scholarly journals LncRNAs interacting with the translation machinery contribute to human neuronal differentiation

2020 ◽  
Author(s):  
Katerina Douka ◽  
Isabel Birds ◽  
Dapeng Wang ◽  
Sophie Clayton ◽  
Abigail Byford ◽  
...  
Keyword(s):  
Neuronal Differentiation ◽  
Selective Constraint ◽  
Ribosome Profiling ◽  
Neuronal Function ◽  
Neurite Length ◽  
Translation Machinery ◽  
Differentiated Cells ◽  
High Sequence Conservation ◽  
Functional Consequences ◽  
Small Orfs

AbstractLncRNAs are less conserved, yet more tissue and developmental-stage specific than mRNAs and are particularly enriched in the nervous system of Drosophila melanogaster, mouse and human. The function of cytoplasmic lncRNAs and their potential translation remains poorly understood. Here we performed Poly-Ribo-Seq to understand the interaction of lncRNAs with the translation machinery and the functional consequences during neuronal differentiation of SH-SH5Y cells. We discovered 237 cytoplasmic lncRNAs upregulated during early neuronal differentiation, most of which are associated with polysome complexes. The majority are cytoplasmically enriched and are intergenic or anti-sense. In addition, we find 45 small ORFs in lncRNAs to be actively translated, 17 specifically upon differentiation. 11 of these smORFs exhibit high sequence conservation across Hominidae suggesting they are under strong selective constraint with putative function in this clade. We discover LINC01116 is induced upon differentiation and contains an 87 codon smORF, which we detect as translated, with increased ribosome profiling signal upon differentiation. The LINC01116 peptide exhibits a cytoplasmic distribution and is detected in neurites. Knockdown of LINC01116 results in significant reduction of neurite length in differentiated cells indicating it contributes to neuronal differentiation. Our findings indicate lncRNAs are a source of non-canonical peptides and contribute to neuronal function.

scholarly journals EFFECT OF M2 MACROPHAGE-DERIVED SOLUBLE FACTORS ON DIFFERENTIATION OF SH-SY5Y CELLS

2021 ◽  
Vol 23 (4) ◽  
pp. 677-684
Author(s):  
I. M. Rashchupkin ◽  
E. Ya. Shevela ◽  
E. R. Chernykh
Keyword(s):  
Retinoic Acid ◽  
Conditioned Medium ◽  
Neuronal Differentiation ◽  
Macrophage Polarization ◽  
Relative Content ◽  
M2 Macrophage ◽  
Neurite Length ◽  
Soluble Factors ◽  
Differentiated Cells ◽  
Morphological Criteria

Macrophages play a key role in triggering and regulation of neuroregeneration. The characteristic feature of macrophages is pronounced plasticity, which manifests itself in the ability of macrophages to change their functional phenotype depending on the micromilieu. Apoptotic cell clearance (efferocytosis) is an important inducer of a macrophage polarization to M2 phenotype under pathological settings. Previously, we have developed an original protocol for the generation of M2-like macrophages, polarized by efferocytosis under serum-deprived conditions (M2 (LS), Low Serum). The present study was aimed to assess a neuroregenerative potential of M2 (LS) macrophages. We studied their effect on the differentiation of SH-SY5Y cells in comparison with retinoic acid (RA). As the morphological criteria of differentiation we have assessed the relative content of differentiated cells, i.e., cells with a neurite length exceeding the cell body length, and the average neurite length on days 3, 7, and 13. The ratio of neuron-like (N-type) and epithelial-like (S-type) cells in cultures was also assessed. SH-SY5Y cells were characterized by a low level of spontaneous differentiation, both under standard conditions (10% FBS) and serum deprivation (1% FBS). Upon RA treatment, SH-SY5Y cells stopped proliferating and underwent neuronal differentiation. Cultivation of SH-SY5Y cells in the presence of M2 (LS) conditioned medium also led to a significant increase in the relative content of differentiated cells, the average length of neurite-like processes, as well as a change in the balance of S- and N-type cells towards a pronounced predominance of the latter. The morphological features of differentiation were significantly less pronounced at early stage (day 3) of differentiation as compared with the RA-induced changes and reached the level of positive control only at later stages (day 13) (p < 0.05). In contrast to retinoic acid, M2 (LS) conditioned medium induced neuronal differentiation of SH-SY5Y cells without suppressing their proliferative activity. The data obtained may indicate a high neuroregenerative potential of M2 macrophages in vitro, which is realized through soluble factors and manifests itself in promoting SH-SY5Y differentiation. 

Comparative analysis of protein-coding and long non-coding transcripts based on RNA sequence features

2018 ◽  
Vol 16 (02) ◽  
pp. 1840013 ◽  
Author(s):  
Oxana A. Volkova ◽  
Yury V. Kondrakhin ◽  
Timur A. Kashapov ◽  
Ruslan N. Sharipov
Keyword(s):  
Position Weight Matrix ◽  
Ribosome Profiling ◽  
Messenger Rnas ◽  
Putative Orfs ◽  
Protein Coding ◽  
Rna Translation ◽  
Cell Life ◽  
Experimental Tool ◽  
Non Coding Rnas ◽  
Small Orfs

RNA plays an important role in the intracellular cell life and in the organism in general. Besides the well-established protein coding RNAs (messenger RNAs, mRNAs), long non-coding RNAs (lncRNAs) have gained the attention of recent researchers. Although lncRNAs have been classified as non-coding, some authors reported the presence of corresponding sequences in ribosome profiling data (Ribo-seq). Ribo-seq technology is a powerful experimental tool utilized to characterize RNA translation in cell with focus on initiation (harringtonine, lactimidomycin) and elongation (cycloheximide). By exploiting translation starts obtained from the Ribo-seq experiment, we developed a novel position weight matrix model for the prediction of translation starts. This model allowed us to achieve 96% accuracy of discrimination between human mRNAs and lncRNAs. When the same model was used for the prediction of putative ORFs in RNAs, we discovered that the majority of lncRNAs contained only small ORFs ([Formula: see text][Formula: see text]nt) in contrast to mRNAs.

scholarly journals MBRS-32. TOPOISOMERASE II β INDUCES NEURONAL, BUT NOT GLIAL, DIFFERENTIATION IN MEDULLOBLASTOMA

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii404-iii404
Author(s):  
Hiroaki Miyahara ◽  
Manabu Natsumeda ◽  
Junichi Yoshimura ◽  
Yukihiko Fujii ◽  
Akiyoshi Kakita ◽  
...  
Keyword(s):  
Histone Modification ◽  
Neuronal Differentiation ◽  
Sonic Hedgehog ◽  
Topoisomerase Ii ◽  
Therapeutic Targets ◽  
Glial Differentiation ◽  
Favorable Prognosis ◽  
Differentiated Cells ◽  
Histone Modification Enzymes ◽  
Hedgehog Signal

Abstract BACKGROUND We previously reported that Gli3, which was a downstream molecule of Sonic Hedgehog signal, induced neuronal and/or glial differentiation in some types of medulloblastoma (desmoplastic/nodular medulloblastoma and medulloblastoma with extensive nodularity), and patients of medulloblastoma with neuronal differentiation showed favorable prognosis, but those with glial differentiation tended to show miserable prognosis (Miyahara H, Neuropathology, 2013). This time, we focused on Topoisomerase II β (Top2β), which was reported to induce neuronal differentiation and inhibit glial differentiation, and examined the expression of Top2β in medulloblastomas with neuronal and glial differentiations. METHODS We assessed the expression of Top2β, NeuN, and GFAP using triple fluorescent immunostaining method in medulloblastoma samples with both neuronal and glial differentiations. Furthermore, the expression of Top2β, H3K4me2, and H3K27me3 were also assessed, because Top2βwas positively or negatively regulated by H3K4me2 and H3K27me3, respectively. RESULTS Many large nuclei in the nodules, in which differentiated cells were seen, was visualized by Top2β. The Top2β signals were seen in NeuN+ cells but not GFAP+ cells. H3K4me2 signals were visualized in Top2β+ large nuclei, but H3K27me3 and NeuN+ large nuclei were distributed independently. CONCLUSIONS These results indicate that Top2β may be a molecule associated with neuronal, but not glial, differentiation of medulloblastoma cells. Drugs targeting histone modification enzymes such as EZH2 inhibitors are possible therapeutic targets as a differentiation-inducing therapy for medulloblastoma.

scholarly journals The Synthetic Cannabinoids THJ-2201 and 5F-PB22 Enhance In Vitro CB1 Receptor-Mediated Neuronal Differentiation at Biologically Relevant Concentrations

2020 ◽  
Vol 21 (17) ◽  
pp. 6277
Author(s):  
João Alexandre ◽  
Rui Malheiro ◽  
Diana Dias da Silva ◽  
Helena Carmo ◽  
Félix Carvalho ◽  
...  
Keyword(s):  
Neuronal Differentiation ◽  
Synthetic Cannabinoids ◽  
In Vitro Differentiation ◽  
Neurite Length ◽  
Neuronal Marker ◽  
Biologically Relevant ◽  
Specific Antagonist ◽  
Recreational Use ◽  
Potential Onset

Recreational use of synthetic cannabinoids (SCs) before and during pregnancy poses a major public health risk, due to the potential onset of neurodevelopmental disorders in the offspring. Herein, we report the assessment of the neurotoxic potential of two commonly abused SCs, THJ-2201 and 5F-PB22, particularly focusing on how they affect neuronal differentiation in vitro. Differentiation ratios, total neurite length, and neuronal marker expression were assessed in NG108-15 neuroblastoma x glioma cells exposed to the SCs at non-toxic, biologically relevant concentrations (≤1 μM), either in acute or repeated exposure settings. Both SCs enhanced differentiation ratios and total neurite length of NG108-15 cells near two-fold compared to vehicle-treated cells, in a CB1R activation-dependent way, as the CB1R blockade with a specific antagonist (SR141718) abrogated SC-induced effects. Interestingly, repeated 5F-PB22 exposure was required to reach effects similar to a single THJ-2201 dose. Cell viability and proliferation, mitochondrial membrane potential, and intracellular ATP levels were also determined. The tested SCs increased mitochondrial tetramethyl rhodamine ethyl ester (TMRE) accumulation after 24 h at biologically relevant concentrations but did not affect any of the other toxicological parameters. Overall, we report firsthand the CB1R-mediated enhancement of neurodifferentiation by 5F-PB22 and THJ-2201 at biologically relevant concentrations.

The role of noradrenaline in the differentiation of amphibian embryonic neurons

Development ◽  
1993 ◽  
Vol 119 (4) ◽  
pp. 1343-1357
Author(s):  
S.J. Rowe ◽  
N.J. Messenger ◽  
A.E. Warner
Keyword(s):  
Nervous System ◽  
Neuronal Differentiation ◽  
Adrenergic Receptor ◽  
Immunocytochemical Staining ◽  
Tail Bud ◽  
Receptor Antagonists ◽  
Alpha Adrenergic Receptor ◽  
Differentiated Cells ◽  
Neurula Stage ◽  
Fertilized Egg

The possibility that monoamines might act as signalling molecules during the early development of the nervous system has been examined in embryos of the amphibian Xenopus laevis. The distributions of 5-hydroxytryptamine, dopamine, noradrenaline and their precursor, dopa, were determined from the fertilized egg up to the late neurula stages using High Performance Liquid Chromatography, formaldehyde-induced fluorescence and antibody staining. 5-hydroxytryptamine was not detected until the tail bud stage. The fertilized egg contained significant concentrations of dopa (10(−6) M) and dopamine (10(−7) M). Both monoamines persisted with little change in concentration up to the late neurula stage. Early neurula stage embryos contained very low levels of noradrenaline. Aldehyde-induced fluorescence showed that monoamines are localized in dorsal regions of the embryo, in ectoderm and mesoderm cells. Monoamines were not present in endoderm cells. Immunocytochemical staining showed dopamine predominantly in the ectoderm, except in future neural regions where it was found also in the mesoderm. Dopamine staining was always most intense in dorsal regions of the embryo. The consequences for subsequent neuronal differentiation of interfering with the biosynthesis and receptor binding of monoamines during neurulation was assayed. Neuronal differentiation was monitored quantitatively in cultures set up as the neural tube closed and qualitatively in intact tadpoles that were left to develop for two days after washout of test reagent. The number of neurons, the number of muscle cells and the total number of differentiated cells were counted after 18–24 hours of culture. Comparison of the number of neurons that differentiated from control and treated embryos showed that inhibition of dopamine beta-hydroxylase, the enzyme catalysing the conversion of dopamine to noradrenaline, during the neural plate stages reduced substantially subsequent neuronal differentiation. The differentiation of myocytes and the total number of differentiated cells were not affected. Exogenous noradrenaline (10(−6) M) or dopamine (10(−6) M) could increase the number of neurons that differentiated subsequently in culture. Interfering with noradrenaline binding to receptors with receptor antagonists during neurulation showed that alpha-adrenergic receptor antagonists reduced substantially the subsequent differentiation of neurons. The differentiation of myocytes and the total number of differentiated cells were not affected. The effect of alpha-adrenergic receptor antagonists was overcome by the simultaneous inclusion of noradrenaline or alpha-receptor agonists, but not agonists at beta-adrenergic receptors. The quantitative reduction in the differentiation of neurons was paralleled by defects in the Central Nervous System of intact tadpoles.(ABSTRACT TRUNCATED AT 400 WORDS)

The bHLH gene Hes6, an inhibitor of Hes1, promotes neuronal differentiation

Development ◽  
2000 ◽  
Vol 127 (13) ◽  
pp. 2933-2943 ◽  
Author(s):  
S. Bae ◽  
Y. Bessho ◽  
M. Hojo ◽  
R. Kageyama
Keyword(s):  
Cell Differentiation ◽  
Neuronal Differentiation ◽  
Rod Photoreceptor ◽  
Bhlh Gene ◽  
Basic Helix Loop Helix ◽  
Helix Loop Helix ◽  
Differentiated Cells ◽  
Loop Region ◽  
The Family ◽  
Enhancer Of Split

We have isolated the basic helix-loop-helix (bHLH) gene Hes6, a novel member of the family of mammalian homologues of Drosophila hairy and Enhancer of split. Hes6 is expressed by both undifferentiated and differentiated cells, unlike Hes1, which is expressed only by the former cells. Hes6 alone does not bind to the DNA but suppresses Hes1 from repressing transcription. In addition, Hes6 suppresses Hes1 from inhibiting Mash1-E47 heterodimer and thereby enables Mash1 and E47 to upregulate transcription in the presence of Hes1. Furthermore, misexpression of Hes6 with retrovirus in the developing retina promotes rod photoreceptor differentiation, like Mash1, in sharp contrast to Hes1, which inhibits cell differentiation. These results suggest that Hes6 is an inhibitor of Hes1, supports Mash1 activity and promotes cell differentiation. Mutation analysis revealed that Hes1- and Hes6-specific functions are, at least in part, interchangeable by alteration of the loop region, suggesting that the loop is not simply a nonfunctional spacer but plays an important role in the specific functions.

scholarly journals Super-resolution ribosome profiling reveals unannotated translation events in Arabidopsis

2016 ◽  
Vol 113 (45) ◽  
pp. E7126-E7135 ◽  
Author(s):  
Polly Yingshan Hsu ◽  
Lorenzo Calviello ◽  
Hsin-Yen Larry Wu ◽  
Fay-Wei Li ◽  
Carl J. Rothfels ◽  
...  
Keyword(s):  
Global Analysis ◽  
Super Resolution ◽  
Mrna Translation ◽  
Ribosome Profiling ◽  
Optimization Strategy ◽  
Plant Genomics ◽  
Reading Frame ◽  
Periodic Property ◽  
Evolutionarily Conserved ◽  
Small Orfs

Deep sequencing of ribosome footprints (ribosome profiling) maps and quantifies mRNA translation. Because ribosomes decode mRNA every 3 nt, the periodic property of ribosome footprints could be used to identify novel translated ORFs. However, due to the limited resolution of existing methods, the 3-nt periodicity is observed mostly in a global analysis, but not in individual transcripts. Here, we report a protocol applied to Arabidopsis that maps over 90% of the footprints to the main reading frame and thus offers super-resolution profiles for individual transcripts to precisely define translated regions. The resulting data not only support many annotated and predicted noncanonical translation events but also uncover small ORFs in annotated noncoding RNAs and pseudogenes. A substantial number of these unannotated ORFs are evolutionarily conserved, and some produce stable proteins. Thus, our study provides a valuable resource for plant genomics and an efficient optimization strategy for ribosome profiling in other organisms.

scholarly journals RiboNT: A Noise-Tolerant Predictor of Open Reading Frames from Ribosome-Protected Footprints

Life ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 701
Author(s):  
Bo Song ◽  
Mengyun Jiang ◽  
Lei Gao
Keyword(s):  
Ribosome Profiling ◽  
Open Reading Frames ◽  
Model Organisms ◽  
Widespread Application ◽  
Optimized Protocol ◽  
Membrane Bound ◽  
Noise Tolerant ◽  
Diverse Plant Species ◽  
Small Orfs ◽  
Reading Frames

Ribo-seq, also known as ribosome profiling, refers to the sequencing of ribosome-protected mRNA fragments (RPFs). This technique has greatly advanced our understanding of translation and facilitated the identification of novel open reading frames (ORFs) within untranslated regions or non-coding sequences as well as the identification of non-canonical start codons. However, the widespread application of Ribo-seq has been hindered because obtaining periodic RPFs requires a highly optimized protocol, which may be difficult to achieve, particularly in non-model organisms. Furthermore, the periodic RPFs are too short (28 nt) for accurate mapping to polyploid genomes, but longer RPFs are usually produced with a compromise in periodicity. Here we present RiboNT, a noise-tolerant ORF predictor that can utilize RPFs with poor periodicity. It evaluates RPF periodicity and automatically weighs the support from RPFs and codon usage before combining their contributions to identify translated ORFs. The results demonstrate the utility of RiboNT for identifying both long and small ORFs using RPFs with either good or poor periodicity. We implemented the pipeline on a dataset of RPFs with poor periodicity derived from membrane-bound polysomes of Arabidopsis thaliana seedlings and identified several small ORFs (sORFs) evolutionarily conserved in diverse plant species. RiboNT should greatly broaden the application of Ribo-seq by minimizing the requirement of RPF quality and allowing the use of longer RPFs, which is critical for organisms with complex genomes because these RPFs can be more accurately mapped to the position from which they were derived.

scholarly journals Genes with 5′ terminal oligopyrimidine tracts preferentially escape global suppression of translation by the SARS-CoV-2 Nsp1 protein

2020 ◽  
Author(s):  
Shilpa Rao ◽  
Ian Hoskins ◽  
P. Daniela Garcia ◽  
Tori Tonn ◽  
Hakan Ozadam ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Ribosome Profiling ◽  
Fine Tuning ◽  
Host Protein ◽  
Structural Protein ◽  
Translation Machinery ◽  
Human Genes ◽  
Repressive Effect

AbstractViruses rely on the host translation machinery to synthesize their own proteins. Consequently, they have evolved varied mechanisms to co-opt host translation for their survival. SARS-CoV-2 relies on a non-structural protein, Nsp1, for shutting down host translation. Despite this, it is currently unknown how viral proteins and host factors critical for viral replication can escape a global shutdown of host translation. Here, using a novel FACS-based assay called MeTAFlow, we report a dose-dependent reduction in both nascent protein synthesis and mRNA abundance in cells expressing Nsp1. We perform RNA-Seq and matched ribosome profiling experiments to identify gene-specific changes both at the mRNA expression and translation level. We discover a functionally-coherent subset of human genes preferentially translated in the context of Nsp1 expression. These genes include the translation machinery components, RNA binding proteins, and others important for viral pathogenicity. Importantly, we uncovered a remarkable enrichment of 5′ terminal oligo-pyrimidine (TOP) tracts among preferentially translated genes. Using reporter assays, we validated that 5’ UTRs of TOP transcripts can drive preferential protein synthesis in the presence of NSP1. Collectively, our study suggests fine tuning of host gene expression and translation by Nsp1 despite its global repressive effect on host protein synthesis.

scholarly journals Neuronal differentiation strategies: insights from single-cell sequencing and machine learning

Development ◽  
2020 ◽  
Vol 147 (23) ◽  
pp. dev193631
Author(s):  
Nikolaos Konstantinides ◽  
Claude Desplan
Keyword(s):  
Machine Learning ◽  
Single Cell ◽  
Neuronal Differentiation ◽  
Cell Types ◽  
Specific Cell ◽  
Single Cell Sequencing ◽  
Differentiated Cells ◽  
History Of ◽  
Induced Pluripotent

ABSTRACTNeuronal replacement therapies rely on the in vitro differentiation of specific cell types from embryonic or induced pluripotent stem cells, or on the direct reprogramming of differentiated adult cells via the expression of transcription factors or signaling molecules. The factors used to induce differentiation or reprogramming are often identified by informed guesses based on differential gene expression or known roles for these factors during development. Moreover, differentiation protocols usually result in partly differentiated cells or the production of a mix of cell types. In this Hypothesis article, we suggest that, to overcome these inefficiencies and improve neuronal differentiation protocols, we need to take into account the developmental history of the desired cell types. Specifically, we present a strategy that uses single-cell sequencing techniques combined with machine learning as a principled method to select a sequence of programming factors that are important not only in adult neurons but also during differentiation.

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