47 High-resolution ribosome profiling reveals translational selectivity in the mammalian blastocyst

2021 ◽  
Vol 33 (2) ◽  
pp. 130
Author(s):  
L. K. Zhu ◽  
H. Ming ◽  
S. C. Liu ◽  
R. Iyyappan ◽  
E. D. Llano ◽  
...  
Keyword(s):  
Ribosome Biogenesis ◽  
Embryo Implantation ◽  
Rna Isolation ◽  
Sucrose Density Gradient ◽  
Ribosome Profiling ◽  
Immunofluorescent Staining ◽  
Transcriptome Data ◽  
Bioinformatics Analyses ◽  
Low Input ◽  
Mammalian Embryos

Transcriptomic analyses of early mammalian embryos from multiple species have been comprehensively conducted in the last decade. However, mRNAs detected from overall transcriptomic profile of an embryo or a single cell do not necessarily represent their functional status, as there is a gap between the overall transcriptome and mRNAs that are actively translated. Ribosome profiling has been developed to infer the translational status of a specific mRNA species and thus analyse the genome-wide translatome. However, the broad application of ribosome profiling has been slowed by its complexity and the difficulty of adapting it to low-input samples such as embryos. In this study, we developed an ultra-low-input ribosome profiling protocol optimized for mammalian embryos and systematically analysed both polysome- and non-polysome-bound mRNA profiles of invitro-produced bovine blastocysts. Ten equal fractions were collected by means of sucrose density gradient and ultracentrifugation of lysates from 100 pooled blastocysts (n=2 pools), and subjected to RNA isolation and RNA sequencing. Our bioinformatics analyses of the mRNA profiles from each fraction along with the whole-transcriptome data revealed that compared with the overall transcriptome, there is a strong selection of mRNAs in the ribosome- and polysome-associated fractions, including transcriptional factors (e.g. POU5F1, ESRRB, AQP3, and APOA1) and genes involved in ribosome biogenesis, oxidative phosphorylation, and metabolic pathways, many of which are essential for the function of embryo implantation. We also identified novel epigenetic regulators selectively translated, including regulatory enzymes on histone modifications and RNA modifications (e.g. JMJD7, ALKBH4, ALKBH7, and METTL26). In addition, we confirmed the translation of the highly expressed, yet developmentally essential pathways in the blastocysts (e.g. Wnt and Notch signalling pathways). The selectively translated mRNAs were further validated by immunofluorescent staining at the protein level and cross-validated in both bovine and mouse blastocysts. Some of these genes show only modest expression in the overall transcriptome data. Their selective translation at the blastocyst stage is only being revealed by the ribosome fractions analyses, and their functions warrant future detailed investigations. In conclusion, this study reveals bona fide active translating mRNAs in the mammalian blastocyst. The low-input ribosome profiling protocol and the data presented here set an example and open future avenues for detailed ribosome fraction–based translatome analyses to reveal novel cellular/embryonic functional regulators beyond transcriptomic data.

scholarly journals Neuronal ribosomes exhibit dynamic and context-dependent exchange of ribosomal proteins

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Claudia M. Fusco ◽  
Kristina Desch ◽  
Aline R. Dörrbaum ◽  
Mantian Wang ◽  
Anja Staab ◽  
...  
Keyword(s):  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Ribosome Profiling ◽  
Metabolic Labeling ◽  
Local Protein Synthesis ◽  
Translational Machinery ◽  
Neuronal Synapses ◽  
Cellular Environment ◽  
Neuronal Processes ◽  
Local Protein

AbstractOwing to their morphological complexity and dense network connections, neurons modify their proteomes locally, using mRNAs and ribosomes present in the neuropil (tissue enriched for dendrites and axons). Although ribosome biogenesis largely takes place in the nucleus and perinuclear region, neuronal ribosomal protein (RP) mRNAs have been frequently detected remotely, in dendrites and axons. Here, using imaging and ribosome profiling, we directly detected the RP mRNAs and their translation in the neuropil. Combining brief metabolic labeling with mass spectrometry, we found that a group of RPs rapidly associated with translating ribosomes in the cytoplasm and that this incorporation was independent of canonical ribosome biogenesis. Moreover, the incorporation probability of some RPs was regulated by location (neurites vs. cell bodies) and changes in the cellular environment (following oxidative stress). Our results suggest new mechanisms for the local activation, repair and/or specialization of the translational machinery within neuronal processes, potentially allowing neuronal synapses a rapid means to regulate local protein synthesis.

scholarly journals Separation and Paired Proteome Profiling of Plant Chloroplast and Cytoplasmic Ribosomes

Plants ◽  
2020 ◽  
Vol 9 (7) ◽  
pp. 892
Author(s):  
Alexandre Augusto Pereira Firmino ◽  
Michal Gorka ◽  
Alexander Graf ◽  
Aleksandra Skirycz ◽  
Federico Martinez-Seidel ◽  
...  
Keyword(s):  
Ribosome Biogenesis ◽  
Gradient Centrifugation ◽  
Leaf Tissue ◽  
Proteome Analysis ◽  
Sucrose Density Gradient ◽  
Cross Linking ◽  
Sucrose Density Gradient Centrifugation ◽  
Cytoplasmic Ribosome ◽  
Plant Chloroplast

Conventional preparation methods of plant ribosomes fail to resolve non-translating chloroplast or cytoplasmic ribosome subunits from translating fractions. We established preparation of these ribosome complexes from Arabidopsis thaliana leaf, root, and seed tissues by optimized sucrose density gradient centrifugation of protease protected plant extracts. The method co-purified non-translating 30S and 40S ribosome subunits separated non-translating 50S from 60S subunits, and resolved assembled monosomes from low oligomeric polysomes. Combining ribosome fractionation with microfluidic rRNA analysis and proteomics, we characterized the rRNA and ribosomal protein (RP) composition. The identity of cytoplasmic and chloroplast ribosome complexes and the presence of ribosome biogenesis factors in the 60S-80S sedimentation interval were verified. In vivo cross-linking of leaf tissue stabilized ribosome biogenesis complexes, but induced polysome run-off. Omitting cross-linking, the established paired fractionation and proteome analysis monitored relative abundances of plant chloroplast and cytoplasmic ribosome fractions and enabled analysis of RP composition and ribosome associated proteins including transiently associated biogenesis factors.

88 MicroRNA profile of invitro bovine embryos cultured in the presence of amniotic extracellular vesicles shifts toward invivo-collected blastocysts

2020 ◽  
Vol 32 (2) ◽  
pp. 170
Author(s):  
A. Lange-Consiglio ◽  
B. Lazzari ◽  
F. Pizzi ◽  
A. Idda ◽  
F. Cremonesi ◽  
...  
Keyword(s):  
Pregnancy Rate ◽  
Extracellular Vesicles ◽  
Embryo Implantation ◽  
Principal Component ◽  
Rna Isolation ◽  
Differentially Expressed ◽  
Bovine Embryos ◽  
Total Rna ◽  
Mirna Profiling ◽  
Genomic Study

The absence of maternal-embryo signals could be an important cause of the poor pregnancy rates of invitro-produced embryos, compared with those collected invivo. In the context of paracrine communication, co-culture of embryo with amniotic-derived extracellular vesicles (EVs) improved their quality compared with control (CTR) (Perrini and Lange Consiglio 2018 Reprod. Fertil. Dev. 30, 658-671), and after cryopreservation, provided higher invitro embryo hatching and recipient pregnancy rate (Lange-Consiglio et al. 2019 Reprod. Fertil. Dev. 31, 155). After these results, the aim of this study was to evaluate microRNA (miRNA) profiling of invitro-produced blastocysts with or without EV supplementation, using invivo-produced blastocysts as CTR. Invitro embryos were produced based on our protocol (Perrini and Lange Consiglio 2018 Reprod. Fertil. Dev. 30, 658-671) with or without 100×106 EVsmL−1 in synthetic oviductal fluid with amino acids (SOFaa) on Day 5 post-fertilisation (Perrini and Lange Consiglio 2018 Reprod. Fertil. Dev. 30, 658-671). Grade 1 blastocysts (B7) were immediately snap frozen in liquid nitrogen for genomic study. These embryos were obtained from three replicates. Invivo embryos were obtained from three cows superovulated by Folltropin and inseminated by the same cryopreserved semen. After flushing, only B7 were snap frozen for genomic study. Samples for RNA isolation were obtained from 3 pools of 10 embryos each for each condition (vivo, vitro-CTR, and vitro+EVs). Total RNA was isolated by a NucleoSpin1 miRNA kit. Concentration and quality of RNA were determined by an Agilent 2100 Bioanalyzer. Libraries were prepared using TruSeq Small RNA Library Preparation kits (Illumina). Differential expression analyses between samples were run with the Bioconductor edgeR package (false discovery rate<0.05). MicroRNA cluster analysis was performed with Genesis. The average quantity of total RNA extracted from each pool was 3.5ng. Our results show that the miRNAs identified were 1.74E5, 2.3E5, and 3.6E5 for vivo, vitro-CTR, and vitro+EVs, respectively. Principal component analysis calculated on differentially expressed miRNAs showed a separation of the three groups with a distinctive miRNA trait. The miRNAs differentially expressed among three comparisons (vivo vs. vitro-CTR, vivo vs. vitro+EVs, and vitro-CTR vs. vitro+EVs) were 20, 15, and 2, respectively. Principal component 1, which explains 62.4% of the variance, clearly separates invivo- and invitro-produced embryos even if EV addition seems to ameliorate the effect of invitro production, and this agrees with the embryo quality and pregnancy rate after EV supplementation (Perrini and Lange Consiglio 2018 Reprod. Fertil. Dev. 30, 658-671; Lange-Consiglio et al. 2019 Reprod. Fertil. Dev. 31, 155). Indeed, vitro-CTR and vitro+EVs embryos differ significantly for two miRNAs (miR-130a, miR-181b) that are found to be higher in our vitro-CTR embryos compared with vitro+EV ones. The miR-181b was also found to be higher in degenerate bovine embryos compared with good blastocysts (Kropp et al. 2014 Front. Genetics 24, 91). In conclusion, this is the first study reporting the complete miRNA profiling of invitro blastocysts compared with those obtained invivo. The addition of EVs during invitro production seems to influence the expression of specific miRNAs involved in the success of embryo implantation.

scholarly journals Neuronal ribosomes dynamically exchange ribosomal proteins in a context-dependent manner.

2021 ◽  
Author(s):  
Claudia M. Fusco ◽  
Kristina Desch ◽  
Aline R. Doerrbaum ◽  
Mantian Wang ◽  
Anja Staab ◽  
...  
Keyword(s):  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Ribosome Profiling ◽  
Metabolic Labeling ◽  
Dependent Manner ◽  
Dense Network ◽  
Translational Machinery ◽  
Neuronal Synapses ◽  
Cellular Environment ◽  
Neuronal Processes

Owing to their morphological complexity and dense network connections, neurons modify their proteomes locally, using mRNAs and ribosomes present in the neuropil (tissue enriched for dendrites and axons). Although ribosome biogenesis largely takes place in the nucleus and perinuclear region, neuronal ribosomal protein (RP) mRNAs have been frequently detected remotely, in dendrites and axons. Here, using imaging and ribosome profiling, we directly detected the RP mRNAs and their translation in the neuropil. Combining brief metabolic labeling with mass spectrometry, we found that a group of RPs quickly associated with translating ribosomes in the cytoplasm and that this incorporation is independent of canonical ribosome biogenesis. Moreover, the incorporation probability of some RPs was regulated by location (neurites vs. cell bodies) and changes in the cellular environment (in response to oxidative stress). Our results suggest new mechanisms for the local activation, repair and/or specialization of the translational machinery within neuronal processes, potentially allowing remote neuronal synapses a rapid solution to the relatively slow and energy-demanding requirement of nuclear ribosome biogenesis.

scholarly journals Optimization of ribosome profiling using low-input brain tissue from fragile X syndrome model mice

2018 ◽  
Vol 47 (5) ◽  
pp. e25-e25 ◽  
Author(s):  
Botao Liu ◽  
Gemma Molinaro ◽  
Huan Shu ◽  
Emily E Stackpole ◽  
Kimberly M Huber ◽  
...  
Keyword(s):  
Fragile X Syndrome ◽  
Brain Tissue ◽  
Fragile X ◽  
Ribosome Profiling ◽  
Low Input

scholarly journals miR-26b-5p/TCF-4 Controls the Adipogenic Differentiation of Human Adipose-derived Mesenchymal Stem Cells

2020 ◽  
Vol 29 ◽  
pp. 096368972093441 ◽  
Author(s):  
Yadong Luo ◽  
Huan Ji ◽  
Yan Cao ◽  
Xu Ding ◽  
Meng Li ◽  
...  
Keyword(s):  
Adipogenic Differentiation ◽  
Negative Regulator ◽  
Immunofluorescent Staining ◽  
Luciferase Reporter ◽  
Bioinformatics Analyses ◽  
Protein Levels ◽  
Pathway Gene ◽  
Adipocytic Differentiation ◽  
The Impact

In this study, we assessed the ability of miR-26b-5p to regulate T cell factor 4 (TCF-4) expression and thereby control human adipose-derived mesenchymal stem cell (hADMSC) adipogenic differentiation. Adipogenic medium was used to induce hADMSC differentiation over a 6-d period. The ability of miR-26b-5p to interact with the TCF-4 mRNA was confirmed through both predictive bioinformatics analyses and luciferase reporter assays. Immunofluorescent staining was used to visualize the impact of miR-26b-5p inhibition or overexpression on TCF-4 and β-catenin levels in hADMSCs. Further functional analyses were conducted by transfecting these cells with siRNAs specific for TCF-4 and β-catenin. Adipogenic marker and Wnt/β-catenin pathway gene expression levels were assessed via real-time polymerase chain reaction and western blotting. β-catenin localization was assessed via immunofluorescent staining. As expected, our adipogenic media induced the adipocytic differentiation of hADMSCs. In addition, we confirmed that TCF-4 is an miR-26b-5p target gene in these cells, and that protein levels of both TCF-4 and β-catenin were reduced when these cells were transfected with miR-26b-5p mimics. Overexpression of this microRNA also enhanced hADMSC adipogenesis, whereas TCF-4 and β-catenin overexpression inhibited this process. The enhanced hADMSC adipogenic differentiation that was observed following TCF-4 or β-catenin knockdown was partially reversed when miR-26b-5p expression was inhibited. We found that miR-26b-5p serves as a direct negative regulator of TCF-4 expression within hADMSCs, leading to inactivation of the Wnt/β-catenin pathway and thereby promoting the adipogenic differentiation of these cells in vitro.

scholarly journals Translation Elongation Factor 4 (LepA) Contributes to Tetracycline Susceptibility by Stalling Elongating Ribosomes

2018 ◽  
Vol 62 (8) ◽  
Author(s):  
Bin Liu ◽  
Chunlai Chen
Keyword(s):  
Protein Synthesis ◽  
Ribosome Biogenesis ◽  
Molecular Mechanisms ◽  
Elongation Factor ◽  
Ribosome Profiling ◽  
Translation Elongation Factor ◽  
Translation Elongation ◽  
Content Type ◽  
Elongation Phase ◽  
Profiling Analysis

ABSTRACTEven though elongation factor 4 (EF4) is the third most conserved protein in bacteria, its physiological functions remain largely unknown and its proposed molecular mechanisms are conflicting among previous studies. In the present study, we show that the growth of anEscherichia colistrain is more susceptible to tetracycline than its EF4 knockout strain. Consistent with previous studies, our results suggested that EF4 affects ribosome biogenesis when tetracycline is present. Through ribosome profiling analysis, we discovered that EF4 causes 1-nucleotide shifting of ribosomal footprints on mRNA when cells have been exposed to tetracycline. In addition, when tetracycline is present, EF4 inhibits the elongation of protein synthesis, which leads to the accumulation of ribosomes in the early segment of mRNA. Altogether, when cells are exposed to tetracycline, EF4 alters both ribosome biogenesis and the elongation phase of protein synthesis.

scholarly journals Ribosome profiling reveals ribosome stalling on tryptophan codons and ribosome queuing upon oxidative stress in fission yeast

2020 ◽  
Author(s):  
Angela Rubio ◽  
Sanjay Ghosh ◽  
Michael Mülleder ◽  
Markus Ralser ◽  
Juan Mata
Keyword(s):  
Oxidative Stress ◽  
Stress Response ◽  
Fission Yeast ◽  
Translational Control ◽  
Ribosome Biogenesis ◽  
Mapk Pathway ◽  
Ribosome Profiling ◽  
Translation Elongation ◽  
Ribosome Stalling ◽  
Response To Stress

Abstract Translational control is essential in response to stress. We investigated the translational programmes launched by the fission yeast Schizosaccharomyces pombe upon five environmental stresses. We also explored the contribution of defence pathways to these programmes: The Integrated Stress Response (ISR), which regulates translation initiation, and the stress-response MAPK pathway. We performed ribosome profiling of cells subjected to each stress, in wild type cells and in cells with the defence pathways inactivated. The transcription factor Fil1, a functional homologue of the yeast Gcn4 and the mammalian Atf4 proteins, was translationally upregulated and required for the response to most stresses. Moreover, many mRNAs encoding proteins required for ribosome biogenesis were translationally downregulated. Thus, several stresses trigger a universal translational response, including reduced ribosome production and a Fil1-mediated transcriptional programme. Surprisingly, ribosomes stalled on tryptophan codons upon oxidative stress, likely due to a decrease in charged tRNA-Tryptophan. Stalling caused ribosome accumulation upstream of tryptophan codons (ribosome queuing/collisions), demonstrating that stalled ribosomes affect translation elongation by other ribosomes. Consistently, tryptophan codon stalling led to reduced translation elongation and contributed to the ISR-mediated inhibition of initiation. We show that different stresses elicit common and specific translational responses, revealing a novel role in Tryptophan-tRNA availability.

scholarly journals Long-Term Effects of Very Low Dose Particle Radiation on Gene Expression in the Heart: Degenerative Disease Risks

Cells ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 387
Author(s):  
Venkata Naga Srikanth Garikipati ◽  
Arsen Arakelyan ◽  
Eleanor A. Blakely ◽  
Polly Y. Chang ◽  
May M. Truongcao ◽  
...  
Keyword(s):  
Metabolic Diseases ◽  
Biological Response ◽  
Rna Isolation ◽  
Degenerative Disease ◽  
Low Doses ◽  
Long Term Effects ◽  
Bioinformatics Analyses ◽  
Particle Radiation ◽  
Tissue Samples ◽  
Linear Pattern

Compared to low doses of gamma irradiation (γ-IR), high-charge-and-energy (HZE) particle IR may have different biological response thresholds in cardiac tissue at lower doses, and these effects may be IR type and dose dependent. Three- to four-month-old female CB6F1/Hsd mice were exposed once to one of four different doses of the following types of radiation: γ-IR 137Cs (40-160 cGy, 0.662 MeV), 14Si-IR (4-32 cGy, 260 MeV/n), or 22Ti-IR (3-26 cGy, 1 GeV/n). At 16 months post-exposure, animals were sacrificed and hearts were harvested and archived as part of the NASA Space Radiation Tissue Sharing Forum. These heart tissue samples were used in our study for RNA isolation and microarray hybridization. Functional annotation of twofold up/down differentially expressed genes (DEGs) and bioinformatics analyses revealed the following: (i) there were no clear lower IR thresholds for HZE- or γ-IR; (ii) there were 12 common DEGs across all 3 IR types; (iii) these 12 overlapping genes predicted various degrees of cardiovascular, pulmonary, and metabolic diseases, cancer, and aging; and (iv) these 12 genes revealed an exclusive non-linear DEG pattern in 14Si- and 22Ti-IR-exposed hearts, whereas two-thirds of γ-IR-exposed hearts revealed a linear pattern of DEGs. Thus, our study may provide experimental evidence of excess relative risk (ERR) quantification of low/very low doses of full-body space-type IR-associated degenerative disease development.

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