disease. The cytoskeleton was not overtly altered in clusters. These results suggest that mutant SOD1 and defective mitochondria create localized dysfunctional domains in motor axons, which may lead to progressive axonopathy in ALS. Antioxid. Redox Signal. 11, 1535–1545.

Background Due to the absence of transcription initiation regulation of protein coding genes transcribed by RNA polymerase II, posttranscriptional regulation is responsible for the majority of gene expression changes in trypanosomatids. Therefore, cataloging the abundance of mRNAs (transcriptome) and the level of their translation (translatome) is a key step to understand control of gene expression in these organisms. Results Here we assess the extent of regulation of the transcriptome and the translatome in the Chagas disease causing agent, Trypanosoma cruzi, in both the non-infective (epimastigote) and infective (metacyclic trypomastigote) insect’s life stages using RNA-seq and ribosome profiling. The observed steady state transcript levels support constitutive transcription and maturation implying the existence of distinctive posttranscriptional regulatory mechanisms controlling gene expression levels at those parasite stages. Meanwhile, the downregulation of a large proportion of the translatome indicates a key role of translation control in differentiation into the infective form. The previously described proteomic data correlate better with the translatomes than with the transcriptomes and translational efficiency analysis shows a wide dynamic range, reinforcing the importance of translatability as a regulatory step. Translation efficiencies for protein families like ribosomal components are diminished while translation of the transialidase virulence factors is upregulated in the quiescent infective metacyclic trypomastigote stage. Conclusions A large subset of genes is modulated at the translation level in two different stages of Trypanosoma cruzi life cycle. Translation upregulation of virulence factors and downregulation of ribosomal proteins indicates different degrees of control operating to prepare the parasite for an infective life form. Taking together our results show that translational regulation, in addition to regulation of steady state level of mRNA, is a major factor playing a role during the parasite differentiation. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1563-8) contains supplementary material, which is available to authorized users.

To better understand the role of protein synthesis in axons, we have identified the source of a portion of axonal RNA. We show that proximal segments of transected sciatic nerves accumulate newly-synthesized RNA in axons. This RNA is synthesized in Schwann cells because the RNA was labeled in the complete absence of neuronal cell bodies both in vitro and in vivo. We also demonstrate that the transfer is prevented by disruption of actin and that it fails to occur in the absence of myosin-Va. Our results demonstrate cell-to-cell transfer of RNA and identify part of the mechanism required for transfer. The induction of cell-to-cell RNA transfer by injury suggests that interventions following injury or degeneration, particularly gene therapy, may be accomplished by applying them to nearby glial cells (or implanted stem cells) at the site of injury to promote regeneration.

Peripheral nerve axons of mammals have been demonstrated to contain ribosomes (Court et al., 2008, 2011, Kun et al. 2007, Li et al. 2005a and 2005b, Sotelo et al. 1999), as well as specific mRNAs that have been shown to concentrate in specific peripheral axonal domains (Koenig & Martin 1996, Koenig et al., 2000, Sotelo-Silveira et al., 2006, 2008), the so called PeriaxoplasmicRibosomal-Plaques (PARPs). Two possible origins have been proposed to supply mRNAs and ribosomes to axons and PARPs: a) from neuronal body axonal transport, or b) Schwann cell to axon trans-cellular transfer (Court et al. 2008, 2011, Sotelo-Silveira et al. 2006, Sotelo et al., to be published elsewhere). We showed that Schwann cell provide newly synthesized RNA (Bromouridine -BrUlabeled RNA) to the axon by a transcellular transfer process. This newly synthesized RNA was provided by Schwann cell nucleus and transported to the axon throughout Schmidt-Lanterman Incisures, and/or Nodes of Ranvier using the actin network, using molecular motors such as Myosin-Va. This was found in normal regenerating nerves disconnected from their neuronal body of origin, meaning that the only possible origin of this axonal RNA is the Schwann cell (to be published elsewhere).

Trypanosoma cruzi is the protozoan parasite causing American trypanosomiasis or Chagas disease, a neglected parasitosis with important human health impact in Latin America. The efficacy of current therapy is limited, and its toxicity is high. Since parasite proliferation is a fundamental target for rational drug design, we sought to progress into its understanding by applying a genome-wide approach. Treating a TcI linage strain with hydroxyurea, we isolated epimastigotes in late G1, S and G2/M cell cycle stages at 70% purity. The sequencing of each phase identified 305 stage-specific transcripts (1.5-fold change, p≤0.01), coding for conserved cell cycle regulated proteins and numerous proteins whose cell cycle dependence has not been recognized before. Comparisons with the parasite T. brucei and the human host reveal important differences. The meta-analysis of T. cruzi transcriptomic and ribonomic data indicates that cell cycle regulated mRNAs are subject to sub-cellular compartmentalization. Compositional and structural biases of these genes- including CAI, GC content, UTR length, and polycistron position- may contribute to their regulation. To discover nucleotide motifs responsible for the co-regulation of cell cycle regulated genes, we looked for overrepresented motifs at their UTRs and found a variant of the cell cycle sequence motif at the 3' UTR of most of the S and G2 stage genes. We additionally identified hairpin structures at the 5' UTRs of a high proportion of the transcripts, suggesting that periodic gene expression might also rely on translation initiation in T. cruzi. In summary, we report a comprehensive list of T. cruzi cell cycle regulated genes, including many previously unstudied proteins, we show evidence favoring a multi-step control of their expression, and we identify mRNA motifs that may mediate their regulation. Our results provide novel information of the T. cruzi proliferative proteins and the integrated levels of their gene expression control. Fil: Chávez, Santiago. Universidad del Uruguay. Facultad de Ciencias; Uruguay Fil: Eastman, Guillermo. Instituto de Investigaciones Biológicas "Clemente Estable"; Uruguay Fil: Smircich, Pablo. Universidad del Uruguay. Facultad de Ciencias; Uruguay Fil: Becco, Lorena Lourdes. Universidad del Uruguay. Facultad de Ciencias; Uruguay Fil: Oliveira Rizzo, Carolina. Universidad del Uruguay. Facultad de Ciencias; Uruguay Fil: Fort, Rafael. Universidad del Uruguay. Facultad de Ciencias; Uruguay Fil: Potenza, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular ; Argentina Fil: Garat, Beatriz. Universidad del Uruguay. Facultad de Ciencias; Uruguay Fil: Sotelo Silveira, Jos¨¦ Roberto. Universidad del Uruguay. Facultad de Ciencias; Uruguay Fil: Duhagon, María Ana. Universidad del Uruguay. Facultad de Ciencias; Uruguay

A new ovarian near-diploid cell line, OVDM1, was derived from a highly aneuploid serous ovarian metastatic adenocarcinoma. A metastatic tumor was obtained from a 47-year-old Ashkenazi Jewish patient three years after the first surgery removed the primary tumor, both ovaries, and the remaining reproductive organs. OVDM1 was characterized by cell morphology, genotyping, tumorigenic assay, mycoplasma testing, spectral karyotyping (SKY), and molecular profiling of the whole genome by aCGH and gene expression microarray. Targeted sequencing of a panel of cancer-related genes was also performed. Hierarchical clustering of gene expression data clearly confirmed the ovarian origin of the cell line. OVDM1 has a near-diploid karyotype with a low-level aneuploidy, but samples of the original metastatic tumor were grossly aneuploid. A number of single nucleotide variations (SNVs)/mutations were detected in OVDM1 and the metastatic tumor samples. Some of them were cancer-related according to COSMIC and HGMD databases (no founder mutations in BRCA1 and BRCA2 have been found). A large number of focal copy number alterations (FCNAs) were detected, including homozygous deletions (HDs) targeting WWOX and GATA4. Progression of OVDM1 from early to late passages was accompanied by preservation of the near-diploid status, acquisition of only few additional large chromosomal rearrangements and more than 100 new small FCNAs. Most of newly acquired FCNAs seem to be related to localized but massive DNA fragmentation (chromothripsis-like rearrangements). Newly developed near-diploid OVDM1 cell line offers an opportunity to evaluate tumorigenesis pathways/events in a minor clone of metastatic ovarian adenocarcinoma as well as mechanisms of chromothripsis.

Background DZIP1 (DAZ-interacting protein 1) has been described as a component of the Hh signaling pathway with a putative regulatory role in ciliogenesis. DZIP1 interacts with DAZ RNA binding proteins in embryonic stem cells and human germ cells suggesting a role in mRNA regulation. Results We investigated DZIP1 function in HeLa cells and its involvement in ribonucleoprotein complexes. DZIP1 was predominantly located in granules in the cytoplasm. Under oxidative stress conditions, DZIP1 re-localized to stress granules. DZIP appears to be important for the formation of stress granules during the stress response. We used immunoprecipitation assays with antibodies against DZIP1 and microarray hybridization to identify mRNAs associated with DZIP1. The genetic networks formed by the DZIP1-associated mRNAs were involved in cell cycle and gene expression regulation. DZIP1 is involved in the Hedgehog signaling pathway. We used cyclopamine, a specific inhibitor of this pathway, to analyze the expression of DZIP1 and its associated mRNAs. The abundance of DZIP1-associated mRNAs increased with treatment; however, the silencing or overexpression of DZIP1 in HeLa cells had no effect on the accumulation of the associated mRNAs. Polysomal profile analysis by sucrose gradient centrifugation demonstrated the presence of DZIP1 in the polysomal fraction. Conclusions Our results suggest that DZIP1 is part of an RNP complex that occupies various subcellular locations. The diversity of the mRNAs associated with DZIP1 suggests that this protein is a component of different RNPs associated with translating polysomes and with RNA granules.

Myosin-Va has been shown to have multiple functions in a variety of cell types, including a role in RNA transport in neurons. Using primary cultures of cells from organs of young dilute-lethal (Myo5ad-l/Myo5ad-l) null mutant mice and wild-type controls, we show that in some, but not all, tissues, RNA distribution is dramatically different in the homozygous null mutant cells. The dependence of RNA localization on myosin-Va correlates with the relative abundance of the brain-specific splicing pattern of the myosin-Va tail. We also show that myosin-Va is involved in RNA localization soon after synthesis, because the effects of its absence are diminished for RNAs that are more than 30 min old. Finally, we show that localization of β-actin mRNA is significantly changed by the absence of myosin-Va. These results suggest that myosin-Va is involved in a transient transport or tethering function in the perinuclear region. Cell Motil.

Background Tc38 of Trypanosoma cruzi has been isolated as a single stranded DNA binding protein with high specificity for the poly [dT-dG] sequence. It is present only in Kinetoplastidae protozoa and its sequence lacks homology to known functional domains. Tc38 orthologues present in Trypanosoma brucei and Leishmania were proposed to participate in quite different cellular processes. To further understand the function of this protein in Trypanosoma cruzi, we examined its in vitro binding to biologically relevant [dT-dG] enriched sequences, its expression and subcellular localization during the cell cycle and through the parasite life stages. Results By using specific antibodies, we found that Tc38 protein from epimastigote extracts participates in complexes with the poly [dT-dG] probe as well as with the universal minicircle sequence (UMS), a related repeated sequence found in maxicircle DNA, and the telomeric repeat. However, we found that Tc38 predominantly localizes into the mitochondrion. Though Tc38 is constitutively expressed through non-replicating and replicating life stages of T. cruzi, its subcellular localization in the unique parasite mitochondrion changes according to the cell cycle stage. In epimastigotes, Tc38 is found only in association with kDNA in G1 phase. From the S to G2 phase the protein localizes in two defined and connected spots flanking the kDNA. These spots disappear in late G2 turning into a diffuse dotted signal which extends beyond the kinetoplast. This later pattern is more evident in mitosis and cytokinesis. Finally, late in cytokinesis Tc38 reacquires its association with the kinetoplast. In non-replicating parasite stages such as trypomastigotes, the protein is found only surrounding the entire kinetoplast structure. Conclusions The dynamics of Tc38 subcellular localization observed during the cell cycle and life stages support a major role for Tc38 related to kDNA replication and maintenance.

In this paper we examine a new distance-based method for identifying and characterizing possible interactions between biological structures and objects, with respect to the initial developmental stages of Echinococcus granulosus. By adopting the surface of the foramen as the distance reference, several interesting results have been identified, including the fact that the cell nuclei tend to be organized with respect to the foramen surface as well as the stability of the spatial distribution of these nuclei along the development stages.

Molecular studies have shown several peculiarities in the regulatory mechanisms of gene expression in trypanosomatids. Protein coding genes are organized in long polycistronic units that seem to be constitutively transcribed. Therefore, post-transcriptional regulation of gene expression is considered to be the main point for control of transcript abundance and functionality. Here we describe the characterization of a 17 kDa RNA-binding protein from Trypanosoma cruzi (TcRBP19) containing an RNA recognition motive (RRM). This protein is coded by a single copy gene located in a high molecular weight chromosome of T. cruzi. Orthologous genes are present in the TriTryp genomes. TcRBP19 shows target selectivity since among the different homoribopolymers it preferentially binds polyC. TcRBP19 is a low expression protein only barely detected at the amastigote stage localizing in a diffuse pattern in the cytoplasm.

Periaxoplasmic ribosomal plaques (PARPs) are periodic cortical ribosomal domains, which are likely focal sites of translational activity along myelinated fibers. b-actin mRNA, and its trans-acting binding factor, ‘zip binding protein-1’ (ZBP-1), were localized in PARP domains of axoplasmic whole-mounts isolated from goldfish Mauthner, rabbit and rat spinal nerve root fibers. Although RNA and ZBP-1 fluorophores showed co-distributions, co-localization signals of overlapped RNA and ZBP-1 pixels were skewed and asymmetric, suggesting possible transport-related RNPs. RT-PCR of b-actin mRNA confirmed its presence in RNA extracted from axoplasm of single Mauthner fibers. A metabolically active in vitro isolated Mauthner fiber system, which required cyclic AMP (cAMP) to activate translation, was developed in order to probe the importance of actin cytoskeletal integrity on cycloheximide-sensitive [35S]met/cys incorporation into axoplasm. Pre-treatment of fiber segments with cytochalasin D inhibited protein synthesis in axoplasm very significantly, while effects on the associated myelin sheath were modest and not significant. We conclude that activation of translation in axoplasm can be modulated by cAMP, and that the integrity of the actin cyoskeleton is essential for optimal translation. We suggest that mechanisms for targeting and localizing b-actin mRNA to PARP domains may be similar to those proposed for dendritic subdomains.

We have established and characterized a new ovarian cancer cell line derived from stage IV serous ovarian adenocarcinoma metastatic tissue from a 47 year old Ashkenazi Jewish patient. The patient developed ovarian cancer at age 44 and died of the disease at age 48. The tissue was obtained after an extensive secondary cytoreductive intraperitoneal surgery. Prior to the surgery the patient underwent primary cytoreduction followed by several systemic therapies with recurrences. A culture of the metastatic tissue was transfected with Simian Virus 40 Large T-Antigen and telomerase. The cell line has been in culture on and off during 3 years. The identity of the cell line was confirmed by Short Tandem Repeat genotyping on genomic DNA extracted from early and late passages as well as from frozen metastatic tissue. The cell line was further characterized by array-Comparative Genomic Hybridization and gene expression microarray; and by Spectral Karyotyping and Fluorescent In Situ Hybridization. The results of these analyses were compared, when appropriate, with those of frozen metastatic tissue. A low level of genetic instability was detected in the cell line, but the karyotypes seem to be near diploid and relatively stable, after several years of cultivation. We believe that this novel ovarian cancer cell line could contribute to the understanding of the molecular genetic basis of the disease in this advanced stage, and be representative of the tumors of a great number of patients that become resistant to the majority if not all known cancer therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3265.

Background The cancer stem cell (CSC) hypothesis proposes that a population of tumor cells bearing stem cell properties is responsible for the origin and maintenance of tumors. Normal and cancer stem cells possess the ability to grow in vitro as self-renewing spheres, but the molecular basis of this phenotype remains largely unknown. We intended to establish a comprehensive culture system to grow prostatospheres (PSs) from both cancer cell lines and patient tumors. We then used gene expression microarrays to gain insight on the molecular pathways that sustain the PS tumor initiating cell (TIC) phenotype. Results Traditional stem cell medium (SCM) supplemented with Knockout™SR (KO) allows the propagation of monoclonal PSs from cell lines and primary cells. PSs display gene expression and tumorigenicity hallmarks of TICs. Gene expression analysis defined a gene signature composed of 66 genes that characterize LNCaP and patient PSs. This set includes novel prostate TIC growth factors (NRP1, GDF1, JAG1), proteins implicated in cell adhesion and cytoskeletal maintenance, transcriptional regulators (MYCBP, MYBL1, ID1, ID3, FOS, ELF3, ELF4, KLF2, KLF5) and factors involved in protein biosynthesis and metabolism. Meta-analysis in Oncomine reveals that some of these genes correlate with prostate cancer status and/or progression. Reporter genes and inhibitors indicate that the Notch pathway contributes to prostatosphere growth. Conclusions We have developed a model for the culture of PSs, and provide a genomic profile that support CSCs identity. This signature identifies novel markers and pathways that are predicted to correlate with prostate cancer evolution.

A growing body of metabolic and molecular evidence of an endogenous protein-synthesizing machinery in the mature axon is a challenge to the prevailing dogma that the latter is dependent exclusively on slow axoplasmic transport to maintain protein mass in a steady state. However, evidence for a systematic occurrence of ribosomes in mature vertebrate axons has been lacking until recently, when restricted ribosomal domains, called "periaxoplasmic plaques," were described in goldfish CNS myelinated axons. Comparable restricted RNA/ribosomal "plaque" domains now have been identified in myelinated axons of lumbar spinal nerve roots in rabbit and rat on the basis of RNase sensitivity of YOYO-1-binding fluorescence, immunofluorescence of ribosome-specific antibodies, and ribosome phosphorus mapping by electron spectroscopic imaging (ESI). The findings were derived from examination of the axoplasm isolated from myelinated fibers as axoplasmic whole mounts and delipidated spinal nerve roots. Ribosomal periaxoplasmic plaque domains in rabbit axons were typically narrow ( approximately 2 microm), elongated ( approximately 10 microm) sites that frequently were marked by a protruding structure. The domain complexity included an apparent ribosome-binding matrix. The small size, random distribution, and variable intermittent axial spacing of plaques around the periphery of axoplasm near the axon-myelin border are likely reasons why their systematic occurrence has remained undetected in ensheathed axons. The periodic but regular incidence of ribosomal domains provides a structural basis for previous metabolic evidence of protein synthesis in myelinated axons.