Axon micro-dissection and transcriptome profiling reveals the in vivo RNA content of fully differentiated myelinated motor axons

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 steady state. Recent studies have begun to identify the mRNAs localized in axons, which could be translated locally under different conditions. Despite that 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 system, we modified the axon micro-dissection 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, is 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 micro-dissection is a valuable method to obtain data at genome-wide levels of 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). Faria

Upstream ORFs influence translation efficiency in the parasite Trypanosoma cruzi

It is generally accepted that the presence of ORFs in the 5′ untranslated region of eukaryotic transcripts modulates the production of proteins by controlling the translation initiation rate of the main CDS. In trypanosomatid parasites, which almost exclusively depend on post-transcriptional mechanisms to regulate gene expression, translation has been identified as a key step. However, the mechanisms of control of translation are not fully understood. In the present work, we have annotated the 5′UTRs of the Trypanosoma cruzi genome both in epimastigotes and metacyclic trypomastigotes and, using a stringent classification approach, we identified putative regulatory uORFs in about 9% of the analyzed 5′UTRs. The translation efficiency (TE) and translational levels of transcripts containing putative repressive uORFs were found to be significantly reduced. These findings are supported by the fact that proteomic methods only identify a low number of proteins coded by transcripts containing repressive uORF. We additionally show that AUG is the main translation initiator codon of repressive uORFs in T. cruzi. Interestingly, the decrease in TE is more pronounced when the uORFs overlaps the main CDS. In conclusion, we show that the presence of the uORF and features such as initiation codon and/or location of the uORFs may be acting to fine tune translation levels in these parasites

Draft genome sequence of Cupriavidus UYMMa02A, a novel beta-rhizobium species

We present the draft genome of Cupriavidus UYMMa02A, a rhizobium strain isolated from root nodules of Mimosa magentea. The assembly has approximately 8.1 million bp with an average G+C of 64.1%. Symbiotic and metal-resistance genes were identified. The study of this genome will contribute to the understanding of rhizobial evolution

vtRNA2-1/nc886 produces a small RNA that contributes to its tumor suppression action through the microRNA pathway in prostate cancer

vtRNA2-1 is a vault RNA initially classified as microRNA precursor hsa-mir-886 and recently proposed as “nc886”, 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 “seed” in their 3′-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 actio

Hypoxia, acidification and oxidative stress in cells cultured at large distances from an oxygen source

Hypoxia is a condition frequently encountered by cells in tissues, whether as a normal feature of their microenvironment or subsequent to deregulated growth. Hypoxia can lead to acidifcation and increased oxidative stress, with profound consequences for cell physiology and tumorigenesis. Therefore, the interplay between hypoxia and oxidative stress is an important aspect for understanding the efects of hypoxic microenvironments on cells. We have used a previously developed variant of the method of coverslip-induced hypoxia to study the process of acidifcation in a hypoxic microenvironment and to simultaneously visualize intracellular levels of hypoxia and oxidative stress. We observed high accumulation of CO2 in hypoxic conditions, which we show is the main contributor to acidifcation in our model. Also, increased levels of oxidative stress were observed in moderately hypoxic cells close to the oxygen source, where the mitochondrial membrane potential was preserved. Conversely, cells at large distances from the oxygen source showed higher levels of hypoxia, milder oxidative stress and reduced mitochondrial membrane potential. Our results contribute to characterize the interplay between reduced oxygen levels, acidifcation and oxidative stress in a simple in vitro setting, which can be used to model cell responses to an altered environment, such as the early tumor microenvironment

Extensive translational regulation through the proliferative transition of Trypanosoma cruzi revealed by Multi-Omics

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 < 0.05), respectively. A major translational remodeling accompanied by an extensive proteome change is found, while the transcriptome remains largely unperturbed at the replicative entrance of the cell cycle. The differentially expressed genes comprise specific cell cycle processes, confirming previous findings while revealing candidate cell cycle regulators that undergo previously unnoticed translational regulation. Clusters of genes showing a coordinated regulation at translation and protein abundance share related biological functions such as cytoskeleton organization and mitochondrial metabolism; thus, they may represent posttranscriptional regulons. The translatome and proteome of the coregulated clusters change in both coupled and uncoupled directions, suggesting that complex cross talk between the two processes is required to achieve adequate protein levels of different regulons. This is the first simultaneous assessment of the transcriptome, translatome, and proteome of trypanosomatids, which represent a paradigm for the absence of transcriptional control. The findings suggest that gene expression chronology along the T. cruzi cell cycle is controlled mainly by translatome and proteome changes coordinated using different mechanisms for specific gene groups

Draft genome sequence and gene annotation of the uropathogenic bacterium Proteus mirabilis Pr2921

Here, we report the genome sequence of Proteus mirabilis Pr2921, a uropathogenic bacterium that can cause severe complicated urinary tract infections. After gene annotation, we identified two additional copies of ucaA, one of the most studied fimbrial protein genes, and other fimbriae related-proteins that are not present in P. mirabilis HI4320

Using a variant of coverslip hypoxia to visualize tumor cell alterations at increasing distances from an oxygen source

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

Estudio de los efectos de un microambiente hipóxico en queratinocitos humanos in vitro y su correlato con alteraciones del microambiente en la patología de liquen plano oral

La hipoxia es un factor fundamental en el proceso de génesis tumoral, así como en patologías precursoras de cáncer, como es el Liquen Plano Oral (LPO). Objetivo: Determinar si es posible establecer una correlación entre las alteraciones que sufren queratinocitos normales en un microambiente hipóxico in vitro y alteraciones que aparecen en los queratinocitos en el epitelio de la mucosa oral en el contexto de la patología LPO. Métodos: Se estudiaron los cambios morfológicos mediante microscopía de contraste de fases, y la detección de marcadores asociados a hipoxia de queratinocitos humanos (HaCaT), como modelo celular oral, en un microambiente hipóxico generado por la variante del método “Hipoxia inducida por cubreobjetos”. Resultados: Mediante microscopía confocal se observó la presencia de los marcadores de hipoxia GLUT-1 y aductos de pimonidazol (Hipoxyprobe) en los cultivos celulares de HaCaT expuestos a un microambiente hipóxico. Además, se observó la presencia del marcador GLUT-1 mediante inmunohistoquímica en tejido epitelial humano derivado de biopsias de la patología LPO. Conclusiones: Se estableció una correlación entre las alteraciones detectadas en queratinocitos humanos inducidos a un microambiente hipóxico in vitro y las alteraciones detectadas in vivo en tejido epitelial de la mucosa oral