The Trypanosoma cruzi nucleic acid binding protein Tc38 presents changes in the intramitochondrial distribution during the cell cycle

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.FIRCAFondo Clemente Estable (DICyT)FAPESConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)PROSULPEDECIBAAMSUD-PasteurFac Ciencias, Lab Interacc Mol, Montevideo, UruguayFac Med, Dept Genet, Montevideo, UruguayFac Ciencias, Dept Biol Celular & Mol, Montevideo, UruguayInst Invest Biol Clemente Estable Montevideo Urug, Dept Neurobiol Celular & Mol, Montevideo, UruguayUniv Nacl Gen San Martin, CONICET, INTECH, Inst Invest Biotecnol, Buenos Aires, DF, ArgentinaUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilSUNY Buffalo, Dept Microbiol & Immunol, Buffalo, NY 14260 USAInst Biol Mol Parana, Curitiba, Parana, BrazilUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilFIRCA: R03 TW05665-01Fondo Clemente Estable (DICyT): 7109Web of Scienc

Distinct small non-coding RNA landscape in the axons and released extracellular vesicles of developing primary cortical neurons and the axoplasm of adult nerves

Neurons have highlighted the needs for decentralized gene expression and specific RNA function in somato-dendritic and axonal compartments, as well as in intercellular communication via extracellular vesicles (EVs). Despite advances in miRNA biology, the identity and regulatory capacity of other small non-coding RNAs (sncRNAs) in neuronal models and local subdomains has been largely unexplored. We identified a highly complex and differentially localized content of sncRNAs in axons and EVs during early neuronal development of cortical primary neurons and in adult axons in vivo. This content goes far beyond miRNAs and includes most known sncRNAs and precisely processed fragments from tRNAs, sno/snRNAs, Y RNAs and vtRNAs. Although miRNAs are the major sncRNA biotype in whole-cell samples, their relative abundance is significantly decreased in axons and neuronal EVs, where specific tRNA fragments (tRFs and tRHs/tiRNAs) mainly derived from tRNAs Gly-GCC, Val-CAC and Val-AAC predominate. Notably, although 5ʹ-tRHs compose the great majority of tRNA-derived fragments observed in vitro, a shift to 3ʹ-tRNAs is observed in mature axons in vivo. The existence of these complex sncRNA populations that are specific to distinct neuronal subdomains and selectively incorporated into EVs, equip neurons with key molecular tools for spatiotemporal functional control and cell-to-cell communication

Brain transcriptomics of agonistic behaviour in the weakly electric fish Gymnotus omarorum, a wild teleost model of non-breeding aggression

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 < 0.05) and numerous genes that co-varied with behavioural traits. We also compared our results with previous reports of differential gene expression in other teleost species. Overall, our study establishes G. omarorum as a powerful model system for understanding the neuromolecular bases of social status independent of reproductive state

PDCD4 regulates axonal growth by translational repression of neurite growth-related genes and is modulated during nerve injury responses

© 2020 Di Paolo et al. 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

Nc886 is epigenetically repressed in prostate cancer and acts as a tumor suppressor through the inhibition of cell growth

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

Following Ribosome Footprints to Understand Translation at a Genome Wide Level

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 profilin

Myosin-Va-dependent cell-to-cell transfer of RNA from Schwann cells to axons.

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

Myosin-Va function is required for transfer of RNA from Schwann cells to axons.

<p>Longitudinal 10-µm sections of transected sciatic nerves from null (d-l) <i>Myo5a</i> mutant mice have reduced axoplasmic levels of newly synthesized RNA. <b>A and C</b>, null mutant; <b>B and D</b>, wild-type control. RNA labeled by BrU is shown in green, the paranodal marker Caspr in red. Panels C and D show higher magnification views of boxed regions in panels A and B respectively. Arrows, nodes of Ranvier; arrowheads, bands of Cajal (compare to arrows in Fig. 6). Micrographs are single optical sections from Z-stacks imaged with a laser scanning confocal microscope. Bar = 5 µm. <b>E</b>, linescan quantitation of abundance of BrU-labeled RNA across fibers from d-l mutant and wild-type control mice. Edges are the outer wraps of Schwann cells; center approximates the location of the axon. Intensity measurements were normalized to the mean of each linescan. Bars represent standard deviations. <b>F</b>, Absolute BrU fluorescence intensities in edges (as shown in <b>E</b>, 4 bins at each end combined; n = 160) and centers (10 central bins combined; n = 200). Error bars represent standard errors.</p